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Departments of
*
Medicine and
Pathology, Northwestern University Medical School, Chicago, IL 60611;
Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
Veterans Administration, Lakeside Division, Chicago Health Care System, Chicago IL 60611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
, macrophage inflammatory
protein-2, and RANTES were decreased in AxCAIL-4 rat ankle homogenates
compared with PBS-treated homogenates. Thus, increased expression of
IL-4 via gene therapy administered in a preventative and/or therapeutic
manner reduced joint inflammation, synovial cellularity, levels of
proinflammatory cytokines, vascularization, and bony destruction in rat
AIA, suggesting that a similar treatment in humans may be
beneficial. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-4 is a pleiotropic cytokine that plays a number of important roles,
including the regulation of inflammation (2). IL-4
production in normal tissue is tightly regulated and mainly occurs in
Th2 cells, mast cells, and basophils (2). The RA synovium,
however, lacks IL-4 (3), in addition to many other T
cell-derived cytokines, despite the abundance of T cells in the
synovial infiltrate (4). IL-4 acts on LPS-stimulated
monocytes in vitro to down-regulate the production of the inflammatory
cytokines TNF-, IL-1, IL-1, IL-6, IL-8, G-CSF, and macrophage
inflammatory protein (MIP)-1 (5). Complementary to its
anti-inflammatory properties on monocytes, IL-4 inhibits RA
synoviocyte proliferation and production of PGE2
and GM-CSF (6). IL-4 enhances monocyte apoptosis, thereby
decreasing monocyte accumulation (7), and acts as an
autocrine growth factor promoting the differentiation of naive T cells
to Th2 cells (2). Interestingly, both IL-4- and
IL-4/IL-13-deficient mice can still employ compensatory mechanisms
enabling a Th2-like response, as demonstrated by the production of IL-5
(8). We have recently found that in a human ex vivo model
of RA, using adenovirally produced human IL-4, we are able to markedly
lower the secretion of proinflammatory mediators in the synovium
(9).
Bypassing the initiating factors in RA and manipulating the cytokine balance may be an effective therapeutic means by which chronic inflammation can be managed (10). The outlook is favorable for the combination of RA treatment with gene therapy as a means for agent delivery, making the appropriate selection of candidate therapeutic proteins essential (11, 12, 13). Here, we use an adenoviral gene therapy approach in the rat AIA model to increase the expression of IL-4 in inflamed joints. We demonstrate that adenovirally produced IL-4 can reduce AIA-induced inflammation and the presence of inflammatory cells in the synovium, decrease bony destruction, reduce the quantity of synovial blood vessels, and attenuate proinflammatory cytokine levels in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two experimental designs, including preventative and therapeutic manipulation of AIA, were used to determine whether an adenovirus containing no inserted gene (AxCAIL-4) could prevent or treat AIA-associated inflammation. Rats for the preventative study were arbitrarily divided into three groups on day 0, including an AxCAIL-4 group as well as two control groups designed to receive PBS or an adenovirus containing no inserted gene (AxCANI; n = 10 rats/group). The PBS group was included to assure that anti-inflammatory IL-4 did not simply decrease inflammation relative to a mildly inflammatory adenoviral control without actual effects on AIA-induced swelling. Previous experiments with the rat AIA model demonstrated that maximal inflammation occurred around day 18 postadjuvant. Therefore, on day 8 after AIA induction, 5 x 106 or 1 x 108 PFUs of the adenoviral vector with or without the IL-4 gene or PBS was administered to each ankle in a 10-µl volume via intra-articular (i.a.) injection. Clinical parameters were assessed on days 0, 2, 4, and 7 after adjuvant injection (before i.a. injection) as well as days 9, 11, 14, 16, and 18 after adjuvant injection. All animals were sacrificed, and ankles were collected for further examination on day 18.
