Original Paper
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file on Synergy |
Acta Biochim Biophys
Sin 2008, 40: 116-124
doi:10.1111/j.1745-7270.2008.00384.x
Co-expression of IL-18 binding
protein and IL-4 regulates Th1/Th2 cytokine response in murine collagen-induced
arthritis
Jianhang Leng1*,
Hangping Yao2, Junya Shen1, Keyi Wang1,
Guangchao Zhuo1, and Ziwei Wang1
1 Center of
Clinical Experimental Medicine, The First People’s Hospital of Hangzhou,
Hangzhou 310006, China
2 The First
Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003,
China
Received: August 4,
2007
Accepted: October
30, 2007
This work was
supported by grants from the Science and Technology Foundation of Zhejiang
Province (No. 2005C33008) and the Medical and Health Science Foundation of
Zhejiang Province (No. 2004B069), China
*Corresponding
author: Tel, 86-571-87065701, ext 10544; Fax, 86-571-87065701, ext 10549;
E-mail, [email protected]
We
constructed a recombinant adenoviral vector containing a murine interleukin
(IL)-18 binding protein (mIL-18BP) and murine IL-4 (mIL-4) fusion gene (AdmIL-18BP/mIL-4)
and used a gene therapy approach to investigate the role of IL-18BP and IL-4 in
modulating the T-helper1 and T-helper2 (Th1/Th2) balance in mice with
collagen-induced arthritis (CIA). Mice with CIA were intra-articularly injected
with 107 pfu/6 ml of either
AdmIL-18BP/mIL-4, or a control adenovirus, or with the control vehicle
(phosphate-buffered saline). After intra-articular gene therapy with
AdmIL-18BP/mIL-4, the serum levels of tumor necrosis factor-a (TNF-a), g-interferon
(IFN-g), IL-4,
IL-10, and IL-18 in mice with CIA were assessed by ELISA. IFN-g-expressing
and IL-4-expressing CD4+ T cells from mice splenocytes were
monitored by flow cytometry. Mice with CIA at weeks 1, 2, and 4 after
intra-articular injection of AdmIL-18BP/mIL-4 showed significantly increased
serum concentrations of IL-4 and IL-10 (P<0.01 at all time points) but greatly decreased serum concentrations of IFN-g, TNF-a, and IL-18 (P<0.01 at all time points) compared to both the control adenovirus and phosphate-buffered saline control groups. The percentage of IFN-g-producing CD4+
T cells was significantly decreased in response to local AdmIL-18BP/mIL-4
treatment. The percentage of IL-4-producing CD4+ T cells
increased significantly at 1 week after local injection of AdmIL-18BP/mIL-4
then returned to normal by week 4. These data indicated the significant
modifying effects on the Th1/Th2 imbalance in murine CIA produced by local
overexpression of IL-18BP and IL-4. Combination treatment with IL-18BP and IL-4
is a promising potential therapy for rheumatoid arthritis.
Keywords interleukin-18 binding protein; interleukin-4; gene
therapy; rheumatoid arthritis
A critical advance in cellular immunology has been the discovery of functionally
distinct T-cell subsets, T-helper1 (Th1) and T-helper2 (Th2), separated on the
basis of their cytokine expression. Th1 cells mainly secrete g-interferon
(IFN-g), interleukin (IL)-2, and tumor necrosis factor-a (TNF-a), whereas Th2
cells generally produce IL-4, IL-5, and IL-10. The balance of Th1 and Th2
subsets is implicated in the regulation of many immune responses [1]. Th1
cytokines have been linked to the pathogenesis of autoimmune diseases,
including animal models [2,3], in which a T-cell response against an unknown
self-antigen might play a role. In contrast, the Th2-like cytokines IL-4 and
IL-10 down-regulate inflammation in these models [4].IL-4 is a pleiotropic cytokine that plays a number of important
roles, including the regulation of inflammation [5]. IL-4 acts as an autocrine
growth factor promoting the differentiation of naive T cells to Th2 cells. It
also inhibits the differentiation of naive T cells to Th1 as well as cytokine
production by Th1 cells [6]. IL-18 is a cytokine with powerful Th1-promoting
