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Inhibition of interleukin-1 but not tumor necrosis factor suppresses neovascularization in rat models of corneal angiogenesis and adjuvant arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 46, No. 10, October 2002, pp. 2604–2612
DOI 10.1002/art.10546
© 2002, American College of Rheumatology
Inhibition of Interleukin-1 but Not Tumor Necrosis Factor
Suppresses Neovascularization in Rat Models of
Corneal Angiogenesis and Adjuvant Arthritis
Angela Coxon, Brad Bolon, Juan Estrada, Stephen Kaufman, Sheila Scully,
Alana Rattan, Diane Duryea, Yi-Ling Hu, Karen Rex, Efrain Pacheco, Gwyneth Van,
Debra Zack, and Ulrich Feige
Objective. To assess the capacities of the cytokine
inhibitors interleukin-1 receptor antagonist (IL-1Ra;
anakinra) and PEGylated soluble tumor necrosis factor
receptor I (PEG sTNFRI; pegsunercept) to suppress
neovascularization.
Methods. A corneal angiogenesis assay was performed by implanting nylon discs impregnated with an
angiogenic stimulator (basic fibroblast growth factor or
vascular endothelial growth factor) into one cornea of
female Sprague-Dawley rats. Animals were treated with
IL-1Ra or PEG sTNFRI for 7 days, after which new
vessels were quantified. In a parallel study, male Lewis
rats with mycobacteria-induced adjuvant-induced arthritis were treated with IL-1Ra or PEG sTNFRI for 7
days beginning at disease onset, after which scores for
inflammation and bone erosion as well as capillary
counts were acquired from sections of arthritic hind
paws.
Results. Treatment with IL-1Ra yielded a dosedependent reduction in growth factor–induced corneal
angiogenesis, while PEG sTNFRI did not. IL-1Ra, but
not PEG sTNFRI, significantly reduced the number of
capillaries in arthritic paws, even though both anticyto-
kines reduced inflammation and bone erosion to a
similar degree.
Conclusion. These data support a major role for
IL-1, but not TNF␣, in angiogenesis and suggest that an
additional antiarthritic mechanism afforded by IL-1
inhibitors, but not anti-TNF agents, is the suppression
of the angiogenic component of pannus.
Inflammatory arthritis is initiated and sustained
by the release of myriad proinflammatory cytokines (for
review, see refs. 1 and 2). In patients with rheumatoid
arthritis (RA), 2 critical proinflammatory cytokines are
interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣)
(3). Inhibition of IL-1 and/or TNF␣ reduces the extent
of inflammation in RA (4,5) and lessens inflammation
and bone destruction in various experimental models of
arthritis (6–12). Therefore, therapeutic agents that inhibit the action of these 2 cytokines are gaining rapid
acceptance as early, aggressive treatments for RA.
IL-1 (13–15) and TNF␣ (16–19) both also have
been implicated as angiogenic factors. This finding is
provocative because angiogenesis is a critical component
in the initiation and maintenance of pannus, the aggressive fibrovascular extension of synovial tissue that is
responsible for the extensive bone and cartilage damage
in RA (20) and experimental models of arthritis (21).
Human vascular endothelial cells activated by exposure
in vitro to either IL-1 or TNF␣ increase their expression
of receptor activator of nuclear factor ␬B ligand
(RANKL, an osteoclast differentiation factor) and its
soluble receptor osteoprotegerin (OPG, an osteoclastogenesis inhibitory factor) (22). However, OPG expression (and thus, RANKL inhibition) peaks early and then
falls, while RANKL increases slowly and is sustained
(22). These in vitro results implicate cytokine-activated
Presented in part at the 65th Annual Scientific Meeting of the
American College of Rheumatology, San Francisco, CA, November
2001.
Angela Coxon, PhD, Brad Bolon, DVM, PhD, Juan Estrada,
MD, Stephen Kaufman, MS, Sheila Scully, BS, Alana Rattan, BS,
Diane Duryea, Yi-Ling Hu, BS, Karen Rex, BS, Efrain Pacheco, BS,
Gwyneth Van, BS, Debra Zack, MD, PhD, Ulrich Feige, PhD: Amgen
Inc., Thousand Oaks, California.
Drs. Coxon and Bolon contributed equally to this work.