Rats for the therapeutic study were likewise divided into three groups on day 0, including an AxCAIL-4 group as well as PBS and AxCANI groups (n = 13 rats/group). Because we were examining the reduction of inflammation, on day 18 we selected the rats with the most inflamed ankles, based on ankle circumference and paw volume in each group, for i.a. injection, thus resulting in a minimum of 10 rats/group. All animals were administered an i.a. injection (1 x 108 PFUs in 10 µl to each ankle) of the appropriate test group or PBS (10 µl) on day 18 to determine whether AxCAIL-4 could attenuate maximal arthritis. Clinical parameters were assessed on days 0, 2, 4, 7, 9, 11, 14, 16, and 18 postadjuvant (before adenoviral/control injection) as well as days 21, 23, and 25 postadjuvant. All animals were sacrificed, and ankles were collected for further examination on day 25.
Preparation, propagation, purification, and titration of adenoviruses
Replication incompetent AxCAIL-4 and AxCANI were prepared via
homologous recombination in 293 cells using methods described
previously (14). In short, expression of the rat IL-4 gene
was directed by the chicken -actin promotor and the CMV enhancer of
pAxCAwt, a 45-kb cosmid containing the full-length sequence of type 5
adenovirus deleted of the E1A, E1B, and E3 regions (15, 16). The production of rat IL-4 protein was examined by ELISA in
conditioned medium of rat synovial fibroblast cultures. Viruses were
propagated through successive infection of 293 cells followed by
harvesting of cell lysates. Purification of virus was accomplished
using cesium chloride density ultracentrifugation followed by dialysis.
Titer was estimated by the number of PFUs of virus in 293 cells.
Induction of rat AIA
Female Lewis rats (100 g) were injected s.c. with 300 µl (5 mg/ml) of lyophilized Mycobacterium butyricum (Difco, Detroit, MI) at the base of the tail. All time points were considered relative to this AIA induction day, which was designated day 0. The time course and the expression of an adenoviral vector in the rat AIA model have previously been examined (14).
Clinical measurements
Clinical parameters were assessed on the days detailed above and
included measurements of body weight, articular index (AI) scores,
ankle circumference, and paw volume. AI scores were assigned to every
joint by a single observer blinded to the group of the animal. Scoring
was performed on a 0–4 scale where 0 = no swelling or erythema,
1 = slight swelling and/or erythema, 2 = low to moderate
edema, 3 = pronounced edema with limited joint usage, and 4 =
excess edema with joint rigidity. For ankle circumference
determination, two perpendicular diameters of the joint were measured
with a caliper (Lange Caliper, Cambridge Scientific Industries,
Cambridge, MA). Ankle circumference was determined using the geometric
formula: circumference = 2
(
(a2 +
b2/2)), where a is the
latero-lateral diameter, and b is the antero-posterior
diameter, as we have previously described (17). The volume
of hind ankles was determined using a paw volume plethysmometer (Kent
Scientific, Litchfield, CT).
Ankle x-rays and radiographic scoring
Upon sacrifice, ankles were promptly removed to ice, and x-rays were taken. Ankles were positioned over a radiographic cassette containing standard veterinary x-ray film (Animal Care Products, St. Paul, MN) to obtain a lateral view. A conventional x-ray source (Mobile 225, General Electric, Milwaukee, WI) was used at exposure factors of 40 kV (peak) and 25 mA for 0.1 s. Radiographs were scored on a scale of 0–3 based on joint space between the tibia and calcaneus, the tibia and talus, and the talus and calcaneus (0 = no increase in space, 3 = maximal increased joint space). Radiographs were also scored for the degree of bony destruction/erosions (from 0–4), assigning a point for an erosion in the tibia, calcaneus, talus, and metatarsals (considered together). For example, the maximum score an ankle could receive was 4 if an erosion were present in the tibia, calcaneus, talus, and any one or more of the metatarsals. Soft tissue swelling was also scored on a scale of 0–3 (0 = mild or no swelling, 2 = moderate swelling, 3 = severe swelling), where the maximum score a single ankle could receive was 3. All scores were calculated by an observer blinded to the experimental groups.
Ankle homogenates and ELISAs
After x-rays were taken, ankles for ELISA use were skinned, weighed, and frozen at -80°C. Ankles were homogenized in a 50-ml conical centrifuge tube containing 3 ml of Complete Mini protease inhibitor cocktail (Roche, Indianapolis, IN) homogenization buffer. Ankle homogenization was completed on ice using a motorized homogenizer, followed by 30 s of sonication. Homogenates were centrifuged at 2000 x g for 10 min, filtered through a 0.45-µm pore size Millipore filter (Bedford, MA), and stored at -80°C until use.
