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*
Unité de Recherches d’Immunopathologie des Maladies Tumorales et Autoimmunes, Institut National de la Santé et de la Recherche Médicale (INSERM) U475, Montpellier;
Institut Pasteur, Unité de Recherche Associée/Centre National de la Recherche Scientifique 1854, Lille; and
Service d’Immunorhumatologie, Hôpital Lapeyronie, Service Rhumatologie, Montpellier, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-
) to a clear Th2 phenotype
(IL-4, IL-5, IL-10) between days 15 and 30 of the development of CIA in
DBA1 mice (1). Although the Th1/Th2 switch occurs, Th2-type cytokines
remain insufficient, and proinflammatory cytokines derived from
macrophages and fibroblasts such as TNF-, IL-1, IL-6, and
granulocyte-macrophage-CSF predominate in the synovial fluid, leading
to CIA symptoms progression.
Numerous studies have indicated that cytokines of the Th2 phenotype or
specific inhibitors of Th1 cytokines (IL-ra, anti-TNF-) play an
important immunoregulatory role in inhibiting the disease process
(2, 3, 4, 5). Among these factors, IL-10 has received growing attention for
its therapeutic potential.
IL-10 is a cytokine synthesis-inhibitory factor produced by Th2 cells
(6) that predominantly mediates immunosuppressive effects through the
down-regulation of macrophage functions (7, 8) and the inhibition of
proinflammatory cytokines produced by Th1 cells, such as IL-1ß, IL-6,
IL-8, TNF-, IFN-, and granulocyte-macrophage-CSF (9, 10, 11).
Endogenous IL-10 expression by T cell clones is enhanced in the
synovium during RA (9, 12, 13), and previous data have suggested that
IL-10 might be the major endogenous mediator of spontaneous
immunosuppression that occurs to prevent disease expression and
development (2, 3). Indeed, twice daily i.p. injections of murine
rIL-10 (2000 U/day) suppressed established CIA in DBA1 mice and
protected against cartilage destruction (2). In contrast,
neutralization with anti-murine IL-10 (anti-mIL-10) Ab further
accelerated the expression of CIA and increased its severity. Others
showed that systemic mIL-10 treatment with miniosmotic pumps (100
µg/day) delayed the onset of arthritis and reduced the severity of
CIA in rats when injected before the symptoms appeared (3). IL-10
activities have also been intensively investigated in a number of in
vivo models including endotoxemia (14, 15, 16, 17, 18), transplantation (19, 20, 21),
autoimmune diseases (22), diabetes in NOD mice (23), and
allergen-induced lung inflammation (24).
However, mIL-10 has some immunostimulatory activities, including enhanced expression of class II MHC molecules on B cells (25) and induction of cytotoxic T cell differentiation. Viral IL-10 (vIL-10), a product encoded by EBV, was found to be highly homologous to both murine and human IL-10. Viral IL-10 shares many biologic properties with murine and human IL-10, but it does not possess the T cell costimulatory activities (10, 19). Moreover, Qin et al. (20) showed that retroviral gene transfer of vIL-10 significantly prolonged murine cardiac allograft survival through induction of local immunosuppression. Suzuki et al. (19) also demonstrated that local vIL-10 but not mIL-10 production can suppress immune rejection of immunogenic tumor cells. Thus, all of these in vitro and in vivo data support vIL-10 as a better candidate for reversing the immune response in the setting of autoimmune disease.
Most of the studies to date have used daily injections of rIL-10 because of its short half-life (26). Gene transfer technologies have developed efficient and prolonged expression of cytokines in vivo. Replication-defective adenoviral vectors are appealing candidates for in vivo gene therapy because of their capacity to transduce a wide variety of cell types, including nonreplicative cells. Recently, Xing et al. described mIL-10 production in the sera of mice after intramuscular injection of mIL-10 recombinant adenovirus (15).
The purpose of this study was to examine the efficiency of vIL-10 adenovirus-mediated (AdvIL-10) gene transfer on the incidence and severity of murine CIA. Our results show that systemic administration of adenovirus expressing vIL-10, but not the control recombinant adenovirus, can inhibit CIA onset and reduce clinical and histopathologic sypmtoms of the autoimmune animal model. This effect is dose dependent and is specifically neutralized by anti-vIL-10 Ab. Finally, these data suggest that adenovirus-mediated vIL-10 gene transfer may be a valuable therapeutic alternative to arthritis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male DBA1 mice were bred in our facilities and immunized at the age of 8 to 10 wk.