activity in synergy with IL-12 [7]. IL-18 induces proliferation, up-regulates
IL-2 receptor antagonist (IL-2Ra) expression, and promotes IFN-g, TNF-a, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) production by Th1
clones [8]. Data indicate that IL-18 is capable of promoting a severe, erosive
inflammatory polyarthropathy in a murine model of inflammatory arthritis. An
IL-18 binding protein (IL-18BP) is a member of a novel family of soluble
proteins that also includes several poxvirus-encoded putative proteins. It is
constitutively expressed in lymphoid tissues and can bind to IL-18, thus
blocking its biological activity and limiting the contribution of IL-18 to Th1
responses [9].Rheumatoid arthritis (RA) is an autoimmune disorder characterized by
chronic synovitis of multiple joints normally leading to destruction of joint
cartilage and erosion of bone. The Th1 and Th2 cytokine balance has attracted
great interest as it is hypothesized that the degree of polarization and
heterogeneity of T-cell lymphocytes could be important to the initiation and
perpetuation of synovial inflammation [10]. Using highly sensitive techniques,
several investigators have found a preferential activation of Th1 cells in
rheumatoid synovium, suggesting that Th1, rather than Th2, cytokines are
involved in the pathogenesis of the disease [11].Bypassing the initiating factors in RA and manipulating the cytokine
balance might be an effective therapeutic means by which chronic inflammation
can be managed [12]. It is promising for the combination of RA treatment with
gene therapy as a means for agent delivery, making the appropriate selection of
candidate therapeutic proteins essential [13–15]. In the present study, we constructed a recombinant adenoviral vector
containing a murine IL-18BP (mIL-18BP) and murine IL-4 (mIL-4) fusion gene
(AdmIL-18BP/mIL-4). We used this adenoviral gene therapy in murine collagen-induced
arthritis (CIA), an autoimmune model of RA, to increase the expression of
IL-18BP and IL-4 in inflamed joints. We postulated that adenovirally-produced
IL-4 and IL-18BP could significantly down-regulate the production of the Th1
cytokines, thus modulating the Th1/Th2 balance in established CIA. Results of
this investigation could support the feasibility of AdmIL-18BP/mIL-4 gene
therapy in the treatment of RA.
Materials and Methods
Animals
Male DBA-1/BOM mice were purchased from the Sipper BK Experimental Animal
Company (Shanghai, China). The mice were housed in filter-top cages, and were
given free access to water and food. The mice were immunized at the age of 10–12 weeks.
Recombinant adenoviral vectors
The recombinant replication-deficient adenoviral vector containing
mIL-18BP and mIL-4 fusion cDNA (AdmIL-18BP/mIL-4) was prepared and the virus
was purified as follows. The two cDNA from mIL-18BP and mIL-4 were cloned by
RT-PCR from total RNA obtained from mouse splenocytes, then connected by a
linker [(Gly-Gly-Gly-Gly-Ser)3]. To ensure the correct nucleotide sequence, the
entire cDNA was subsequently sequenced. The fusion cDNA of mIL-18BP and mIL-4
was inserted into the p-Shuttle-CMV vector (Stratagene, La Jolla, USA). The
recombinant replication-deficient adenovirus AdmIL-18BP/mIL-4 was generated by
homologous recombination after electrotransforming BJ5183-AD-1 Electroporation
Competent Cells (pre-transformed with pAdEasy-1 adenoviral vector backbone
for 3-fold improvement of recombinant adenovirus production) (Stratagene) with
p-Shuttle-CMV-mIL-18BP/mIL-4. p-Shuttle-CMV-LacZ (Stratagene) was used as a
control vector. High titers of recombinant adenoviruses were amplified in
adenoviral E1-transformed human embryonic kidney 293 cells (Stratagene). Purification
of the virus was accomplished using cesium chloride density ultracentrifugation
followed by dialysis, and the viral titer (pfu/ml) was determined by plaque
assay in 293 cells as described previously [16].