Cytokine levels in ankle homogenates were determined using commercially
available ELISA kits that specifically recognize the rat cytokines
TNF-, IL-1, monocyte chemoattractant protein-1 (MCP-1), MIP-2,
and RANTES (BioSource International, Camarillo, CA) according to the
manufacturer’s procedure. Rat IL-4 levels were determined using an
ELISA designed with matched Ab pairs available from PharMingen (San
Diego, CA). Briefly, 96-well plates were coated with mouse anti-rat
IL-4 (2 µg/ml) overnight at 4°C in coat buffer (50 mM
H3BO3 and 120 mM NaCl, pH
8.6) and washed with wash buffer (PBS and 0.05% Tween-20). Wells were
blocked with block buffer (PBS and 2% BSA) for 1 h at 37°C and
washed. Recombinant rat IL-4 (R&D Systems, Minneapolis, MN) and ankle
homogenates were added in duplicate to wells for 3 h at 37°C,
followed by washing. Biotinylated rabbit anti-rat IL-4 (1 µg/ml)
in block buffer was incubated for 1 h at 37°C and washed,
followed by incubation with streptavidin-HRP (PharMingen; 1/10,000) in
block buffer. After 30 min wells were washed, and HRP was detected with
3,3',5,5'-tetramethyl-benzidine liquid substrate system (Sigma, St.
Louis, MO). Color development was terminated with 0.5 M
H2SO4, and plates were read
at 450 nm.
Histologic analysis of tissue sections
After x-rays were taken, ankles for sectioning were skinned, embedded in OCT (Miles, Elkhart, IN), and frozen at -80°C until sectioning. Sections (4 µm) were cut using a knife suitable for bone cutting and were stained with hematoxylin and eosin. The synovial infiltrate, including monocyte/macrophages, lymphocytes, and polymorphonuclear cells, was determined, based on characteristic morphologic features, by a pathologist, blinded to the experimental groups. The sum of cell counts as well as the number of blood vessels in three x1000 microscopic fields were determined for each section.
Statistical analysis
Rats treated in the preventative and therapeutic manner described above received identical injections in each ankle based on their group assignment (PBS, AxCANI, or AxCAIL-4). As in previous studies, AIA rats often developed inflammation to different degrees in each of the hind limbs, as demonstrated by different AI scores. For this reason, each ankle was treated independently for statistical purposes. By Student’s t test, p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several concentrations of AxCANI, namely 5 x
106, 1 x 107, 5
x 107, and 1 x 108
PFU, were injected i.a. into AIA as well as adjuvant-naive rats to
assess induction of ankle inflammation. Adjuvant-naive rats were
injected with AxCANI at various doses on day 0, and their weights, AI
scores, and ankle circumferences were measured over a 19-day period.
Every AI score recorded over the 2.5-wk period for each ankle at every
dose received a zero, indicating that no dose of AxCANI induced
inflammation (data not shown). Similarly, ankle circumferences measured
over this period did not vary significantly among the groups (Fig. 1
A). Further, rat body weight
was not significantly altered among the groups (data not shown). In
contrast, when AIA rats were injected with the same doses of virus 8
days postadjuvant injection, but before signs of swelling, AxCANI
clearly induced ankle inflammation when examining ankle circumference
(Fig. 1B) and AI scores (Fig. 1C). It appears
that a cut-off point exists between the lowest dose used (5 x
106 PFU), which does not appear to induce
inflammation, and the 1 x 107 PFU dose,
which does.