Materials
Bovine type II collagen was purchased from Institute Jacques Boy (Reims, France). PBS was obtained from Eurobio (Les Ulis, France). LPS (Eschrichia coli 0111:B4), BSA, Freund’s complete adjuvant, Tween 20, orthophenylene diamine, and all chemicals were purchased from Sigma (St. Louis, MO). Monoclonal anti-human and anti-viral IL-10 Abs for ELISA and in vivo neutralization were obtained from PharMingen (San Diego, CA). The 96-well flat-bottom Maxisorb microtiter plates were obtained from Nunc (Life Technologies, Cergy Pontoise, France). Avidin-peroxidase was purchased from Immunotech, (Marseille, France).
Recombinant adenoviral vectors
Replication-defective type 5 adenovirus vector was deleted for sequences E1a and E1b, impairing the ability of the virus to replicate into cell or transform them. Construction of the recombinant AdvIL-10 and AdvIL-10 mut was described in detail elsewhere (C.V., manuscript in preparation). Briefly, the BCRF1-coding gene (-16, +625), flanked in 5' with the promoter of the cytomegalovirus and in 3' with a SV40 polyadenylation sequence, was inserted in place of E1 region into the adenoviral genome. In the AdvIL-10 mut control, a mutation was introduced by inserting a cytosine at position +71, just after the sequence coding for the signal peptide, leading to a modification of the reading frame. High titer recombinant adenoviruses were prepared by amplification in 293 cells and purified according to established methods.
Induction of arthritis
Bovine type II collagen was diluted at 2 mg/ml with 0.1 M acetic acid overnight at 4°C, then emulsified with an equal volume of CFA before use. CIA was induced by intradermal injection at the base of the tail with 100 µl of solution (100 µg collagen) at day 0. On day 21, the animals were boosted with an i.p. injection of 100 µg type II collagen at 1 mg/ml in PBS. On day 28, the onset of arthritis was initiated by a single i.p. injection of 40 µg of LPS (in 100 µl of PBS). This resulted in the onset of CIA within a week, as previously described (2, 27, 28, 29).
Treatment of CIA
Before the onset of arthritis, animals were divided into five groups. On day 30 after immunization, mice were either untreated or injected i.v. with 109 or 1010 pfu of adenoviral vector expressing the native or mutated vIL-10 gene. On day 29, one group received 0.2 mg of rat anti-human and vIL-10 mAb i.v. (clone JES3-19F, PharMingen). This group was then injected on day 30 with 109 pfu of AdvIL-10. Blood samples were taken on days 0, 28, 33, 37, and 45 following arthritis induction.
Assessment of CIA
From day 21, the paw widths were measured with a micrometer. Swelling, redness, or the presence of erythema or wound were visually evaluated for macroscopic scoring of arthritis as previously described (30). The severity of arthritis was graded on a scale of 0 to 3 for each paw, where 0 = normal, 1 = swelling and moderate redness, 2 = marked edema, and 3 = severe edema with wound. The macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 12. Scoring was done by two independent observers, without knowledge of the experimental groups. Mice were sacrificed at day 50, and the hind paws from five to nine animals were randomly collected from two independent experiments for radiography, then fixed in 4% paraformaldehyde, decalcified in 5% formic acid, embedded in paraffin, and 2-µm sections were stained with hematoxylin/eosin/safranin O. Sections and x-rays were examined by two independent, blind observers, who were different from the persons who had grouped and clinically scored the animals. X-rays performed using Kodak film were scored for the tibioastragalus and tarsum joints from 0 to 5 according to the degree of destruction: 0, normal; 1, demineralization; 2, narrowing <50% of joint space; 3, narrowing <50% of joint space and joint erosion; 4, loss of joint space; 5, complete joint destruction. Two different areas of each paw (midfoot and hindfoot joints) were scored on four successive sections for overall histopathologic analysis as previously described (31): 0, normal; 1, inflammatory infiltrates and synovial hyperplasia; 2, pannus formation and cartilage erosion; 3, important cartilage erosion and bone destruction; 4, loss of joint integrity.
