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*
Rheumatology Research Lab, Department of Rheumatology, University Hospital Nijmegen, Nijmegen, The Netherlands; and
Department of Pathology, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß were suppressed as well, and IL-1ß and nitric
oxide production by arthritic synovial tissue were strongly reduced.
Our data show an impressive cartilage-protective effect of local IL-4
and underline the feasibility of local gene therapy with this cytokine
in arthritis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now generally accepted that the cytokines TNF- and IL-1 are
principal mediators in RA (3, 4). These proinflammatory
cytokines are produced in increased quantities by RA synovium and
detected in synovial fluid. IL-1, in particular, is a key cytokine in
cartilage destruction, whereas TNF is a major cytokine in the
inflammatory process. Treatment with TNF Abs or soluble receptors
markedly reduced the inflammatory signs in RA patients
(4, 5, 6). Apart from direct interference with TNF/IL-1,
regulation can be exerted at the level of modulatory cytokines such as
IL-10 and IL-4. IL-4 has the potential to effectively antagonize
inflammatory and destructive mediators of the RA process
(7). Induced synovial production of IL-1 and TNF is
strongly reduced; IL-1Ra is enhanced; and spontaneous production of
IL-6, TNF, leukemia-inhibitory factor, and PGE2
is also inhibited by IL-4 (8, 9). Moreover, IL-4 regulates
expression and release of soluble receptors, including IL-1RII and the
TNF receptors (10, 11). Finally, in vitro studies
demonstrated suppressive effects of IL-4 on Th1 activity (12, 13) and beneficial effects on cartilage matrix degradation
(14, 15, 16, 17). Of great importance, IL-4 could not be detected
in synovial fluid, synovial supernatants, or synovium of RA patients
(13). This lack of IL-4 is likely to contribute to the
uneven Th1/Th2 balance and to the chronic nature of RA (13, 18).
In addition to the in vitro work, therapeutic studies have been done with IL-4 in animal models of arthritis. Due to the short t1/2, treatment was systemic and injections were given daily. Suppression of collagen arthritis and streptococcal cell wall arthritis was demonstrated, whereas synergy with IL-10 was evident (19, 20). However, a major risk of prolonged systemic treatment is the induction of generalized immunosuppression or allergic reactions at multiple sites. Local gene transfer with a viral vector might circumvent these problems. Evidence for the potential of successful gene delivery of other cytokine-related constructs to synovial tissue has already been obtained (21, 22, 23, 24, 25).
In the present study, we examined the effects of local IL-4 overexpression, through a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen arthritis. We found that this treatment greatly protects against cartilage erosions, despite ongoing inflammation. The protective effect was associated with a reduction of PMNs in the synovial joint space, decreased NO synthesis, down-regulation of IL-1ß, and a reduction of the MMP-3/TIMP disbalance in the synovium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male DBA-1/BOM mice were purchased from Bomholdgärd (Ry, Denmark). The mice were housed in filter-top cages. The mice were immunized between 10 and 12 wk of age. Water and food were provided ad libitum.
Adenoviral vectors
The recombinant replication-deficient adenovirus Ad5E1 mIL-4 was generated by homologous recombination after cotransfecting 293 cells with PACCMVmIL-4 and a virus-rescuing vector pAdBHG10, as described (26). The empty recombinant replication-deficient adenovirus Ad5de170-3 was used as a control vector throughout the study. High titers of recombinant adenoviruses were amplified, purified, titered, and stored, as described (27).