Address correspondence and reprint requests to Ulrich Feige,
PhD, Amgen, One Amgen Center Drive, M/S 29-M-B, Thousand
Oaks, CA 91320-1799. E-mail: ufeige@amgen.com.
Submitted for publication October 30, 2001; accepted in
revised form June 18, 2002.
2604
INHIBITION OF IL-1, NOT TNF, SUPPRESSES NEOVASCULARIZATION
endothelial cells in newly formed blood vessels as direct
participants in the bone erosion by pannus.
Numerous animal studies indicate that antiangiogenic treatments effectively reduce both the incidence and the severity of collagen-induced arthritis
(CIA) (23–27) and adjuvant-induced arthritis (AIA)
(28,29). The present study compared the antiangiogenic
activities of anti–IL-1 and anti-TNF␣ biologic agents in
conventional models of arthritis and vascular growth
factor–induced neovascularization. Our data indicate
that therapy targeting IL-1, but not TNF␣, will reduce
angiogenesis in arthritic joints.
MATERIALS AND METHODS
Animals. Rats (Charles River, Wilmington, MA) were
acclimated for 1 week, after which they were randomly assigned to treatment groups. Animals were given tap water and
fed pelleted rodent chow (8640; Harlan Teklad, Madison, WI)
ad libitum; calcium and phosphorus contents were 1.2% and
1.0%, respectively. Surgical procedures were performed using
isoflurane anesthesia and standard operating practices and
sterile technique. All animals were killed by CO2 inhalation.
These studies were conducted in accordance with federal
animal care guidelines and were preapproved by the Amgen
Institutional Animal Care and Use Committee.
Treatments. The treatments assessed in this study were
the recombinant human anticytokine biologics IL-1 receptor
antagonist (IL-1Ra) (anakinra; Amgen, Thousand Oaks, CA)
and PEGylated soluble TNF receptor I (PEG sTNFRI) (pegsunercept; Amgen). IL-1Ra was administered at 0, 0.5, 1.5, or
5 mg/kg/hour in cortical somatosensory evoked potential vehicle (140 mM saline containing 10 mM sodium citrate, 0.5 mM
EDTA, and 0.1% [weight/volume] Tween 80) by subcutaneous
(SC) infusion using implanted osmotic minipumps (model
2ML1, delivery rate 10 ␮l/hour; Alza, Palo Alto, CA), while
PEG sTNFRI was given at 0 or 4 mg/kg/day in phosphate
buffered saline (PBS) by SC bolus. Minipumps were implanted
in the dorsal subcutis, and wounds were sealed using steel clips.
Doses of IL-1Ra and PEG sTNFRI were selected based on
previous studies (12) demonstrating that these doses, routes,
and schedule cover the region of the dose-response curve
ranging from (almost) inactive to fully active against the severe
polyarthritis characteristic of the AIA model in Lewis rats. In
particular, we have shown that IL-1Ra (5 mg/kg/hour) and
PEG sTNFRI (4 mg/kg/day) yield comparable antiinflammatory and bone-sparing effects when given for 7 days.
Corneal implant angiogenesis assay. The vascular
growth factor–induced corneal neovascularization bioassay
was performed in triplicate using a standard experimental
design (30), with the modification that a porous solid was used
to dispense protein (rather than cells or a slow-release polymer) (31). Briefly, circular discs (0.6 mm diameter) were
punched from nylon filter paper (Nylaflo; Gelman, Ann Arbor,
MI) using a 20-gauge needle with a squared-off end, after
which they were placed in PBS containing either 0.1% bovine
serum albumin (BSA) alone (vehicle) or 0.1% BSA in combination with 1 of 2 recombinant human vascular growth factors
2605
(R&D Systems, Minneapolis, MN): 3.75 ␮M basic fibroblast
growth factor (bFGF) or 10 ␮M vascular endothelial growth
factor (VEGF). Discs were incubated for 1 hour at 4°C before
use; each disc absorbed ⬃0.1 ␮l of solution.
Adult female Sprague-Dawley rats weighing 250–300
gm (n ⫽ 8 per group) were anesthetized with isoflurane, after
which a vertical incision 0.8 mm in length was made on the
cornea. A pocket was formed in the corneal stoma, and a single
disc was inserted. The margin of each disc was located
⬃1.8–2.0 mm from the blood vessels of the lateral limbus.