Immunoassay of serum vIL-10
Sera were collected from blood samples on days 0, 28, 33, 37,
and 45 and stored at -20°C until tested. The circulating vIL-10
levels were measured by specific ELISA for each individuals. Wells of
flat-bottom Maxisorb microtiter plates were coated with 100 ng of
monoclonal rat Ab specific for human and viral IL-10 (clone JES3-9D7,
PharMingen) in 50 µl of carbonate buffer, pH 8.2, at 4°C overnight.
Following two washes with PBS-0.05% Tween 20, the plates were blocked
with 200 µl PBS-3% BSA 2 h at room temperature (RT). Then,
blocking buffer was eliminated, and 50 µl of IL-10 standard (2226-01,
Genzyme, Cambridge, MA) or serum samples were added. After 2 h of
incubation at RT, wells were washed four times with PBS-0.05% Tween 20
and then incubated 1 h at RT with 100 µl of biotinylated
anti-human and anti-viral IL-10 Ab (18562D, PharMingen) diluted
at 1 µg/ml in PBS-3% BSA. After four washes, wells were incubated
with 100 µl of a 1/10,000 dilution (in PBS-3% BSA) of
avidin-peroxidase for 30 min at RT. Finally, wells were washed six
times, and visualization was achieved by adding 100 µl of solution
containing 5 mg orthophenylene diamine, 12.5 ml 0.1 M sodium acetate,
pH 5.5, and 5 µl 30% hydrogen peroxide. The incubation was performed
in the dark for 
10 min and reaction stopped by the addition of 100
µl of 1N HCl. Absorbance was measured at 492 nm.
Anti-type II collagen (CII) ELISA
Sera collected from blood samples on days 0, 28, 33, 37, and 45 were separately stored at -20°C. Individual sera were analyzed for anti-CII Abs using an isotype-specific ELISA as previously described (1). In brief, 50 µl of 5 µg/ml bovine type II collagen solution was coated in 96-well microtiter plates. After overnight incubation at 4°C, plates were washed and blocked for 1 h at RT with 2% casein (100 µl/well). All samples were tested in duplicate at at a 1/300 dilution. The detection of IgG subclasses was obtained after incubation with a goat anti-mouse IgG1 or IgG2a Ab (02003E or 02013E, PharMingen) conjugated to alkaline phosphatase (50 µl/well of a 1/4000 stock solution). The quantification of bound enzyme was performed with 50 µl/well of paranitrophenyl phosphate (N2640, Sigma) at 405 nm.
Statistics
Statistical analysis was done using Fisher’s exact test for contingency group comparisons and the nonparametric Mann-Whitney test or Student t test, as appropriate according to data distribution. All data were analyzed by the Instat2.1 software program for Macintosh.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the effect of IL-10 gene therapy on the incidence of
disease, mice were primed with intradermal CII on day 0. Recombinant
AdvIL-10 was given i.v. at a dose of 109 pfu/mouse on
day 30. This treatment resulted in inhibition of arthritis onset, only
20 to 30% of the mice having developed arthritis by the end of the
experiment (Fig. 1
). The difference was
statistically significative from the control group
(p < 0.01 on day 44, and p <
0.04 on day 47). These results cannot be attributed to the vector,
since mice that received the same inoculum of the non-vIL-10-expressing
recombinant adenovirus (AdvIL-10 mut) developed an arthritic profile
similar to that of untreated mice. In both groups, 100% of the animals
progressively reached arthritis by day 50. Moreover, when
anti-IL-10 Ab (0.2 mg/mouse) was administered i.v. 24 h before
the injection of 109 pfu AdvIL-10, the incidence of
arthritis was enhanced, with 50% of the treated animals developing
arthritic symptoms on day 30. On the contrary, arthritis onset was
markedly delayed in the group injected with 1010 pfu of
AdvIL-10, suggesting a dose-dependent effect. On day 48, 22% of the
mice presented clinical symptoms, as did mice treated with
109 pfu AdvIL-10. These results suggested that vIL-10 could
prevent the onset of arthritis with a single injection of the
adenoviral vector on day 30, when clinical symptoms of arthritis
start.
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From day 24 to day 47 after induction of arthritis, thickness of
the paws was measured and macroscopic arthritis score was determined in
all groups as defined (see Materials and Methods).