Materials
Freund’s complete adjuvant and Mycobacterium tuberculosis (strain H37Ra) were obtained from Difco (Detroit, MI). Bovine type II collagen (CII) was prepared as described (20). RPMI 1640 was obtained from Life Technologies (Breda, The Netherlands). ELISA plates (Maxisorb) were purchased from Nunc (Copenhagen, Denmark). The following mAbs were used in the cytokine ELISAs: anti-murine IL-4 Abs (capture 18031D, detection 18042D) were purchased from PharMingen (San Diego, CA). Anti-murine IL-1ß Abs (capture PM-425-B, detection MM-425-B) were obtained from Endogen (Cambridge, MA). Anti-murine IL-1Ra Abs (capture MAB480, detection BAF480) were from R&D Systems (Minneapolis, MN). Streptavidin-polyperoxidase conjugate was obtained from CLB (Amsterdam, The Netherlands). Murine rIL-4 was a kind gift of Dr. S. Smith (Schering-Plough, Kenilworth, NJ). Murine rIL-1ß was from R&D Systems. NIMP-R14 (rat IgG2b anti-mouse mAb) was kindly provided by Dr. M. Strath (National Institute for Medical Research, London, U.K.). Anti-VDIPEN IgG Abs were a generous gift of Drs. I. Singer and E. Bayne (Merck Research Laboratories, Rahway, NJ).
Induction of CIA
Bovine CII was diluted in 0.05 M acetic acid to a concentration of 2 mg/ml and was emulsified in equal volumes of Freund’s complete adjuvant (2 mg/ml of M. tuberculosis). The mice were immunized intradermally at the base of the tail with 100 µl of emulsion (100 µg of collagen). On day 21, mice were given an i.p. booster injection of 100 µg of CII diluted in PBS, and normally arthritis onset occurs at about day 28.
Study protocol
CIA was induced in male DBA-1 mice, as described above. Just before expected onset of CIA, mice were scored visually for the appearance of arthritis. Mice without macroscopic signs of arthritis in the paws were selected. Mice were anesthetized with ether, and a small aperture in the skin of the knee was performed for the intraarticular (i.a.) injection procedure. When absence of arthritis was confirmed in the knee joint, i.a. injections were performed with 106 or 107 PFU/6 µl of either an IL-4-expressing (Ad5E1 mIL-4) or an empty control (Ad5del70-3) recombinant human type 5 adenovirus vector or with saline. Seven days after the i.a. injection of the viral vector, mice were sacrificed by cervical dislocation and the skin of the knee joint was removed. The appearance of arthritis in the injected joints was assessed and severity score was recorded as previously described (20). Thereafter, knee joints were isolated and processed for light microscopy.
Assessment of arthritis
Mice were considered to have arthritis when significant changes in redness and/or swelling were noted in the digits or in other parts of the paws. Knee joint inflammation was scored visually after skin dissection, using a scale of noninflamed (0), mild (1), marked (1.5), or severe (2) inflammation. Scoring was done by two independent observers, without knowledge of the experimental groups.
Determination of IL-4, IL-1ß, and IL-1Ra protein
To determine the levels of IL-4, IL-1ß, and IL-1Ra in patella washouts, patellae were isolated in a standardized manner from knee joints, as previously described (28). Patellae were incubated in RPMI 1640 medium with 0.1% BSA, gentamicin (50 µg/ml), and L-glutamine (2 mM) (200 µl/patella) for 1 h at RT. After supernatant was harvested, the IL-4, IL-1ß, and IL-1Ra levels were measured by ELISA. Briefly, ELISA plates were coated with the capture Ab (3 µg/ml) by overnight incubation at 4°C in carbonate buffer (pH 9.6). Nonspecific binding sites were blocked by 1-h incubation at 37°C with 1% BSA in PBS/Tween. The supernatants from the patella cultures were tested by 3-h incubation at 37°C. The plates were then incubated for 1.5 h at 37°C with the biotinylated second Ab, followed by a 30-min incubation at 37°C with streptavidin-polyperoxidase conjugate. Bound complexes were detected by reaction with orthophenylenediamine and H2O2. Absorbance was measured at 492 nm using an ELISA plate reader (Titertek Multiscan MCC/340). The cytokine concentration in the samples was calculated as pg/ml using recombinant murine IL-4, IL-1ß, or IL-1Ra as a standard. The sensitivity of the IL-4 and IL-1ß ELISA is 10 pg/ml, and of the IL-1Ra ELISA is 160 pg/ml.