Prior immunohistochemical work in our laboratory has shown
that the vascular growth factor persists in and around the
corneal disc for more than 7 days (Coxon A: unpublished
observations).
Treatment with IL-1Ra (0, 0.5, 1.5, or 5 mg/kg/hour) or
PEG sTNFRI (0 or 4 mg/kg/day) was initiated on the day of
surgery and continued for 7 days. An additional cohort to
control for nonspecific anti-angiogenic effects of stress associated with implantation of the osmotic minipump was infusion
of BSA at 5 mg/kg/hour. All groups were run in parallel to limit
variability associated with interstudy comparisons. At necropsy, the eyes were enucleated and immersed overnight in
zinc formalin. The cornea and associated implant were removed, placed in distilled water, transilluminated, and photographed at 5⫻ using a Sony CatsEye DKC 5000 digital camera
(A.G. Heinze, Lake Forest, CA) mounted on a Nikon SMZ-U
stereomicroscope (A.G. Heinze); a reference stage micrometer was photographed for image calibration. Numerical data
were generated from the digital images using Metamorph
image analysis software (v4.5; Universal Imaging, Downingtown, PA) installed on a Windows NT workstation (Microsoft,
Redmond, WA). Three end points were analyzed on each
corneal image: 1) disc placement distance from the limbal
vessels, 2) the number of vessels intersecting a 2.0 mm–long
perpendicular line drawn across the midpoint of the shortest
line between the disc and the limbus, and 3) blood vessel area
(as determined by digital thresholding and automated pixel
counting).
After 7 days of treatment with PEG sTNFRI or
vehicle, selected corneas were stained with a proprietary
mouse monoclonal antibody (mAb) specific for human
sTNFRI (Amgen). Briefly, acetone-fixed 6 ␮M–thick frozen
sections were blocked with CAS Block (Zymed, South San
Francisco, CA) and incubated with either the anti-sTNFRI
antibody or an isotype-matched mAb control. Binding of the
primary antibody was detected using a biotinylated horse
anti-mouse secondary antibody (Vector, Burlingame, CA).
Slides were quenched with 3% H2O2 followed with avidin–
biotin–peroxidase complex (Vector). Reaction sites were visualized with diaminobenzidine (Dako, Carpinteria, CA) and
counterstained with hematoxylin.
Selected corneas were processed for in situ hybridization. The eyes were harvested, after which the corneal region
containing the disc was removed and fixed by immersion in
zinc formalin (Z-Fix; Anatech, Battle Creek, MI). Tissue was
processed in paraffin by routine methods, after which antisense
RNA probes for rat CD31 (platelet endothelial cell adhesion
molecule [PECAM], corresponding to nucleotides 220–474 of
the mouse sequence; GenBank accession no. L06039), rat
IL-1RI (nucleotides 777–1126; GenBamk accession no.
M95578), and rat IL-1RII (nucleotides 683–933; GenBank
2606
COXON ET AL
Table 1. Semiquantitative criteria for histopathologic lesion scores
Score
Inflammation
0
1
2
3
4
Bone erosion
0
1
2
3
4
5
Grading scale
Normal
Few inflammatory cells
Mild inflammation
Moderate inflammation (often but not always
diffuse)
Marked inflammation (diffuse and dense,
with large periarticular abscesses)
Normal
Minimal loss of cortical or trabecular bone at
a few sites
Mild loss of cortical or trabecular bone at
modest numbers of sites (generally tarsals)
Moderate loss of bone at many sites (usually
the trabeculae of the tarsals, but
sometimes the cortex of the distal tibia)
Marked loss of bone at many sites (usually as
extensive destruction of trabeculae in the
tarsals, but sometimes with partial loss of
cortical bone in the distal tibia)
Marked loss of bone at many sites (with
fragmenting of tarsal trabeculae and fullthickness penetration of cortical bone in
the distal tibia)
accession no. Z22812) labeled with 33P-rUTP (Amersham,
Arlington Heights, IL) were applied to 4 ␮M–thick sections
according to standard protocols (32). Following emulsion
autoradiography, sections were counterstained with hematoxylin and eosin (H&E) and examined under both darkfield and
standard illumination.