The injection of 109 pfu of AdvIL-10 on day 30 had a
suppressive and significant (p < 0.03) effect
on joint thickness (Fig. 2). Moreover, in
the group treated with an increasing dose of adenoviral vector
(1010 pfu AdvIL-10), a marked reduction was observed when
compared with the CIA-untreated group (p <
0.0007 on day 35). When a more complete picture of arthritis was
considered, both AdvIL-10 treatments were beneficial, since macroscopic
scoring of arthritis revealed significant improvement (Fig. 3; p < 0.001 and
p < 0.008, respectively, on day 37).
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The control group of DBA1 mice treated on day 30 with the control
adenoviral vector (AdvIL-10 mut) developed full arthritis on days 35
through 37. This profile of CIA course was similar to that in untreated
CIA mice: respectively, 1.80 ± 0.09 mm vs 1.81 ± 0.05 mm
for paw thickness on day 37 (Fig. 2
).
At the arthritis peak (day 37), changes measured in joint thickness and
arthritic score were significantly different (p
< 0.0032 and p < 0.01, respectively) between mice
treated with 109 pfu of AdvIL-10 or AdvIL-10 mut (Figs. 2 and 3).
To assess specificity of observed effects in AdvIL-10-treated groups,
we then neutralized the expression of vIL-10 with i.v. injection of a
rat anti-vIL-10 Ab 1 day before AdvIL-10 injection. Figure 2 shows
an earlier onset and enhanced magnitude of joint swelling in the group
(AdvIL-10 + anti-IL-10), even though differences were never
significant when compared with control groups (CIA ± AdvIL-10
mut). However, inhibition of exogenous vIL-10 significantly enhanced
the expression of CIA when compared with the AdvIL-10-treated group
(p < 0.002). The same results could be
observed with macroscopic scores (Fig. 3) (p <
0.001). Figures 1 through 3 represent pooled data of two independent
experiments, as can be seen in detail Table I.
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Histologic analysis of the ankle joint was performed at day 50.
Figure 4 shows representative pictures of
untreated and AdvIL-10-treated CIA animals, and Table II summarizes the respective scores. The
effects observed in untreated and AdvIL-10 mut-treated mice were
infiltration of mononuclear cells into the joint cavity, synovial
hyperplasia, pannus formation, and cartilage erosion (Fig. 4, A and B). Only minor thickening of the
synovium can be seen in the AdvIL-10-treated group (Fig. 4C). As shown previously, treatment with
anti-IL-10 Ab reversed the beneficial effects of AdvIL-10
injection, and joint destruction was dramatically increased (Fig. 4D). The histopathology results for the
AdvIL-10-treated groups were comparable to those in age-matched animals
that had not been immunized (data not shown).
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). Thus,
AdvIL-10-treated groups were significantly protected against synovial
hyperplasia, cartilage erosion, bone destruction, and the overall
histopathology as observed in the control groups. Radiography analysis
The scoring of x-rayed paws on day 50 was correlated significantly
to histology. When compared, CIA and AdvIL-10 mut radiologic scoring
were not significantly different (Table II). However, AdvIL-10
treatment significantly decreased x-ray scoring
(p < 0.024), and this effect was significantly
reversed by anti-IL-10 treatment (p <
0.023).
AdvIL-10 modifies IgG isotype autoantibody levels
To determine whether the AdvIL-10 treatment of CIA was associated
with a modified humoral response to CII, levels of anti-CII
isotypes produced during the course of arthritis were measured by ELISA
in serum samples at different times after immunization. Both IgG2a and
IgG1 anti-CII Abs were present in the sera of the prearthritic (day
28) and arthritic (day 37) DBA1 mice (Table III). The relative isotype
abundance was IgG1 > IgG2a in all groups. However, the ratio of
IgG1:IgG2a increased in AdvIL-10-treated groups. The differences
between day 37 and 28 of the isotype IgG1:IgG2a ratio (Th2:Th1) showed
that AdvIL-10-treated groups presented a more pronounced Th2 phenotype
on day 37 than on day 28, compared with the AdvIL-10 mut or untreated
groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These results were obtained not only at the level of macroscopic analysis, but were also confirmed at radiologic and histologic levels. Higher doses of AdvIL-10 (1010 pfu/mice) totally prevent the onset of arthritis for 15 days, with a significant protective effect found on joint histopathology (p < 0.0001). The majority of mice treated with 109 pfu AdvIL-10 did not show any clinical and histologic evidence of CIA, while the remaining 22% (animals developing arthritic symptoms) presented macroscopic and x-ray scores that were significantly reduced (p < 0.001 and p < 0.024), respectively; and significant protection of cartilage damage (p < 0.0001). We showed that the beneficial action of AdvIL-10 injection could be abolished by pretreating the mice with an anti-vIL-10 Ab. Although differences were never statistically significant, we observed that anti-IL-10-treated mice presented earlier and more severe clinical symptoms than untreated or AdvIL-10 mut-treated mice. This result was related to the cross-reactivity of the neutralizing vIL-10 Ab for mIL-10, due to high sequence homology between murine and viral cytokines. Accelerated expression of arthritis resulting from neutralization of both exogenous and endogenous IL-10 emphasizes the important role of IL-10 in preventing CIA progression and has already been noted by other groups as occurring after mIL-10 neutralization (2).