Functional analysis of
AdmIL-18BP/mIL-4
Synovial fibroblasts of mice with CIA were isolated as previously
described [17]. One million synovial fibroblasts seeded in a 6 cm culture dish
were infected with AdmIL-18BP/mIL-4 or a control adenovirus (AdLacZ) at a
multiplicity of infection of 100. After incubation at 37 ?C in 5% CO2 for 30 min, the supernatants were replaced with RPMI 1640 complete
medium (supplemented with 10% fetal calf serum, 100 U/ml penicillin, 50 mg/ml
streptomycin, and 2 mM glutamine). Supernatant were collected at specified time
points and analyzed for IL-4 release by ELISA (R&D Systems, Minneapolis,
USA).Synovial fibroblasts transfected or not transfected were collected
and lysed by the addition of a lysis buffer consisting of 20 mM Tris-HCl (pH
7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton
X-100, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM
leupeptin and 1 mM phenylmethylsulphonyl fluoride for 30 min at 4 ?C. Cellular
proteins (40 mg/sample) were subjected to Western blot analysis using primary
antibody specific to IL-18BP (R&D Systems) followed by horseradish
peroxidase-conjugated secondary antibody. The reaction was visualized with
enhanced chemiluminescence reagents and the signal was captured on X-ray film.Synovial fibroblasts transfected or not transfected were collected
and lysed by the addition of a lysis buffer consisting of 20 mM Tris-HCl (pH
7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton
X-100, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM
leupeptin and 1 mM phenylmethylsulphonyl fluoride for 30 min at 4 ?C. Cellular
proteins (40 mg/sample) were subjected to Western blot analysis using primary
antibody specific to IL-18BP (R&D Systems) followed by horseradish
peroxidase-conjugated secondary antibody. The reaction was visualized with
enhanced chemiluminescence reagents and the signal was captured on X-ray film.
Induction of collagen-induced
arthritis
Bovine type II collagen was prepared according to the method of
Miller and Rhodes [18], diluted in 0.05 M acetic acid to a concentration of 2
mg/ml, and emulsified in an equal volume of Freund? complete adjuvant (2 mg/ml Mycobacterium
tuberculosis, strain H37Ra; Difco Laboratories, Detroit, USA). The mice
were immunized intradermally at the base of the tail with 100 ml of emulsion
(100 mg collagen). On day 21, mice were given an intraperitoneal booster
injection of 100 mg type II collagen dissolved in phosphate-buffered saline (PBS) (pH
7.4). Arthritis onset typically occurred by day 25–28.
Assessment of arthritis
Mice were examined for the visual appearance of arthritis in peripheral
joints, and assigned a severity score (arthritis score) as previously described
[19]. Mice were considered arthritic when significant changes in redness
and/or swelling were noted in digits or in other parts of the paws. At later
time points, ankylosis was also included in the arthritis score. The clinical
severity of arthritis was graded on a scale of 0–2 for each paw, according
to changes in redness and swelling: 0, no changes; 0.5, slight; 1.0, moderate;
1.5, marked; and 2.0, maximal swelling and redness, and eventually ankylosis.
Arthritis score (mean±SD) was expressed as the cumulative value for all paws,
with a maximum of 8 and expressed as a percentage of the initial score at the
beginning of treatment.
Treatment of CIA with
mIL-18BP/mIL-4 co-expressing recombinant adenovirus vector
To evaluate the effects of AdmIL-18BP/mIL-4 to Th1 and Th2 cytokine
responses on established CIA, mice with CIA were selected with similar
arthritis scores at day 28 and divided into three groups. Thereafter,
intra-articular injections in the right knee joint of mice were carried out
with 107 pfu/6 ml of either the mIL-18BP and mIL-4 fusion expressing recombinant
adenovirus vector (AdmIL-18BP/mIL-4), or a control replication-defective
recombinant adenovirus vector (AdLacZ), or with PBS. At weeks 1, 2, and 4 after
the intra-articular injection of the viral vector, mice serum was collected for
the examination of Th1/Th2 cytokines. Mice splenocytes were isolated for the
measurement of IFN-g-expressing and IL-4-expressing CD4+ T cells.
Measurement of Th1/Th2
cytokines in serum
At weeks 1, 2, and 4 after the intra-articular injection of the
viral vector, six mice from each group were bled and killed by cervical
dislocation. The serum levels of TNF-a, IFN-g, IL-4, IL-10, and IL-18
were measured by ELISA using commercially available kits (R&D Systems).