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Adenovirus was initially investigated at 1 x
108 PFUs because this quantity of IL-4-producing
virus has previously been shown to significantly reduce the production
of proinflammatory mediators in an ex vivo model of RA (9)
and because this dose was used effectively in previous rat models of
arthritis (18, 19). Rat ankle homogenates were analyzed
for IL-4 production by ELISA on day 18, 10 days after i.a. injections
of virus. The mean IL-4 levels of preventatively treated AxCAIL-4
ankles (5.3 ± 1.1 ng/ml; n = 9) were 6.6- and
8.8-fold higher than the mean levels in PBS (0.8 ± 0.05 ng/ml;
n = 6) and AxCANI (0.6 ± 0.05 ng/ml;
n = 5) groups, respectively. Animal body weights were
determined three times per week in the 18-day study (Fig. 2). Mean body weights of rats in all
three groups increased through day 7, before i.a. injections. After day
8 the mean body weights of PBS-injected rats remained consistent for
about 6 days and then decreased. Weight loss before detection of
clinical signs of inflammation in the hind limbs is one of the systemic
AIA effects and was an anticipated result. In contrast, animals that
did not receive adjuvant continued to increase in body weight
throughout this period following a sham injection (20).
Mean body weights of AxCANI rats declined immediately after adenoviral
administration. In contrast, mean body weights of AxCAIL-4 rats
remained approximately constant for 3 days postinjection, and then
increased and remained consistent for the duration of the study. By
comparison with the AxCANI group, mean AxCAIL-4 rat weights were
significantly higher just 1 day after adenoviral administration and
remained significantly higher for the duration of the study
(p < 0.05). Compared with weights in the
control group that received AIA and a sham PBS i.a. injection, mean
AxCAIL-4 rat weights were significantly higher from days 14 through 18,
perhaps indicative of healthier animals.
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A) show that AIA-induced
swelling began around day 10 and increased through day 18. By
comparison, 1 x 108 PFU of adenovirus
induced an inflammatory response immediately after injection, as mean
AI scores on day 9 for both AxCANI- and AxCAIL-4-injected groups were
higher than that in the PBS-injected group (p
< 0.05). After day 9, however, mean AI scores for the AxCANI and
AxCAIL-4 groups quickly diverged as scores for ankles receiving IL-4
leveled off for the remainder of the study. From days 11 through 18,
the mean AI score of AxCAIL-4-injected animals was significantly lower
than that of the AxCANI control (p < 0.05).
Further, on day 16 the mean AI score of AxCAIL-4 rats was significantly
below that of the PBS group.
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B). On day 9, 1 day after
ankle injections, the mean ankle circumferences for the
AxCAIL-4 group were significantly higher than those for the
PBS group (p < 0.05). By day 11, however, mean
ankle circumferences for PBS and AxCAIL-4 animals were identical.
Beyond day 11, the mean ankle circumference of AxCAIL-4 rats was lower
than that of the PBS group, attaining statistical significance on day
16 (p < 0.05). Comparing the mean ankle
circumference of AxCAIL-4 rats with that of the AxCANI group
demonstrates that IL-4-treated rats had significantly smaller ankles
from days 9 through 18 (p < 0.05).
Plethysmometer measurements of paw volume were also used to compare the
relative quantity of inflammation among groups. This measurement was
used because it provides an objective physical measurement of total paw
swelling, whereas ankle circumference does not take swelling in the
lower foot into account, and the AI scores are a subjective
measurement. Mean paw volume measurements on days 11 and 16
demonstrated that AxCAIL-4 conferred an anti-inflammatory effect
less than that in the PBS-injected group (Fig. 3C). Further,
mean paw volumes of the AxCAIL-4 group were significantly less than
those in the AxCANI group from days 14 through 18. These results and
the consistency of the three methods for measuring rat paw inflammation
strongly suggest that IL-4 is capable of reducing clinical symptoms of
rat AIA.
An examination of rat ankle x-rays for bony erosion/destruction and
soft tissue swelling was also performed on day 18. When x-rays from the
preventatively treated group were graded for bone erosions alone, the
mean score for the AxCAIL-4-treated group (0 ± 0) was
significantly lower than that for the PBS (0.8 ± 0.3) or AxCANI
(2.1 ± 0.3) group (p < 0.05). As
indicated by the mean, no AxCAIL-4 preventatively treated ankles were
found to contain bone erosions. When scored for joint space narrowing
alone, the mean score for the AxCAIL-4-injected group (0.3 ± 0.1)
was also lower than that for the PBS-injected (1.1 ± 0.2) or
AxCANI-injected (1.9 ± 0.2) group (p <
0.05). When scored for soft tissue swelling alone, the mean AxCAIL-4
score (1.5 ± 0.1) was significantly lower than that of the
AxCANI-injected group (2.5 ± 0.2; p < 0.05), but
not that of the PBS group (1.5 ± 0.2). Fig. 4 depicts the sum of all the x-ray scores
(bony erosion, joint space narrowing, and soft tissue
swelling), showing mean AxCAIL-4 scores 48 and 73% lower than those of
PBS and AxCANI controls, respectively
(p < 0.05). The data suggest that the
production of IL-4 in the AIA joint can prevent both AIA- and
AIA/adenovirus-induced destruction of bone.