Earlier studies had already suggested that IL-10 was one candidate for
experimental CIA treatment (2, 3). Twice a day i.p. injection of murine
rIL-10 suppressed established CIA and decreased levels of TNF- and
IL-1 mRNA in synovial tissue and articular cartilage, while levels of
IL-1ra mRNA remained elevated. In contrast, neutralization of mIL-10
further accelerated the expression of CIA (2).
IL-10 is abundantly expressed in the joints of RA patients (9, 12, 13), and there is evidence that IL-10 is an important
down-regulator of Th1 cells (11) and a deactivator of macrophage
functions. In vitro studies have suggested that IL-10 could mediate
immunosuppressive effects by: 1) inhibiting proinflammatory cytokines
produced by Th1 cells and macrophages such as TNF-, IL-1ß, and
IL-8 (9, 10, 11) implicated in lymphocyte migration into the synovium; 2)
up-regulating soluble TNF- receptor and IL-1ra monocyte production
(32); 3) down-regulating metalloproteases and preferentially
stimulating TIMP-1 production by macrophages, which participate in the
degradation and remodeling of extracellular matrix in acute and chronic
inflammation situations (7, 8); and 4) inhibiting costimulatory
molecule expression on synovial cells (33). Thus, the effects observed
with AdvIL-10 treatment could be explained by a powerful
anti-inflammatory action of vIL-10 that reduces lymphocyte
infiltrates and local immune response within the joint, doubled by a
chondroprotective action of the joint via the alteration of
macrophage-degradative proteinase/antiproteinase balance.
As cellular IL-10, vIL-10 possesses the anti-inflammatory functions listed above; however, the T cell costimulatory activities were not demonstrated. First, vIL-10 does not costimulate the proliferation of murine thymocytes (34) and mast cell lines (10). Second, vIL-10 reduces the Ag-specific human T cell proliferative response by down-regulating APC class II MHC expression (35). Third, vIL-10 converts activated APCs into resting APCs by down-regulating the expression of costimulatory molecules (CD86) implicated in the interaction with CTLA-4 and CD28 on synovial cells (33, 36, 37). Fourth, vIL-10 has no chemotactic effects on CD8 cells, since infiltration by CD4+ and CD8+ lymphocytes was markedly reduced in CL8–1-vIL-10 tumors compared with nontransduced CL8–1 or CL8–1-mIL-10 tumors (19).
Finally, a comparative study using a syngeneic murine tumor model showed that local vIL-10 gene transfer inhibited rejection of tumor, while mIL-10 gene transfer accelerated tumor rejection (19). Retrovirally mediated gene transfer and expression of vIL-10 significantly prolonged murine cardiac allograft survival (20). Together with in vitro results, these data demonstrated that vIL-10 was a better candidate for immunosuppression. On the other hand, IL-10 has been shown to be very promising in treating a number of other inflammatory (16, 17, 18) and immune diseases (14, 15, 22), as well as allergies (24), diabetes (23), and transplant rejection (19, 20, 21).