Isolation of RNA
Mice were sacrificed by cervical dislocation, and the patellae and adjacent synovium were immediately dissected (29). Synovium biopsy tissue was taken from 6 of 10 patella specimens. Two biopsy specimens with a diameter of 3 mm were punched out, using a biopsy punch (Stifle, Wachtersbach, Germany): one from the lateral side and one from the medial side. Three lateral and three medial biopsy samples were pooled to yield two samples per group. The synovium samples were immediately frozen in liquid nitrogen. Ten patella specimens per experimental group were taken. Patellae were transferred to a 5% EDTA solution and kept on ice for 4 h. Thereafter, the cartilage layer was stripped, as previously described (20). This procedure does not affect mRNA isolation or amplification efficiency. Total RNA from a pool of 10 cartilage samples from a particular group was extracted with 1 ml of Trizol reagent, an improved single-step RNA isolation method based on the method described by Chomczynski and Sacchi (30). Synovium biopsy samples were ground to powder using a microdismembrator II (B. Braun, Melsungen, Germany). Total RNA was extracted in 1 ml of Trizol reagent in a manner similar to that used for cartilage samples.
PCR amplification
One microgram of synovial RNA and the total amount of cartilage
RNA (pool of 10 cartilage layers) was used for RT-PCR. mRNA was reverse
transcribed to cDNA using oligo(dT) primers, and one-twentieth of the
cDNA was used in one PCR amplification. PCR was performed at a final
concentration of 200 µM dNTPs, 0.1 µM of each primer, and 1 U of
Taq polymerase (Life
Technologies) in standard PCR buffer. The mixture was overlaid with
mineral oil and amplified in a thermocycler (Omnigene, Hybaid, U.K.).
Message for GAPDH, IL-1, TNF-, IL-1Ra, and inducible NO synthase was
amplified using the primers described elsewhere (20).
Primers for MMP-3, TIMP-1, macrophage-inflammatory protein-2, and
monocyte-chemotactic protein-1 were designed using Oligo 4.0 and Primer
Software.
Samples (5 µl) were taken from the reaction tubes after a certain number of cycles. PCR products were separated on 1.6% agarose and stained with ethidium bromide.
Histology
Mice were sacrificed by cervical dislocation. Thereafter, whole knee joints were removed and fixed for 4 days in 10% formalin. After decalcification in 5% formic acid, the specimens were processed for paraffin embedding (31). Tissue sections (7 µm) were stained with hematoxylin and eosin (H&E) or Safranin O. Histopathological changes were scored using the following parameters. Infiltration of cells was scored on a scale of 0–3, depending on the amount of inflammatory cells in the synovial cavity (exudate) and synovial tissue (infiltrate). Proteoglycan depletion was determined using Safranin O staining. The loss of proteoglycans was scored on a scale of 0–3, ranging from fully stained cartilage to destained cartilage or complete loss of articular cartilage. A characteristic parameter in CIA is the progressive loss of articular cartilage. This destruction was graded separately on a scale of 0–3, ranging from the appearance of dead chondrocyte (empty lacunae) to complete loss of the articular cartilage. Histopathological changes in the knee joints were scored in the patella/femur region on five semiserial sections of the joint, spaced 70 µm apart. Scoring was performed by two observers without knowledge of the experimental group, as described earlier (20).
For autoradiographic analysis, radiolabeled sulfate (50 µCi/mouse) was injected i.p. 2 h before dissection of the stifle joints. 35S-sulfate was incorporated into the cartilage layer, but not the underlying subchondral bone, and its incorporation reflects newly synthesized proteoglycans (32). Sections (7 µm) were mounted on gelatin-coated slides, which were immersed in K5 emulsion (Ilford; Basildon, Essex, U.K.) and exposed for several weeks before being developed and stained with H&E.