Induction of AIA. Adjuvant arthritis was induced in
adult male Lewis rats weighing 180–200 gm (n ⫽ 6 per group)
on day 0 as described (12), by a single intradermal injection, at
the tail base, of heat-killed Mycobacterium tuberculosis H37Ra
(0.5 mg; Difco, Detroit, MI) suspended in 0.05 ml paraffin oil
(Crescent Chemical, Islandia, NY). A refined volume displacement method (12) was used beginning on day 8 to measure
hind paw volume to determine the clinical onset of arthritis. At
onset (typically, day 9), a 7-day course of therapy was initiated
using IL-1Ra (5 mg/kg/hour) or PEG sTNFRI (4 mg/kg/day).
A concurrent control group consisted of untreated arthritic
rats; additional control cohorts treated with vehicle(s) were not
included in this study because our past experience with this
model has shown that clinical and histopathologic responses of
untreated and vehicle-treated animals are equivalent (Feige U:
unpublished observations). All group studies were run in
parallel. The small group size was used because interindividual
variability between untreated arthritic rats is minimal (12).
At necropsy (day 16 postimmunization), hind paws
were removed at the fur line (just proximal to hock), fixed in
70% ethanol, decalcified, divided longitudinally along the
median axis, and processed in paraffin. One 4 ␮M–thick
section was stained with H&E. As previously described (12),
inflammation and bone erosion scores were acquired in a
“blinded” analysis using semiquantitative grading scales (Table
1). In addition, a serial section of each hind paw was stained
using a commercial indirect immunoperoxidase kit (Vectastain
Elite ABC Kit; Vector) with a rabbit anti-human mAb (Dako)
directed against the endothelial marker von Willebrand factor
(also known as factor VIII–related antigen). Labeled capillaries were enumerated in periarticular soft tissues at 4 sites
(Figure 1) because these zones exhibited a marked leukocyte
infiltrate in untreated arthritic animals. Counts were made at
200⫻ using an ocular reticule with 100 square divisions. The
Figure 1. Photomicrograph of a hind paw from a rat with adjuvant-induced arthritis, compared
with a control rat. Four sites at which capillary counts were acquired are denoted (hematoxylin and
eosin stain; original magnification ⫻ 6).
INHIBITION OF IL-1, NOT TNF, SUPPRESSES NEOVASCULARIZATION
2607
total area counted per site was 0.5 mm2. An attempt to assess
messenger RNA expression levels for selected vascular markers (PECAM), vascular growth factors (bFGF, VEGF), and
IL-1RI and IL-1RII in normal and arthritic joints was thwarted
by degradation of nucleic acid integrity during processing.
Statistical analysis. Statistical significance for the corneal angiogenesis assay was assessed by analysis of variance
followed by Fisher’s exact test. Histopathologic data were
compared using conservative nonparametric tests, the chisquare test for inflammation and erosion scores, and Wilcoxon’s rank sum test for the capillary counts. P values less than
0.05 were considered significant.
RESULTS
Figure 2. Effect of interleukin-1 receptor antagonist (IL-1Ra) on
vascular endothelial growth factor (VEGF)–induced corneal neovascularization. Infusion of IL-1Ra (5 mg/kg/hour for 7 days) yielded
almost complete inhibition of VEGF-induced corneal neovascularization
between the implanted disc (top) and the limbus (bottom). In contrast,
PEGylated soluble tumor necrosis factor receptor I (PEG sTNFRI; 4
mg/kg/day for 7 days) had essentially no effect. A–D show representative
corneas from each of the 4 treatment groups. BSA ⫽ bovine serum
albumin. Bar ⫽ 500 ␮m.
Corneal angiogenesis assay. Seven days after
corneal implantation of discs, neovascularization extending from the limbus to the implant was grossly
prominent in all rats in which discs contained either
VEGF (Figures 2B and 3) or bFGF (Figure 3). As
expected, angiogenesis was absent if vascular growth
factors were not present in the disc (Figure 2A).