IL-10 has a short circulating half-life (26, 38). Administration of
IL-10 produced potent, albeit short, therapeutic effects in murine CIA
models, requiring sustained injections (2, 3). We choose gene transfer
technology because it developed efficient and prolonged expression of
cytokines in vivo. With a single i.v. administration of
1010 pfu AdvIL-10, we could reach significant levels of
circulating protein after 1 wk (
30 ng/ml) and maintain detectable
levels for 2 wk (1 ng/ml). We also demonstrated the absence of
nonspecific effect due to the virus, since animals receiving the same
dose of adenovirus expressing no vIL-10 (AdvIL-10 mut) developed an
arthritic profile similar to that of untreated CIA controls. The use of
systemic vs local delivery was intended to target all of the inflamed
joints. Although it was debatable whether to express a cytokine
systemically, transgene expression was demonstrated to be transient,
with 90% of the Ad genome lost from the liver in the first 24 h
(39). Thus, although the activity of vIL-10 is compromised by its
immunogenicity in the mouse, we can expect the production of
neutralizing Abs to be short, since the vector is rapidly eliminated
from the blood, and to be weak, because of the high sequence homology
between vIL-10 and cIL-10.
Direct injection of vectors into the joints of DBA1 mice was not possible; for technical reasons, injection could only be periarticular. That being the case, injection of first-generation AdvIL-10 into the paws provoked local inflammation (data not shown). Recent work from Whalen et al. showed that with the same model of CIA DBA1 mice only 28.5% of those injected in the rear footpads with 105 pfu of AdvIL-10 developed arthritis (40). Only the noninjected front paws were arthritic, whereas the AdvIL-10-injected rear paws were protected from CIA for up to 10 wk postimmunization.
A critical role of TNF- and IL-1ß in CIA in the DBA1 mouse model
has also been showed by several groups using anti-cytokine therapy
(5) and IL-1ra (4, 5, 41), but the reduction is transient and dependent
on continued suppression. Anti-TNF- treatment appeared to be
efficient shortly after the onset of CIA and less so at later stages,
while anti-IL1 ameliorated both early and full-blown CIA (5).
Fibroblasts transfected in vitro with the retroviral vector expressing
human IL-1ra and then injected intra-articularly into the DBA1 mouse
knee joint prevent the onset of CIA (4), attenuating but not abolishing
the erosion of cartilage (42). These data suggest that the use of ex
vivo gene transfer to obtain local and prolonged expression may lead to
improved results in arthritis treatment. However, this approach seems
to be utopian in a human RA application perspective because of the
risk, the cost, and the time-consuming aspects. Moreover, it is
debatable whether using retroviral vectors would achieve gene delivery
directly into the resident synovial cells. Its low titer and weak
frequency of transfection limits its application in in vivo delivery
(42). In contrast, the recombinant adenovirus vector is characterized
by high titers, lack of potential risk of insertional mutagenesis, and
efficient gene transfer in a wide variety of cell types in vivo,
including in nonreplicative cells such as synovial fibroblasts (43).
Thus, replication-defective adenoviruses appear more suitable than
retrovirus vectors for efficient gene therapy in RA. Particularly in
that type of autoimmune disease characterized by chronic phases of
joint inflammation, transient expression of the transgene is an ideal
method.
Nonetheless, two major issues remain to be solved to optimize the use of adenovirus vectors in gene therapy: 1) stimulation of the host immune response to the vector with a second adneovirus injection, and 2) transgene expression at low levels, in only a small percentage of target cells, without ways to regulate promoter transcription. Better vectors will be available in the near future to optimize expression, targeting, and tolerance.
The AdvIL-10 treatment of CIA was associated with a modified humoral
response to CII during the course of arthritis. Although the relative
isotype abundance was IgG1 > IgG2a in all groups, the ratio of
IgG1:IgG2a increased in AdvIL-10-treated groups between day 28 and 37.
The Th1/Th2 switch observed between days 15 and 30 of CIA development
in DBA1 mice occurred (1), but was more marked on day 37 in AdvIL-10
groups than in control or AdvIL-10 mut groups. Thus, AdvIL-10 affects
Th2/Th1 balance by orienting the CII immune response toward a stronger
Th2-type cytokine profile, since it has been shown that IgG2a
production is associated with a Th1 response, while IgG1 production is
associated with a Th2 response. This stronger switch might be due to
different regulation of pro- vs anti-inflammatory cytokine
production. However, because of the physical limitation of using a CIA
mice model, we could not get access to the locally secreted cytokines
within the joints. Moreover, ELISA analysis of blood samples from
different groups after treatment showed no significant levels for
murine IL-1, IL-4, IL-10, TNF-, or IFN- (data not shown).