Assessment of chondrocyte proteoglycan synthesis
Patellae (10 pieces), in a minimal amount of adjoining soft tissue, were placed in 2 ml RPMI 1640 medium with gentamicin (50 µg/ml), L-glutamine (2 mM), and 20 µCi [35SO4]sulfate. At the end of the 3-h incubation period, patellae were washed in saline three times, fixed in 4% formalin, and subsequently decalcified in formic acid (5%) for 4 h. Patellae were punched out of the adjacent tissue, and dissolved in 0.5 ml Luma solve (Omnilabo, Breda, The Netherlands). The 35S-sulfate content of each patella was measured by liquid scintillation counting and expressed as cpm.
NIMP-R14 staining
Influx of PMNs was analyzed on knee joint sections. Briefly, sections were deparaffinized, and preincubated for 15 min by RT with 20% normal rabbit serum. Thereafter, sections were incubated with NIMP-R14 (25–30 kDa epitope mainly present on neutrophils) Ab for 1 h. After incubation with the second rabbit anti-rat peroxidase Ab for 30 min, sections were incubated with AEC substrate in the dark by 37°C for 10 min. Thereafter, sections were stained with hematoxylin for 30 s. As a negative control, sections were incubated with normal rat Ig instead of NIMP-R14 Abs.
VDIPEN analysis
VDIPEN expression was analyzed as described (33). Briefly, sections were deparaffinized, rehydrated, and digested with chondroitinase ABC (0.25 U/ml 0.1 M Tris-HCl, pH 8) for 1 h at 37°C to remove chondroitin sulfate from the proteoglycans. Sections were then treated with 1% H2O2 in methanol for 20 min, and subsequently with 0.1% Triton in PBS for 5 min. After incubation with 1.5% normal goat serum for 20 min, sections were incubated overnight at 4°C with affinity-purified anti-VDIPEN IgG. This Ab recognizes the neoepitope specifically and does not recognize intact proteoglycan (33). Subsequently, sections were incubated with biotinylated goat anti-rabbit IgG and stained with avidin-peroxidase (Elite kit; Vector, Burlingame, CA). Development of the peroxidase product was done using nickel enhancement to increase sensitivity. Counterstaining was done using orange G (2%) for 5 min. As a negative control, sections were incubated with normal rabbit IgG instead of anti-VDIPEN Abs.
Assessment of nitrite levels in 48-h patella washouts
Patellae with adjacent synovium were isolated in a standardized manner from knee joints, as previously described (28), and incubated in RPMI 1640 medium with 0.1% BSA, gentamicin (50 µg/ml), and L-glutamine (2 mM) (200 µl/patella) for 24 h at 37°C. Thereafter, supernatant was harvested and new medium was added and incubated for another 24 h at 37°C. The concentration of NO2- (a stable breakdown product of NO) was determined by Griess reaction using NaNO2 standards (Merck, Darmstadt, Germany) in RPMI 1640 tissue culture medium with 0.1% BSA. Briefly, 100 µl of conditioned medium of 24 and 48 h was mixed with 100 µl of Griess reagent (0.1% naphthylethylenediamine dihydrochloride (Sigma, St. Louis, MO), 1/1 diluted with 1% sulfanilamide (Sigma) in 5% H3PO4) in a flat-bottom microtiter plate (Costar, Cambridge, MA), and the OD at 545 nm was measured using an ELISA plate reader (Titertek Multiscan MCC/340).
Statistical analysis
Differences between experimental groups were tested using the Mann-Whitney rank sum test, unless stated otherwise.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Different doses of Ad5E1 mIL-4 were intraarticularly injected into
the right knee joint of naive and CII-immunized DBA-1 mice, and IL-4
protein levels were measured at day 7 in washouts of joint tissue.
Considerable IL-4 levels were found after a single injection of
107 PFU (Fig. 1
),
whereas roughly a 10-fold lower IL-4 level was seen after injection of
106 PFU. No major differences were found between
IL-4 levels in naive and CII-immunized mice. No detectable IL-4 was
noted in washouts of normal knees or knee joints injected with a
control vector (Ad5del70-3).