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), while normalization to prehomogenized
ankle weight yielded similar results (data not shown). There were
obvious differences in ankle sizes among the various treatment groups,
which was reflected in ankle weights. Mean ankle weights (±SE) of
preventatively treated PBS, AxCANI, and AxCAIL-4 rats were 1.0 ±
0.2, 1.3 ± 0.1, and 0.8 ± 0.1 g, respectively. The
mean total protein concentrations (±SE) of preventatively treated PBS,
AxCANI, and AxCAIL-4 rat ankles were 5.4 ± 0.8, 8.2 ± 0.4,
and 4.0 ± 0.2 mg/ml, respectively. It is noteworthy that the
protein concentration of AxCANI ankles was significantly higher than
those in the PBS and AxCAIL-4 groups (p <
0.05). There was no significant difference between the protein
concentration of PBS- and AxCAIL-4-treated rat ankles, and
consequently, cytokine data for the AxCANI group were not
included in Table I, because reliable normalization was not possible.
Mean MCP-1 levels were decreased by 44% in AxCAIL-4 rat ankle
homogenates compared with levels in PBS-injected ankle homogenates
(p < 0.05). Quantitation of TNF-, IL-1,
MIP-2, and RANTES was performed in a similar manner. Mean levels of
these cytokines did not differ when normalized to protein
concentration between AxCAIL-4- and PBS-injected ankles (Table I).
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). AxCAIL-4 reduced the mean number of
monocytes by 27 and 19% compared with counts from the PBS-treated
(p < 0.05) and AxCANI-treated groups,
respectively. Mean lymphocyte counts were likewise affected by
AxCAIL-4, being reduced by 41 and 30% compared with mean counts for
PBS-injected (p < 0.05) and AxCANI-injected
joints, respectively. The largest decrease in cell counts was seen in
mean polymorphonuclear cell numbers, where AxCAIL-4 decreased
PBS-injected (p < 0.05) and AxCANI-injected
counts by 64 and 48%, respectively. Finally, AxCAIL-4 reduced the mean
number of blood vessels by 28 and 36% compared with mean vessel
numbers from PBS and AxCANI (p < 0.05)
sections.
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Rat ankle homogenates were analyzed for IL-4 production by ELISA on day 25 postadjuvant injection, 7 days after i.a. injection. Mean IL-4 levels of therapeutically injected AxCAIL-4 ankles (2.4 ± 0.4 ng/ml; n = 12) were 3.4- and 4.0-fold higher than those of PBS-treated (0.7 ± 0.05 ng/ml; n = 6) and AxCANI-treated (0.6 ± 0.06 ng/ml; n = 6) groups, respectively.
Rat weights were determined three times per week for the duration of
the 25-day study. By day 18, the day of i.a. injection, there was a
discrepancy among groups with respect to mean rat weights despite
random assignment of animals to experimental groups. The PBS-injected
group of rats had higher mean weights than rats of the AxCAIL-4 and
AxCANI groups. Therefore, all therapeutic data are presented as a
percentage of the respective group’s day 18 values. On days 23 and 25
AxCAIL-4 rats amassed significantly more weight than PBS or AxCANI
rats, when determined relative to day 18 values (Fig. 6; p < 0.05).
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Ankle circumferences were significantly decreased at all time points
measured after therapy was initiated (Fig. 7A). Mean ankle circumference
values of AxCAIL-4 rats, relative to day 18 values, were 9, 12, and 6%
below similar values for the PBS group on days 21, 23, and 25,
respectively (p < 0.05 at all time points).