The LPS acceleration of CIA used in our protocol may also participate in the shift in Th2 cytokine balance observed in all groups by day 21 postimmunization. One explanation for the enhanced type 2 response in CIA groups treated with AdvIL-10 vector could be an IL-10 selective blockage of IL-12 synthesis by APCs (44).
Thus, we have demonstrated that replication-defective adenoviral vector delivery of vIL-10 transgene achieves therapeutic effects in murine CIA. Several mechanisms may contribute to this effect: vIL-10 may inhibit cytokines important for lymphocyte migration toward the joint by inhibiting proinflammatory synthesis of cytokines (9, 10, 11) and up-regulating production of their endogenous inhibitors (soluble TNFR and IL-1ra) (32). vIL-10 could also inhibit the specific proliferative response of T cells to CII Ag by down-regulating APC class II MHC molecule expression (33). Moreover, vIL-10 might convert activated into nonreactive APCs by down-regulating the expression of costimulatory molecules (CD86) implicated in the interaction with CTLA-4 and CD28 on synovial cells (33, 36). Finally, protection against cartilage destruction might be due to inhibition of metalloproteinases coupled to stimulation of TIMP-1 production induced by vIL-10 (7).
Cytokines other than IL-10 have natural anti-cytokine or anti-inflammatory activity and are in clinical development. Each of these agents has to be evaluated separately, even if optimal treatment of RA may require a combination of treatments because of the complex, intertwined nature of the cytokine network. However, vIL-10 gene therapy may represent one of the more efficient therapeutic treatments because of its combined anti-inflammatory and chondroprotective actions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Florence Apparailly, INSERM U475, 99 rue Puech Villa, 34197 Montpellier cédex 5, France. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; CIA, collagen-induced arthritis; CII, type II collagen; vIL-10, viral IL-10; AdvIL-10, adenoviral vector expressing vIL-10; mIL-10, murine IL-10; pfu, plaque-forming unit; RT, room temperature; IL-1ra, IL-1 receptor antagonist.
Received for publication October 20, 1997. Accepted for publication January 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß, not IL-6, in the newborn endotoxemic mouse. J. Pediatr. Surg. 131:411.
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 P Mukherjee, B Wu, L Mayton, S-H Kim, P D Robbins, and P H Wooley TNF receptor gene therapy results in suppression of IgG2a anticollagen antibody in collagen induced arthritis Ann Rheum Dis, August 1, 2003; 62(8): 707 - 714. [Abstract] [Full Text] [PDF]
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S. S. Shin, M. Jin, H. J. Jung, B. Kim, H. Jeon, J. J. Choi, J. M. Kim, B. W. Cho, S. H. Chung, Y. W. Lee, et al. Suppressive effects of PG201, an ethanol extract from herbs, on collagen-induced arthritis in mice Rheumatology, May 1, 2003; 42(5): 665 - 672. [Abstract] [Full Text] [PDF]
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A. C. M. Johansson, A.-S. Hansson, K. S. Nandakumar, J. Backlund, and R. Holmdahl IL-10-Deficient B10.Q Mice Develop More Severe Collagen-Induced Arthritis, but Are Protected from Arthritis Induced with Anti-Type II Collagen Antibodies J. Immunol., September 15, 2001; 167(6): 3505 - 3512. [Abstract] [Full Text] [PDF]
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J. O. Lindsay, C. J. Ciesielski, T. Scheinin, H. J. Hodgson, and F. M. Brennan The Prevention and Treatment of Murine Colitis Using Gene Therapy with Adenoviral Vectors Encoding IL-10 J. Immunol., June 15, 2001; 166(12): 7625 - 7633. [Abstract] [Full Text] [PDF]
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E. Quattrocchi, M. J. Dallman, A. P. Dhillon, A. Quaglia, G. Bagnato, and M. Feldmann Murine IL-10 Gene Transfer Inhibits Established Collagen-Induced Arthritis and Reduces Adenovirus-Mediated Inflammatory Responses in Mouse Liver J. Immunol., May 15, 2001; 166(10): 5970 - 5978. [Abstract] [Full Text] [PDF]