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).
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DBA-1 mice were immunized with CII, and shortly before expected
onset of collagen arthritis (day 25) a single injection of Ad5E1 mIL-4,
control vector, or saline was given in the right knee joint. Seven days
later, a 100% arthritis incidence was noted in the right knee joints
of the Ad5E1 mIL-4 groups, both after injection of
107 or 106 PFU. In
contrast, 89% and 50% incidence was seen in joints injected with
107 or 106 PFU control
vectors, respectively. The latter was not different from the incidence
observed after saline injection (Fig. 2).
Of note, a single injection of 107 adenovirus did
not induce joint inflammation in a knee joint of naive mice. This
indicates that adenovirus itself can accelerate expression of collagen
arthritis, whereas this expression is further increased when IL-4 is
overexpressed.
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B, severity of arthritis is also
markedly enhanced, even with the low dose of 106
PFU Ad5E1 mIL-4. Furthermore, histologic analysis showed more
inflammatory infiltrate in the synovial tissue and exudate in the
synovial cavity in the low dose IL-4 group as compared with the control
vector group (Fig. 3A).
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Apart form the analysis of inflammatory aspects, the histologic
knee joint sections were stained for proteoglycan content in the
articular cartilage. Furthermore, semiserial sections were scored for
the degree of chondrocyte death and cartilage surface erosions in the
patella and femur region. Profound proteoglycan depletion was found in
the control, arthritic group, and the depletion was not different in
the IL-4 groups (Fig. 3B). However, despite the
pronounced inflammation in the IL-4 groups, the degree of
chondrocyte death and cartilage erosion was highly reduced (Fig. 4). A second experiment is shown in Fig. 4, in which the effect on the chondrocyte death and cartilage damage of
a single injection of 107 PFU is shown. As in the
first experiment, 100% arthritis incidence was noted in the right knee
joint of the IL-4 group with an arthritis score of 2 ± 0.2
compared with 91% incidence in the adenoviral control group with an
arthritis score of 1.8 ± 0.6. Again a strong reduction of the
degree of chondrocyte death and cartilage erosion was found in the IL-4
group. Mean values were 61% and 77% reduced for these
parameters in the high dose IL-4 group as compared with its respective
adenoviral control group (Figs. 4 and 5).
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). Whereas the majority of mice in the
high dose control group were ranked as having severe chondrocyte death,
none of the mice of the IL-4 group was in this catagory, whereas 13 of
21 were ranked as having no or mild chondrocyte death. When cartilage
erosion was scored, the differences between IL-4 and control were even
more impressive. All mice in the control group were ranked as moderate
or severe, whereas all mice in the IL-4 group were nonerosive or mildly
erosive.
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).
None of the nine arthritic mice of the low dose IL-4 group ended up in
this category, again proving that IL-4 provides protection against
progression to major erosion. Local IL-4 overexpression prevents VDIPEN neoepitope expression
VDIPEN neoepitope is a marker of metalloproteinase (MMP)-mediated
cleavage of aggrecan, the major proteoglycan of articular cartilage,
and earlier studies revealed that expression mainly occurred at sites
and stages of advanced damage (33, 34, 35). To further
demonstrate the protective effect of local IL-4, sections were stained
for this neoepitope. As a typical example, VDIPEN was highly expressed
at the edges of the pattela and throughout the femoral cartilage in
sections of control, arthritic mice with moderate erosions (Fig. 6). In contrast, VDIPEN staining was
markedly reduced in the IL-4 group and only present at the margins of
the cartilage.
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). TIMP-1 was equally expressed in the
synovium of control and IL-4 group, implying down-regulation of the
MMP-3/TIMP-1 balance in the synovium of the IL-4-treated mice.
Up-regulation of TIMP-1 was noted in the cartilage of the IL-4
group.