Similarly, mean ankle circumference values of AxCAIL-4 rats, relative
to day 18 values, were 10, 14, and 10% lower than values for the
AxCANI group on days 21, 23, and 25, respectively
(p < 0.05 at all time points).
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B).
An analysis of bony erosions for therapeutically treated rats was also
performed. The mean bony erosion score of AxCAIL-4-treated ankles
(0.6 ± 0.2) was significantly lower than that of AxCANI-treated
ankles (1.5 ± 0.2; p < 0.05), but not that of
PBS-treated ankles (1.0 ± 0.3). When scored for joint space
narrowing, the mean AxCAIL-4 score (0.9 ± 0.2) was again
significantly lower than that of AxCANI-treated ankles (1.6 ±
0.2; p < 0.05), but not that of PBS-treated ankles
(1.0 ± 0.2). Mean soft tissue swelling scores of AxCAIL-4-treated
ankles were not significantly different from those of the PBS or AxCANI
groups. Fig. 8 depicts the sum of the all
x-ray scores (bone erosion, joint space, and soft tissue swelling),
showing mean AxCAIL-4 scores 40% lower than those of the AxCANI
control (p < 0.05) and only 5% lower than
those of the PBS control.
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, because reliable
normalization was not possible. Mean AxCAIL-4-treated rat ankle
homogenate levels of TNF-, IL-1, MIP-2, and RANTES (normalized to
protein content) were decreased by 73, 76, 66, and 71%, respectively,
compared with those in PBS-treated ankles (p <
0.05). In contrast to preventatively treated ankles, AxCAIL-4 had no
effect on mean MCP-1 levels when given after the development of
clinical symptoms. Histologic assessment of tissue sections from therapeutically injected rat ankles were also examined. Although AxCAIL-4 reduced the number of lymphocytes by 19% compared with that in the PBS group, therapeutic injections did not yield statistically significant reductions in any of the cell types or the number of blood vessels (data not shown).
AxCAIL-4 at 5 x 106 PFU
Because the data in Fig. 1 demonstrated that 5 x
106 PFU AxCANI did not induce an
inflammatory response in AIA rats, we also examined the effect of this
dose of AxCAIL-4. In contrast to our data for 1 x
108 PFU, Fig. 9
demonstrates that the lower dose of AxCAIL-4 induces inflammation when
administered before arthritis onset. The AI scores (Fig. 9A)
and ankle circumferences (Fig. 9B) showed a similar trend.
In contrast, the same dose given therapeutically did not have any
effect on ankle circumference (Fig. 9C) or AI scores (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-,
enhanced onset and aggravated synovial inflammation were found
(21). In contrast, retrovirally produced IL-4 was
effective when administered before arthritis onset in reducing paw
swelling (22). Similar to the previous study, the latter
data supported a reduction in bone destruction (22).
Herein, we investigated both preventative and therapeutic approaches
with AxCAIL-4 and focused on inflammatory parameters of AIA.
Preventative AxCAIL-4 at 1 x 108 PFU
decreased ankle inflammation, increased rat body weight, reduced
synovial cellularity and blood vessel numbers, and lowered levels of
the proinflammatory cytokine MCP-1. In addition, in our study ankles
receiving virally produced IL-4 maintained better bone integrity than
AIA- or AIA/adenovirus-treated ankles, as suggested by radiographic
data. Therapeutic AxCAIL-4 at 1 x 108 PFU
likewise inhibited AIA-associated weight loss and reduced ankle
inflammation, as demonstrated by reductions in ankle circumference and
paw volume. In addition, the latter treatment with AxCAIL-4
significantly reduced ankle homogenate levels of the proinflammatory
cytokines IL-1, TNF-, MIP-2, and RANTES.
The actions of endogenous and exogenous IL-4 on inflammation in vivo
are not clear. DBA/1J mice do not develop arthritis when induced with
Mycobacterium tuberculosis over a 120-day period
(23). However, administration of a neutralizing IL-4 Ab
once daily for 10 days i.p. initiated at the time of M.
tuberculosis immunization induced arthritis in 83% of mice by day
28 (23). Similar results occur when IL-4 is neutralized in
T cell-mediated arthritis (24). These studies imply that
endogenous IL-4 may inhibit arthritis development, but do not
demonstrate directly that IL-4 can achieve this effect. Similarly, in
CIA, injections of anti-IL-4 i.p. for 10 days initiated on the day
of immunization with type II articular cartilage collagen markedly
augmented the incidence and severity of arthritis (25).