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J. L. Croxford, M. Feldmann, Y. Chernajovsky, and D. Baker Different Therapeutic Outcomes in Experimental Allergic Encephalomyelitis Dependant Upon the Mode of Delivery of IL-10: A Comparison of the Effects of Protein, Adenoviral or Retroviral IL-10 Delivery into the Central Nervous System J. Immunol., March 15, 2001; 166(6): 4124 - 4130. [Abstract] [Full Text] [PDF]
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M. M. Fort, R. Lesley, N. J. Davidson, S. Menon, F. Brombacher, M. W. Leach, and D. M. Rennick IL-4 Exacerbates Disease in a Th1 Cell Transfer Model of Colitis J. Immunol., February 15, 2001; 166(4): 2793 - 2800. [Abstract] [Full Text] [PDF]
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K. Setoguchi, Y. Misaki, Y. Araki, K. Fujio, K. Kawahata, T. Kitamura, and K. Yamamoto Antigen-Specific T Cells Transduced with IL-10 Ameliorate Experimentally Induced Arthritis Without Impairing the Systemic Immune Response to the Antigen J. Immunol., November 15, 2000; 165(10): 5980 - 5986. [Abstract] [Full Text] [PDF]
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K.-N. Kim, S. Watanabe, Y. Ma, S. Thornton, E. H. Giannini, and R. Hirsch Viral IL-10 and Soluble TNF Receptor Act Synergistically to Inhibit Collagen-Induced Arthritis Following Adenovirus- Mediated Gene Transfer J. Immunol., February 1, 2000; 164(3): 1576 - 1581. [Abstract] [Full Text] [PDF]
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R. M. Minter, J. E. Rectenwald, K. Fukuzuka, C. L. Tannahill, D. La Face, V. Tsai, I. Ahmed, E. Hutchins, R. Moyer, E. M. Copeland III, et al. TNF-{alpha} Receptor Signaling and IL-10 Gene Therapy Regulate the Innate and Humoral Immune Responses to Recombinant Adenovirus in the Lung J. Immunol., January 1, 2000; 164(1): 443 - 451. [Abstract] [Full Text] [PDF]
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J. P. Brown, J. F. Zachary, C. Teuscher, J. J. Weis, and R. M. Wooten Dual Role of Interleukin-10 in Murine Lyme Disease: Regulation of Arthritis Severity and Host Defense Infect. Immun., October 1, 1999; 67(10): 5142 - 5150. [Abstract] [Full Text] [PDF]
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E. R. Lechman, D. Jaffurs, S. C. Ghivizzani, A. Gambotto, I. Kovesdi, Z. Mi, C. H. Evans, and P. D. Robbins Direct Adenoviral Gene Transfer of Viral IL-10 to Rabbit Knees with Experimental Arthritis Ameliorates Disease in Both Injected and Contralateral Control Knees J. Immunol., August 15, 1999; 163(4): 2202 - 2208. [Abstract] [Full Text] [PDF]
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J. D. Whalen, E. L. Lechman, C. A. Carlos, K. Weiss, I. Kovesdi, J. C. Glorioso, P. D. Robbins, and C. H. Evans Adenoviral Transfer of the Viral IL-10 Gene Periarticularly to Mouse Paws Suppresses Development of Collagen-Induced Arthritis in Both Injected and Uninjected Paws J. Immunol., March 15, 1999; 162(6): 3625 - 3632. [Abstract] [Full Text] [PDF]
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C. Jorgensen, F. Apparailly, and J. Sany Immunological evaluation of cytokine and anticytokine immunotherapy in vivo: what have we learnt? Ann Rheum Dis, March 1, 1999; 58(3): 136 - 141. [Full Text]
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 R. M. Minter, M. A. Ferry, J. E. Rectenwald, F. R. Bahjat, A. Oberholzer, C. Oberholzer, D. La Face, V. Tsai, C. M. I. Ahmed, B. Hutchins, et al. Extended lung expression and increased tissue localization of viral IL-10 with adenoviral gene therapy PNAS, January 2, 2001; 98(1): 277 - 282. [Abstract] [Full Text] [PDF]
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) or received a single
injection of 109 pfu of AdvIL-10 expressing native (
,
n = 8) or mutated (
, n = 8) vIL-10.
One group was pretreated with 0.2 mg monoclonal rat anti-vIL-10 Ab
on day 29 (
, n = 6) or injected with
1010 pfu AdvIL-10 (n = 5,
).