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The above studies revealed that IL-4 did prevent chondrocyte
death. To investigate whether these chondrocytes are still
metabolically active and produce proteoglycans, we measured
35S-sulfate incorporation in the whole patellar
cartilage, ex vivo. As shown in Fig. 7,
the 35S-proteoglycan synthesis is markedly
enhanced in the IL-4 group as compared with the arthritic control,
despite the fact that joint inflammation in these mice was
undiminished. Because we noted above that local erosions and
chondrocyte death were a major phenomenon in the control group, we also
performed autoradiography on whole joint sections, after
35S-sulfate labeling in vivo. This showed
variable but low metabolic activity of the chondrocytes in the control
group. The IL-4 treatment induced a general increase in chondrocyte
proteoglycan synthesis in chondrocytes throughout the whole cartilage
(Fig. 8).
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Histologic analysis revealed differences in nature of the synovial
infiltrate between the IL-4 group compared with the control vector
group. In the control arthritic joint, numerous granulocytes adhered to
the cartilage, whereas this was absent in the IL-4-treated joint (Fig. 9, A and B).
Moreover, local IL-4 overexpression markedly diminished the influx of
PMNs in the synovial joint space. This was confirmed by
immunolocalization studies on knee joint sections using NIMP-R14
staining (Fig. 9, C and D) and diminished mRNA
expression of macrophage-inflammatory protein-2 in the synovial tissue
(Table III).
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Previously, we have shown the pivotal role of IL-1 in cartilage
destruction in CIA (36, 37, 38). In addition, IL-1 is a key
mediator in the regulation of the expression of MMP-induced neoepitope
(33). Furthermore, it has been shown that IL-1 together
with NO played an important role in the inhibition of the chondrocyte
metabolic activity (39, 40). Furthermore, it has been
shown that IL-4 up-regulates IL-1Ra, the receptor antagonist of IL-1
(9, 19). We therefore examined the effects of Ad5E1 mIL-4
on the local IL-1 expression in the synovium and cartilage and the
local NO synthesis production. In addition, we analyzed the effects of
local IL-4 on IL-1Ra and the balance IL-Ra/IL-1ß. RT-PCR revealed
major down-regulation of mRNA expression of IL-1ß in the synovium and
also in articular cartilage (Table III). In line with this, pronounced
suppression of IL-1ß protein levels (76%) in the synovium was found
(Table IV). Although no up-regulation of
IL-1Ra mRNA levels (Table III) or protein levels (AdControl,
3831 ± 1329 pg/ml vs AdIL-4, 1959 ± 131 pg/ml) was found,
the balance between IL-1Ra/IL-1ß protein levels was slightly enhanced
by local IL-4 treatment (IL-1Ra/IL-1ß ratio for AdControl, 12 vs
AdIL-4, 26). NO levels in the synovium were also decreased by Ad5E1
mIL-4 (Table IV). This indicates that the cartilage-protective effect
of local overexpression of IL-4 in the synovium of mice with collagen
arthritis might be due to suppression of local IL-1 production and
subsequent reduction of MMP activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we showed the pivotal role of IL-1 in cartilage destruction
in murine collagen arthritis (36). Both IL-1 and
IL-1ß appear to be involved in cartilage destruction in Ag-induced
arthritis, whereas dominant involvement of IL-1ß was observed in
collagen arthritis (37). This autoimmune model is driven
by the combination of cellular and humoral immunity against cartilage
CII and is characterized by rapid and severe erosions of cartilage and
bone (41, 42, 43, 44). The onset of CIA is dependent on IL-12,
TNF, and IL-1, and under the stringent control of endogenous IL-10
(45, 20). The fact that the local IL-4 treatment strongly
reduced IL-1 makes it likely that the protection against erosions is at
least partly achieved through this pathway.