However, anti-IL-4 Ab did not affect CIA incidence or macroscopic
arthritis evaluation in a similar study (26). Together,
the previous studies suggest that a possible explanation for the
preventative characteristics of IL-4 may be its immunoregulatory
ability to decrease a Th1 and/or increase a Th2 cell profile. It is
noteworthy that RA is also a Th1-dominant disease in which IL-4 levels
are limited and IFN-
secretion may be increased (3, 27, 28, 29).
In the present study preventative AxCANI induced inflammation above
that produced by AIA alone, as represented by the PBS group (Fig. 3).
Fig. 3 also demonstrates that production of IL-4 by adenovirus
eliminated adenovirally induced inflammation, as the swelling of
AxCAIL-4 ankles was less than that in the sham-injected PBS control.
This suggests that inclusion of the IL-4 gene in inflammation-inducing
viruses may counteract the immune response induced by the virus and its
related proteins.
The retention of bone integrity by AxCAIL-4-injected rats before disease onset was an additional benefit conferred by IL-4. It is possible that IL-4 reduced bony destruction by inhibiting bone resorption through its actions on osteoclasts and proinflammatory cytokines, as it does ex vivo (30). This result is in agreement with a recent paper that demonstrated that local IL-4 production by virus injection prevented joint damage and bone erosion in mice with CIA (31).
Ample evidence supports a role for IL-4 in modulating established
autoimmune diseases. These include studies in nonobese diabetic mice
(32), experimental allergic encephalomyelitis
(33), CIA (34, 35, 36), and streptococcal cell
wall arthritis (37). Herein, we demonstrate that a single
AxCAIL-4 injection administered near the peak of inflammation can
significantly reduce inflammation and proinflammatory cytokine
production associated with rat AIA. This approach may confer some
advantage over administration of cytokine via a pump. Fig. 6
demonstrates that after disease onset AxCAIL-4 significantly increased
body weights 5 and 7 days after i.a. injection compared with control
weights. Animals receiving AxCANI before disease onset lost more weight
than PBS controls (Fig. 2), whereas the same dose of adenovirus did not
have this effect after inflammation was established (Fig. 6).
Similarly, Fig. 7, A and B, demonstrates that the
dichotomy between the AxCANI and PBS groups was not as disparate as
that with preventative administration (Fig. 3). This suggests that
adenovirus does not induce much additional inflammation beyond that of
AIA, once the disease is at its peak. Measurements of AxCAIL-4-injected
ankles (Fig. 7) clearly demonstrate that IL-4 production can
significantly improve AIA- and AIA/adenovirus-induced inflammation.
X-ray analysis of animals treated after arthritis onset (Fig. 8)
demonstrated that IL-4 could significantly improve
AIA/adenovirus-induced radiographic scores. However, IL-4 could not
statistically improve scores compared with AIA-induced damage alone. It
should be noted that the day 25 analysis of bones in the therapy model
was performed only 7 days after the i.a. injection. Preventatively,
x-ray analysis was performed on day 18, which was 10 days after the
i.a. injection on day 8. It is possible that the prolonged IL-4 ankle
exposure of our preventatively designed experiment may account for the
statistical improvement demonstrated when treatment was administered
before arthritis onset.
In the therapeutic setting the reduction of inflammation may be
attributed to the ability of IL-4 to abrogate production of
proinflammatory cytokines. We recently investigated whether an
adenovirus producing human IL-4 could reduce the production of
proinflammatory mediators from RA synovial tissue explants ex vivo
(9). Virally produced IL-4 significantly reduced the
secretion of IL-1, TNF-, IL-8, MCP-1, epithelial neutrophil
activating peptide-78, growth-related gene product-, and
PGE2, all of which are present in the RA synovium
(9). In the present study there were some differences
between the effects of IL-4 on proinflammatory cytokines in ankles when
administered before and after disease onset. Preventative
administration of AxCAIL-4 significantly decreased MCP-1 levels.