Measurement of IFN-g-expressing and IL-4-expressing CD4+
T cells
At weeks 1, 2, and 4 after the intra-articular injection of the
viral vector, viable mice splenocytes were isolated from the various animal
groups (six mice from each group). The cells were washed with PBS, then
resuspended in RPMI 1640 culture medium (2?106 cells/ml). They were stimulated with phorbol
12-myristate 13-acetate (Sigma, St Louis, USA) and ionomycin (Sigma) for 5 h at
37 ?C in 5% CO2 at final concentrations of 25 ng/ml and 1 mg/ml. Monensin
(Sigma) with a final concentration of 1.7 mg/ml was also added into
the culture to block the intracellular cytokine transport processes for the
final 4 h of the 5 h activation period. After stimulation, the cells were
washed and incubated for 15 min with allophycocyanin-conjugated rat anti-mouse
CD4 antibody (Caltag Laboratories, Burlingame, USA), then fixed and
permeabilized using the Fix & Perm cell permeabilization kit (Scandic,
Vienna, Austria). During permeabilization, the cells were incubated with
fluorescein-isothiocyanate (FITC)-conjugated rat anti-mouse IFN-g (Caltag
Laboratories) and phycoerythrin (PE)-conjugated rat anti-mouse IL-4 (Caltag Laboratories)
antibodies for 15 min. Isotype-matched controls rat IgG1-FITC and rat IgG1-PE
were used to assess non-specific binding. Following a final washing step, the
cells were resuspended in 500 ml PBS and subjected to flow cytometry. All incubations were carried
out at room temperature in the dark. The labeled cells were analyzed by
FACSCalibur four-color flow cytometry (Becton Dickinson Immunocytometry
Systems, San Jose, USA) using CellQuest software (Becton Dickinson). The
cytometer was calibrated with CaliBRITE beads (Becton Dickinson) using
FACSComp software (Becton Dickinson) and with QC-4 beads according to the
manufacturer’s recommendations (Flow Cytometry Standards, San Juan, Puerto
Rico). For the purpose of analysis, 50,000 events were acquired because the
IFN-g-producing cells were expected to be present at low frequencies.
T-helper cells were selected using a forward and side scatter gate for
lymphocytes in combination with a gate on CD4+ cells. This specific
cell population was then analyzed for intracellular cytokines using dual color
dot plots of cytokine IFN-g FITC or IL-4 PE versus CD4+ allophycocyanin.
Non-specific staining and autofluorescence were determined by the
isotype-matched controls.
Statistical analysis
Data were expressed as the mean±SD. Differences were determined by
one-way ANOVA with Bonferroni multiple comparison tests and Student? t-test. Differences were accepted as significant when P<0.05.
Results
IL-4 expression in synovial fibroblasts infected with AdmIL-18BP/mIL-4
In order to establish the function of AdmIL-18BP/mIL-4, synovial
fibroblasts of mice with CIA were isolated and infected with AdmIL-18BP/mIL-4
or a control adenovirus, AdLacZ. Two hours after being infected, IL-4 was
released in a conditioned medium of mouse synovial fibroblast cultures. As
shown by ELISA [Fig. 1(A)], strong IL-4 production was observed at 72 h,
beginning at a concentration of 47.6 ng/ml, followed by a steady increase. One
week after being infected, the expression of IL-4 reached a peak concentration
of 76.4 ng/ml. No detectable levels of IL-4 were found in the culture
supernatants of synovial fibroblasts infected with AdLacZ or pure synovial
fibroblasts (data not shown).
IL-18BP protein expression in synovial fibroblasts infected with
AdmIL-18BP/mIL-4
As there is presently no commercially available ELISA kit for the
measurement of IL-18BP, IL-18BP protein expression in synovial fibroblast
lysates was measured by Western blot analysis [Fig. 1(B)]. These
experiments suggested that the recombinant adenoviral vector AdmIL-18BP/mIL-4
is very effective in vitro.
Regulatory effects on Th1 and Th2 cytokine expression in serum after
AdmIL-18BP/mIL-4 therapy
In order to examine the effects of AdmIL-18BP/mIL-4 on Th1 and Th2 serum
cytokines, mice with CIA were bled and killed at weeks 1, 2, and 4 following
intra-articular injection of AdmIL-18BP/mIL-4, AdLacZ, or PBS. Serum cytokines
were detected by ELISA. As shown in Fig. 2, mice with CIA at weeks 1, 2,
and 4 after injection of AdmIL-18BP/mIL-4 showed significantly increased mean
serum concentrations of IL-4 and IL-10 (P<0.01 at all time points) but greatly decreased mean serum concentrations of IFN-g and TNF-a (P<0.01 at all time points) compared with the control groups that had received either AdLacZ or PBS. At weeks 1, 2, and 4 following injection of AdmIL-18BP/mIL-4, the serum concentrations of IL-4 and IL-10 were 2.3, 2.3, and 2.1 times higher and 2.2, 2.0, and 2.3 times higher, respectively, than those in mice that had received AdLacZ. However, the serum concentrations of IFN-g and TNF-a at 1, 2, and 4
weeks following the injection of AdmIL-18BP/mIL-4 were only 53%, 45%, 42% and
47%, 41%, and 40%, respectively, of the levels found in mice that had received
AdLacZ. In addition, there were no significant differences in the serum
concentrations of the four cytokines between the two control groups at any time
points.The results of serum levels of IL-18 at weeks 1, 2, and 4 following
injection of AdmIL-18BP/mIL-4 are shown in Fig. 3. The serum levels of
IL-18 at weeks 1, 2, and 4 following injection of AdmIL-18BP/mIL-4 were
decreased by 45%, 53%, and 63%, respectively, compared to the serum levels
found in mice that had received AdLacZ (P<0.01 at all time points). The serum levels of IL-18 were the same in both control groups (P>0.05
at all time points).