It has been shown that IL-4 up-regulates IL-1Ra (9, 19). In the present study, no direct up-regulation of IL-1Ra by local IL-4 overexpression was found. One explanation could be that IL-1Ra levels are high in arthritic tissue, as a feedback reaction to IL-1. When prolonged treatment with IL-4 highly reduces the IL-1 levels, this will result in significant down-regulation and this will outbalance the mere up-regulation by IL-4. Interestingly, because of the marked suppression of IL-1ß, we found that the IL-1Ra/IL-1ß balance was slightly up-regulated by local IL-4. Another explanation that could be involved is the fact that local IL-4 markedly prevents cell influx of granulocytes into the joint space. It is known that PMNs are important producers of IL-1Ra. The strong reduction of granulocytes in the exudate by local IL-4 may play an important role in the explanation that IL-1Ra is not up-regulated.
Protection against erosion was also substantiated by lack of VDIPEN neoepitope expression in the cartilage of the IL-4-treated mice. Breakdown of aggrecan, the major proteoglycan of articular cartilage, predominantly occurs through two enzymatic pathways: aggrecanase attack, resulting in NITEGE neoepitopes, and MMP-mediated degradation, resulting in VDIPEN neoepitopes. Previous work from our group revealed that NITEGE expression is an early phenomenon in proteoglycan depletion and that VDIPEN epitope expression was mainly found at particular sites in the articular cartilage, associated with severe proteoglycan depletion and occurrence of chondrocyte death (J. B. J. Van Meurs, manuscript in preparation). Moreover, although inhibition of proteoglycan depletion was not always achieved with blocking of IL-1 in the various arthritis models, IL-1 blocking always fully prevented VDIPEN expression and late erosions. The present study further supports this link between erosions and VDIPEN expression. Although IL-4 treatment did not diminish proteoglycan depletion, it did prevent VDIPEN expression and surface erosions. It is known that IL-1 is a potent inducer of stromelysin (MMP-3) and that MMP-3 can induce the VDIPEN epitopes. The present study further revealed that MMP-3 mRNA levels were highly reduced by IL-4, compatible with a role of MMP-3 in erosion. It is not yet clear whether the reduction of IL-1 was indirectly responsible for the lowered MMP-3 levels or whether the IL-4 directly reduced the enzyme. In vitro studies have suggested that IL-4 is capable of direct reduction of these metalloproteinases (46, 47).
Apart from major reduction in cartilage erosions, we also noted an amelioration of the inhibition of chondrocyte proteoglycan synthesis, by direct measurement and autoradiography. Decreased 35S-proteoglycan synthesis is a hallmark of events in the articular cartilage during joint inflammation, and IL-1 as well as NO, as a secundary mediator, were shown by us to be pivotal mediators in this process, in vivo (39, 40). The lower IL-1 and NO levels in the IL-4-treated mice are in line with these previous findings. Despite the recovery of synthetic activity, pronounced net proteoglycan loss was still observed in the cartilage of these mice, suggesting that there is overruling breakdown due to the inflammatory process, independent of IL-1 and NO. Of high interest, we found a similar uncoupling of NO dependence of proteoglycan synthesis and breakdown in recent studies in inducible NO synthase-deficient mice (40).
The prevention of cartilage damage in CIA was IL-4 dose dependent. Both the high and low dose of IL-4 vector significantly enhanced the expression of collagen arthritis, in line with potent proinflammatory potential of IL-4 (7). The best protection against cartilage erosion was obtained with the high dose, suggesting that substantial levels of IL-4 are needed to control erosion. Of interest, net proteoglycan depletion was not different between the two IL-4 dose groups, underlining uncoupling between depletion and erosion and suggesting that different mediators are involved in these processes.
Enhancement of synovial inflammatory mass by local IL-4 expression is a significant but not unexpected side effect. IL-4 has been shown to be a chemoattractant for macrophages and fibroblast and inducer of fibroblast proliferation (48, 49, 50). Increased cell influx and tissue proliferation have been noted after local IL-4 overexpression in the pancreas, trachea, and liver (51, 52, 53). Recently, up-regulation of ß integrin, VCAM-1, IL-6, and monocyte-chemotactic protein-1 was reported after exposure of lung fibroblast to IL-4 (54).