Systemic administration of IL-1Ra for the duration of the study produced a significant, dose-dependent
decrease in corneal neovascularization induced by either
bFGF (Figure 3) or VEGF (Figures 2C and 3). Counts
of both the new capillary profiles and the total vascularized area were lowered; in animals treated with 5
Figure 3. Effect of IL-1Ra on basic fibroblast growth factor (bFGF)– and VEGF-induced corneal
angiogenesis. IL-1Ra infusion produced a significant (ⴱ ⫽ P ⱕ 0.05) dose-dependent decrease in
both bFGF-induced (a) and VEGF-induced (b) corneal angiogenesis relative to rats treated with
vehicle or with an inactive protein (BSA, a control for stress-induced anti-angiogenic effects
associated with protein infusion). Representative results from 1 of 3 experiments are shown; values
are the group mean ⫾ SEM (n ⫽ 8). CON ⫽ control (see Figure 2 for other definitions).
2608
COXON ET AL
Figure 4. Effect of PEG sTNFRI on VEGF-induced corneal neovascularization. Administration
of PEG sTNFRI (4 mg/kg/day for 7 days) had no impact on VEGF-induced corneal neovascularization. This result was obtained despite the fact that the cornea was permeated with PEG sTNFRI
(upper photomicrograph), as indicated by uniform distribution of immunoreactivity following
application of a mouse monoclonal antibody specific for human sTNFRI. In contrast, corneal
control sections from vehicle-treated rats labeled with anti-sTNFRI (middle panel) and from PEG
sTNFRI–treated animals labeled with an isotype control (CON) antibody (lower panel) exhibited
no reactivity. Arrows indicate VEGF-induced blood vessels. Representative results from 1 of 3
experiments are shown; values are the group mean ⫾ SEM (n ⫽ 8). Bar ⫽ 50 ␮M. See Figure 2
for other definitions.
mg/kg/hour of IL-1Ra, group mean values were comparable with those observed in rats given implants that
lacked vascular growth factors. In contrast, neither PEG
sTNFRI nor BSA affected the angiogenic response
elicited by bFGF (data not shown) or VEGF (Figures 2
and 4). The presence of PEG sTNFRI throughout the
corneal stroma surrounding implants was confirmed by
immunohistochemistry analysis (Figure 4).
The expression patterns of CD31 (PECAM),
IL-1RI, and IL-1RII were determined by in situ hybridization on corneas harvested 7 days after the induction
of angiogenesis (Figure 5). CD31 expression was higher
near the limbus and occurred only in endothelial cells,
including those located at the leading edge of new
capillary branches. Both IL-1RI and IL-1RII were substantially up-regulated in corneas near VEGFcontaining implants. IL-1RI exhibited a more diffuse
pattern, though most expressing cells were located near
the implant. The major cell types labeled with IL-1RI
were mononuclear inflammatory cells (with small to
large nuclei) and corneal stromal cells (with fusiform or
serpentine nuclei), although large endothelial cells occasionally expressed this receptor. The IL-1RII signal
was higher near the implant and was found only in
mononuclear inflammatory cells and a few corneal stromal cells.
AIA. Adjuvant-induced arthritis is characterized
by a clearly defined pattern of structural lesions combining both destructive and reparative processes. Dense
aggregates of mixed inflammatory cells and numerous
osteoclasts were present in association with erosions of
cortical and trabecular regions of the tarsal bones and
tibiae. Production of new bony trabeculae along periosteal and endosteal surfaces was extensive. Joint cartilages often were isolated by erosion of the subjacent
epiphyseal bone, but remained intact. Neutrophils,
newly formed capillaries (i.e., angiogenesis), hyperplastic stromal cells, and sometimes edema were the most
INHIBITION OF IL-1, NOT TNF, SUPPRESSES NEOVASCULARIZATION
2609
Figure 5. In situ hybridization for CD31 (platelet endothelial cell adhesion molecule), IL-1RI, and IL-1RII in the rat corneal angiogenesis model.
Corneal implants impregnated with VEGF induced both angiogenesis and intrastromal leukocyte infiltration (A), while implants with an inactive
protein (BSA) did not (B). Introduction of growth factor substantially up-regulated expression of CD31, as well as IL-1RI and IL-1RII, though their
patterns of expression were different. CD31, an endothelial cell marker, was expressed in blood vessels extending from the limbus toward the disc.