Effects of AdmIL-18BP/mIL-4 on IFN-g-producing and
IL-4-producing CD4+ T cells
At weeks 1, 2, and 4 after intra-articular injection of
AdmIL-18BP/mIL-4, viable mice splenocytes were isolated from six mice from
each group. IFN-g-producing and IL-4-producing CD4+ T cells
were investigated by flow cytometry. Results are shown in Fig. 4.When CD4+ T cells were analyzed 1 week after injection
of AdmIL-18BP/mIL-4 [Fig. 5(A)], the percentage of IFN-g-positive CD4+
T cells (7.03%±2.05%) was lower than that after injection of AdLacZ
(17.10%±3.74%, P<0.01) or PBS (10.74%±3.92%, P<0.05). However, the difference was not significant (P>0.05) when compared to
the normal group (4.58%1.43%). At weeks 2 and 4 after injection of
AdmIL-18BP/mIL-4, the percentage of IFN-g-positive CD4+ T cells was 5.28%±2.29% and 3.99%±0.91%, respectively. However, at
these different time points, no significant difference in the percentage of
IFN-g-positive CD4+ T cells was noticed in the
therapy group or in either of the control groups. As shown in Fig. 5(B),
1 week after injection of AdmIL-18BP/mIL-4, the percentage of IL-4-positive CD4+
T cells (47.56%±6.14%) was higher than after injection of AdLacZ (32.98%±10.00%,
P<0.05) or PBS (14.27%±2.68%, P<0.01). This then decreased gradually. By weeks 2 and 4 after injection of AdmIL-18BP/mIL-4, the percentage of IL-4-positive CD4+ T cells was 9.56%±2.72% and 6.99%±1.37%,
respectively. At week 4, when the therapy group was compared to the normal
group (5.58%±1.95%), the percentage of IL-4-positive CD4+ T cells
showed no significant difference (P>0.05), nor was there any
significant difference for either of the control groups.
Discussion
Treatment of RA requires therapeutic measures over a prolonged
period, therefore gene therapy has been considered to be an interesting
approach that might have advantages over conventional therapies that use
soluble receptor, antagonizing protein, or blocking antibodies. Indeed, gene
therapy has been proven to be useful in various animal models of arthritis
[20]. In the present study we constructed a recombinant adenoviral vector
containing the mIL-18BP and mIL-4 fusion gene (AdmIL-18BP/mIL-4). We evaluated
the effect of IL-18BP/IL-14 gene therapy on modifying the immune response in
murine CIA and identified possible pathways mediating this process in vivo.
We found significantly increased serum levels of IL-4 and IL-10 but greatly
reduced serum levels of IFN-g and TNF-a after intra-articular injection of AdmIL-18BP/mIL-4 in murine CIA. It is now generally accepted that TNF-a plays a key role in the
inflammation and joint damage that occurs in RA [21]. TNF-a controls in
part the production of IL-1 and other pro-inflammatory cytokines, including
IL-6 and IL-8. Furthermore, it increases the expression of adhesion molecules,
chemokines, prostaglandin E2, and matrix metalloproteinases. The importance of
TNF-a in RA has been established by several experimental and clinical observations
[21,22]. TNF-a blockade after the onset of disease resulted in amelioration of
clinical symptoms and prevention of joint destruction. TNF-a has been shown
to be pivotal in the pathogenesis and progression of the disease and has been
successfully targeted in the treatment of RA patients [23].The production of IL-10 is one of the anti-inflammatory mechanisms
used to control a dysregulated immune response in reaction to the activity of
pro-inflammatory cytokines in RA patients. IL-10 can suppress TNF-a and IL-1
production by activated macrophages and can enhance the production of TNF
inhibitors, acting as a potent anti-inflammatory cytokine [24]. Some investigations of IL-4 treatment in murine CIA focused on
synovial and cartilage destruction. In this model, IL-4 displayed marked
protection against cartilage and bone erosion, and promoted tissue repair [25].