The inflammation of collagen arthritis is characterized by a florid exudate in the joint space, containing numerous amounts of granulocytes, and a progressive destruction of the articular cartilage. Apart from the high numbers of granulocytes in the joint cavity, the synovial tissue contains large numbers of macrophages and lymphocytes, but also in this compartment granulocytes are prominent in the first 2 wk after onset. The most characteristic feature of collagen arthritis is the aggressive attack of the inflammatory process at the articular cartilage. In this model, heavy sticking of granulocytes at the cartilage surface is a common finding. Granulocytes may play an active role in the cartilage destruction, linked to sticking to anti-CII immune complexes in the surface layers. Igs adherent to cartilage surfaces have been identified in rheumatoid joints (55, 56), which can provide an anchorage and trigger for granulocyte activation (57). Reactive oxygen species and proteolytic enzymes present in the PMNs can be released directly into the surface of the cartilage, thereby escaping inhibitors present in the synovial fluid (58). PMNs need this close contact to cartilage to inflict cartilage damage (59, 60, 61). In the present study, we found numerous granulocytes adhered to the cartilage layer in the control arthritis joint, whereas this was absent in the IL-4-treated joint. This indicates that the marked reduction of granulocytes (PMNs) in the synovial joint space by local IL-4 may play an important role in the prevention of cartilage destruction.
The above paragraphs discussed various pathways of cartilage destruction in collagen arthritis. Apart from IL-1-mediated regulation of granulocyte influx, IL-1 is crucial in induction of latent enzymes in the articular cartilage, still needing further activation by other enzymes. Granulocytes may provide elastase, which is a potent activator of latent stromelysin. IL-4 can interfere with cartilage destruction at multiple levels. It reduces IL-1, it reduces stromelysin, and it prevents activation of latent stromelysin, through reduction of granulocyte influx and adherence to the cartilage surface. Although IL-4 enhances the inflammatory mass in the joint, cells do not show aggressive behavior. It is intriguing to note that joint inflammation in various patient groups can show a destructive and nondestructive phenotype. It is tempting to speculate that relative levels of IL-4 might make a difference.
It is known that IL-4 has a suppressive effect on Th1 activity and is a crucial factor in differentiation of naive T cells to the Th2 phenotype (12). In the present study, no substantial IL-10 expression was found in tissue washouts of the IL-4-treated group (data not shown). In contrast, preliminary studies on local IL-12 mRNA expression in the synovial tissue suggested marked reduction of this cytokine, implying that lower Th1 rather than enhanced Th2 activity contributes to the net protection. Changes in activity of CII-specific T cells in such an infiltrate of IL-4-treated mice are at present under investigation.
In conclusion, this study represents the first demonstration of cartilage-protective effects of local IL-4 gene therapy in experimental arthritis, despite ongoing inflammation. It furthermore illustrates the feasibility of gene transfer. Apart from overexpression of inhibitors, probably asking for high and prolonged expression levels (38), the introduction of modulators such as IL-4 may provide a promising alternative to treat destructive arthritis. Our findings also underline the often suggested, but hardly proven concept that the balance of destructive and protective mediators determines the relative erosive nature of a given arthritis, rather than the bulk of the inflammatory mass. The main clinical problem of chronic arthritides is the destruction of cartilage and bone. Our data make it clear that IL-4 is a promising regulator of these elements.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Erik Lubberts, University Hospital Nijmegen, Department of Rheumatology, Rheumatology Research Lab, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; CIA, collagen-induced arthritis; CII, collagen type II; H&E, hematoxylin and eosin; i.a., intraarticular; IL-1Ra, IL-1 receptor antagonist; MMP, metalloproteinase; PMN, polymorphonuclear cell; RT, room temperature; TIMP-1, tissue inhibitor of metalloproteinase.
Received for publication February 22, 1999. Accepted for publication August 2, 1999.
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