Treatment after arthritis onset, in contrast, decreased TNF-,
IL-1, MIP-2, and RANTES levels, but not those of MCP-1. It is
possible that preventative IL-4 effects were partially achieved by
inhibiting an early and essential monocyte-dependent stage of rat AIA
that is MCP-1 reliant. Once the inflammatory response is established,
however, IL-4 selectively inhibited a broader array of inflammatory
mediators. An additional possibility for differences between the
outcomes when treating before or after disease onset may be related to
the differences in the amount of IL-4 produced. Comparing ankle
homogenate concentrations of IL-4 in the two studies demonstrates that
preventatively injected ankles contain 2.2-fold higher mean
concentrations of IL-4 than ankles injected after disease onset. This
is probably the result of the disparity in the paw volume that is being
injected between days 8 and 18. For example, inflamed joints on day 18
(therapeutic study) had 2.0-fold greater paw volume than ankles
injected on day 8 (preventative study). Therefore, optimizing the
quantity of AxCAIL-4 used in therapeutic injections to produce an
equivalent concentration of rat IL-4 as in preventatively injected
ankles may have resulted in additional significant changes, including a
decrease in synovial cellularity. Despite this, the concentration of
IL-4 produced was sufficient to significantly improve body weight,
reduce ankle circumference, lower paw volume, and decrease quantities
of proinflammatory cytokines.
A possible mechanism that could account for the actions of IL-4 may be via inhibiting neovascularization into synovial tissue. IL-4 has a biphasic dose-response curve with regard to endothelial cell migration in vitro and neovascularization in vivo (38). For example, we have shown previously that low doses of IL-4 are proangiogenic, while higher doses can inhibit neovascularization induced by basic fibroblast growth factor (38, 39). When 1 x 108 PFU AxCAIL-4 was administered before arthritis onset, IL-4 production reduced the mean number of blood vessels in synovial sections by 39 and 36% vs those in PBS and AxCANI tissue sections, respectively. This could in part contribute to the decreased inflammation and cellularity of IL-4-treated synovium. When 5 x 106 PFU AxCAIL-4 were administered before arthritis onset, IL-4 induced inflammation. This is in agreement with recently published results showing that 1 x 106 PFU of virally produced IL-4 increased clinical and histological signs of inflammation in CIA in mice when administered preventatively, but a dose of 1 x 107 PFU did not (21, 31). In short, these results underscore the critical nature of proper dosing with IL-4.
While this paper was under review a study was published that
investigated virally produced IL-4 in murine CIA in a therapeutic
manner (40). Local injections of 5 x
108 viral particles producing murine IL-4 were
shown to partially reverse the progression of established disease
(40). Our results suggest that low doses (5 x
106 PFU) of virally produced IL-4 in rat AIA are
proinflammatory, whereas higher doses (1 x
108 PFU) are anti-inflammatory when
administered before arthritis onset. We show that joint vascularity as
well as cytokine levels of TNF-, IL-1, MCP-1, RANTES, and MIP-2
are all modulated by IL-4. We demonstrate that adenovirally produced
IL-4 administered before or after AIA induction can ameliorate
arthritis. In comparison with animals given AIA or adenoviral/AIA
treatment, animals injected with virally produced IL-4 had higher body
weights, reduced inflammation, decreased synovial cellularity, and
reduced bony destruction. Optimizing the proper vector, dose, and route
of administration of IL-4 may result in an efficient therapeutic
modality for the treatment of RA patients.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alisa E. Koch, Department of Medicine, Section of Rheumatology, Northwestern University Medical School, 303 East Chicago Avenue, Ward Building 3-315, Chicago, IL 60611.
3 Abbreviations used in this paper: RA, rheumatoid arthritis; AxCAIL-4, adenovirus containing the IL-4 gene; AxCANI, adenovirus containing no inserted gene; AIA, adjuvant-induced arthritis; MIP, macrophage inflammatory protein; i.a., intra-articular; AI, articular index; MCP-1, monocyte chemoattractant protein-1; CIA, collagen-induced arthritis.
Received for publication November 15, 1999. Accepted for publication October 23, 2000.
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References |
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