Expression of IL-1RI was more diffuse, was concentrated near the disc, and was found in leukocytes, corneal stromal cells, and some large
endothelial cells. IL-1RII expression was also highest near the implanted disc but was restricted to leukocytes and some stromal cells. Sections are
oriented with the limbus (L) on the left and the implanted disc (D) on the right. Dashed lines in insets A1 and B1 denote the plane of section. A
and B are hematoxylin and eosin–stained brightfield images. The 3 panels below A and B show isotopic in situ hybridization under darkfield
illumination, with higher-magnification insets below A1 and B1. Black arrowheads indicate capillaries; red arrows indicate nonvascular (leukocytic
or stromal) cells. See Figure 2 for definitions.
prominent inflammatory changes in the periarticular
soft tissues.
Semiquantitative grades of both inflammation
and bone erosion generally were of marked severity in
rats with untreated arthritis, while changes were absent
in nonarthritic controls (Figure 6). Therapy with either
IL-1Ra (5 mg/kg/hour) or PEG sTNFRI (4 mg/kg/day)
significantly reduced inflammation and bone erosion to
a similar degree (Figure 6). However, despite their
similar antiinflammatory and bone-protective effects,
IL-1Ra reduced the angiogenic response in arthritic
joints, while PEG sTNFRI did not. The mean number of
capillary profiles per mm2 in rats given IL-Ra was
modestly but not significantly increased by 13% relative
to counts in nonarthritic controls (Figure 7). In contrast,
both untreated arthritic rats and arthritic animals
treated with PEG sTNFRI had 35% more capillaries in
periarticular soft tissues than did nonarthritic controls
(Figure 7).
DISCUSSION
The development of new therapies that combat
the angiogenic component of pannus could have a
significant impact on the health and quality of life for
RA patients. This treatment paradigm is supported by
numerous animal studies demonstrating that antiangiogenic agents significantly suppress both the incidence and the severity of disease (23–29). The most
efficient means of adding angiogenesis inhibitors to the
clinical armamentarium for RA would be to determine
whether current therapies exhibit anti-angiogenic properties. For example, 2 current RA therapies, methotrexate (33) and D-penicillamine (34), have been shown to
inhibit neovascularization in vitro and in vivo, although
the mechanisms by which this effect is mediated are not
known. A new paradigm in this therapy is the early and
aggressive deployment of biologic response-modifying
agents to specifically inhibit the actions of proinflamma-
2610
tory cytokines in RA. Both IL-1 (13–15) and TNF␣
(16,17), 2 master cytokines that play a significant role in
RA (3), are known to promote angiogenesis. Given this
intense interest in anticytokine biologics, our experiments provide important new information regarding the
ability of such novel therapeutic molecules to regulate
neovascularization in the arthritic joint.
One key finding afforded by our present data is
that administration of IL-1Ra to block IL-1 activity
significantly inhibits angiogenesis in vivo. This effect was
observed in both the corneal implant system (a noninflammatory setting) and the AIA model (an inflammatory condition). IL-1Ra previously has been found to
inhibit angiogenesis in a model of inflammatory corneal
neovascularization in mice (15). The exact mechanism
by which IL-1Ra exerts this effect is unknown. IL-1 has
many pro-angiogenic activities, including increased expression of vascular growth factors (35,36), endothelial
Figure 6. Effect of IL-1Ra and PEG sTNFRI on inflammation and
bone erosion. Infusion of IL-1Ra or injection of PEG sTNFRI reduced
inflammation (A) and bone erosion (B) to a comparable degree in
Lewis rats with adjuvant-induced arthritis. ⴱⴱ ⫽ P ⱕ 0.05 versus
untreated arthritic animals; the percent reduction is indicated. Values
are the mean and SEM (n ⫽ 6). See Figure 2 for definitions.
COXON ET AL
Figure 7. Effect of IL-1Ra on the number of capillaries in periarticular soft tissues of Lewis rats. Infusion of IL-1Ra reduced the number
of capillaries in periarticular soft tissues of Lewis rats with adjuvantinduced arthritis relative to untreated arthritic rats. In contrast,
injection of PEG sTNFRI had no effect on blood vessel numbers. ⴱⴱ ⫽
P ⱕ 0.05 versus normal animals; the percent difference is indicated.
Values are the mean and SEM (n ⫽ 6). See Figure 2 for definitions.
mitogenesis (37), and induction of matrix metalloproteinases (36); IL-1Ra presumably can negate all of them.