The mechanisms of action of endogenous and exogenous IL-4 on inflammation in
vivo are not clear. Previous studies suggest that a possible explanation
for the tissue-protective effect of IL-4 might be its immunoregulatory ability
to play a critical role in the Th2 reaction and to modulate the IL-1- and TNF-a-mediated
inflammatory responses [26]. IL-1 has also been proven to be a critical cytokine
involved in synovitis in RA [27]. In the RA synovium, an imbalance in this
system exists because the relative levels of production of IL-1Ra are not
adequate to effectively block the pro-inflammatory effects of IL-1 [28]. Some
studies revealed that IL-4 treatment in animal models and in RA patients can
enhance levels of IL-1Ra or suppress levels of IL-1b [29]. Recently, it was
shown in rat adjuvant-induced arthritis that IL-4 gene delivery
resulted in anti-angiogenic effects in vitro and in vivo. IL-4
reduced synovial tissue vascularization through angiostatic effects, mediated
inhibition of angiogenesis by an association with altered pro- and
anti-angiogenic cytokines. This study implied that IL-4 gene therapy
might be a useful approach to the reduction of neovascularization in arthritis
[30].We also found the serum levels of IL-18 were significantly
down-regulated in response to local AdmIL-18BP/mIL-4 treatment in murine CIA.
IL-18, initially described as an IFN-g-inducing factor, is a pro-inflammatory
cytokine that plays an important role in the Th1-type immune response through
the induction of IFN-g synthesis in T cells and natural killer cells, T-cell
proliferation, and cytokine production [31]. Significant levels of expression
of IL-18 have been shown previously in the synovium of RA patients [32]. In
vivo observations further support a pro-inflammatory role in articular
inflammation. Thus, IL-18 can replace the requirement for Freund’s complete
adjuvant to induce arthritis in collagen-immunized DBA/1 mice [33]. Using
adenoviral delivery of IL-18 and TNF-a/IL-1 deficient mice, Joosten and colleagues
subsequently showed that, although IL-18-induced joint inflammation is
independent of IL-1, cartilage degradation requires IL-18 induced IL-1b production
[34]. Furthermore, they suggested that TNF is partly involved in IL-18-induced
joint swelling and influx of inflammatory cells, but that cartilage
proteoglycan loss occurs independent of TNF. These findings indicate that
IL-18, in contrast to TNF, contributes through distinct pathways to joint
inflammation and cartilage destruction. IL-18-deficient DBA/1 mice have a
reduced incidence and severity of CIA associated with amelioration of articular
damage [35]. IL-18BP, a constitutively expressed and secreted protein, has
been identified [9]. IL-18BP binds IL-18 with high affinity (400 pM) and
blocks its biological activity at a 1:1 molar ratio [36]. Such a naturally
occurring molecule represents an interesting inhibitor for testing in
experimental models of disease. IL-18 is an early signal leading to Th1
cytokine responses that are essential for the cytotoxic T cell response.
Therefore, IL-18BP could modulate one of the earliest phases of the Th1 immune
response. Moreover, local overexpression of IL-18 binding protein C by
adenoviral delivery also ameliorates articular destruction [37]. Therefore, the
therapeutic use of IL-18BP might modulate the cytokine balance, ameliorating
established arthritis.The investigation of intracellular cytokine profiles is not
influenced by the soluble cytokine receptors or inhibitors in serum or plasma.
Hence, we analyzed IFN-g-producing and IL-4-producing CD4+ T cells by flow
cytometry. A previous study showed that the mean percentage of IFN-g-producing CD4+
T cells in patients with RA was almost 4-fold higher than the number of
IL-4-producing CD4+ T cells [38]. We showed that the mean percentage
of IFN-g-producing CD4+ T cells in murine CIA was significantly
decreased and seemed to reach a normal level after injection of
AdmIL-18BP/mIL-4, whereas the mean percentage of IL-4-producing CD4+
T cells was remarkably increased at week 1, then reduced to a normal group
level at week 4. Our data show for the first time the marked effect of
modifying Th1/Th2 imbalance in murine CIA after local AdmIL-18BP/mIL-4 gene
therapy.RA is a systemic disease and its joint manifestation depends, at
least in part, on systemic immune dysfunction. The significant modificatory
effects on Th1/Th2 imbalance in murine CIA after local AdmIL-18BP/mIL-4 gene
therapy support the view that combination treatment with IL-18BP and IL-4 is a
promising therapeutic target for RA if overexpressed locally.
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