However, our data indicating that IL-1Ra blocks
bFGF- and VEGF-induced angiogenesis in the cornea
are particularly intriguing in that increasing evidence
suggests that these vascular growth factors play a prominent role in the pathogenesis of RA. For example,
VEGF levels in serum (38,39) and synovial fluid (40–42)
are substantially elevated in RA and are correlated with
disease activity. Furthermore, VEGF is expressed widely
in arthritic joints, especially macrophages and fibroblasts
in RA patients (40,43), as well as mice with CIA (25) and
rats with AIA (42). Significantly, IL-1 induces VEGF
expression in cultured fibroblast-like synoviocytes isolated from RA synovial tissue (35,44). Similarly, IL-1,
acting via cyclooxygenase 2 (45,46) and nitric oxide (36),
also mediates the expression of bFGF. Notwithstanding
a conflicting report that IL-1 can inhibit bFGF-induced
angiogenesis in a rabbit corneal angiogenesis model
(47), the preponderance of evidence, including the data
acquired in our corneal angiogenesis model, suggests
that IL-1 plays some part in vivo in the induction of
angiogenesis by bFGF and VEGF. More important, our
findings clearly show that inhibition of IL-1 suppresses
angiogenesis in arthritic joints.
Surprisingly, another significant finding afforded
by our present data is that administration of a TNF
inhibitor, PEG sTNFRI, has no impact on angiogenesis
in vivo. Again, this outcome was apparent in both the
corneal implant system and the Lewis rat model of AIA.
This finding was unanticipated because TNF␣ has been
reported to initiate angiogenesis in the rabbit (16) and
rat (17) models of corneal neovascularization, and anti-
INHIBITION OF IL-1, NOT TNF, SUPPRESSES NEOVASCULARIZATION
TNF␣ therapy significantly decreases serum VEGF in
RA (38). The explanation for this phenomenon is unclear at this point. It is unlikely that the process of
growth factor–induced neovascularization that occurs in
the cornea is mediated by different biochemical events
than pathologic angiogenesis in the inflamed joint. This
inference is supported by a study documenting sustained
elevation in circulating TNF␣ concentrations in conjunction with falling tissue levels of VEGF in arthritic rats
treated with an angiogenic inhibitor (24). However, our
data do not support this interpretation because PEG
sTNFRI did not exhibit an anti-angiogenic effect
whether applied in either the arthritic joint or the
cornea.
Alternatively, this discrepancy might reflect a
difference in the arthritogenic functions governed by
IL-1 and TNF␣. While many proinflammatory activities
of these 2 cytokines are shared, their functions are not
identical (2). Thus, the conclusion that best fits our
present data is that the proinflammatory and jointdamaging effects of IL-1 and TNF␣ in arthritis represent
functions held in common, while IL-1 alone controls the
angiogenic response. This hypothesis is supported by
numerous experimental studies of arthritis that suggest a
central role for TNF␣ in mediating inflammation, while
IL-1 controls bone and cartilage destruction as well as
inflammation (48). The importance of IL-1 but not
TNF␣ to angiogenesis in the joints of rats with AIA
suggested by our present data could help to explain the
better efficacy afforded by IL-1 inhibition with respect to
preserving joint integrity.
In conclusion, our experiments indicate that IL-1,
but not TNF␣, plays a critical role in neovascularization,
including pathologic angiogenesis that occurs in arthritic
joints of rats with AIA. This important new finding
suggests that IL-1 inhibitors, but not anti-TNF agents,
will affect the process of arthritis not just by downregulating the inflammatory cascade, but also by thwarting the invasive, highly vascular pannus reaction that is
responsible for much of the bone and cartilage destruction characteristic of RA. These data are significant
because agents that block IL-1 should preserve joint
integrity by 2 distinct mechanisms: averting pathologic
angiogenesis (as demonstrated by our results) and moderating osteoclast expansion (49,50). Given the high
treatment costs and lost productivity associated with the
crippling bone sequelae of chronic RA, the capacity of
IL-1Ra to have an effect on bone and cartilage destruction via 2 pathways should afford a central place for this
agent as well as other IL-1 inhibitors in the anti-RA
armamentarium.
2611
ACKNOWLEDGMENT
The authors thank Dr. Robert Radinsky for critical
review of the manuscript.
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