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*
Department of Medicine, Northwestern University Medical School, Chicago, IL 60611; and
Veteran’s Administration Chicago Health Care System, Lakeside Division, Chicago, IL 60611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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v
3 integrin. To test whether IL-18
induced endothelial cell tube formation in vitro, we quantitated the
degree of tube formation on Matrigel matrix. IL-18, 1 or 10 nM,
resulted in a 77% or 87% increase in tube formation compared with
control (p < 0.05). To determine whether IL-18 may
be angiogenic in vivo, we implanted IL-18 in Matrigel plugs in mice,
and IL-18 at 1 and 10 nM induced angiogenesis (p <
0.05). The angiogenesis observed appears to be independent of the
contribution of local TNF-, as evidenced by adding neutralizing
anti-TNF- Ab to the Matrigel plugs. In an alternative in vivo
model, sponges embedded with IL-18 or control were implanted into mice.
IL-18 (10 nM) induced a 4-fold increase in angiogenesis vs the control
(p < 0.05). These findings support a novel
function for IL-18 as an angiogenic factor in RA and may elucidate a
potential therapeutic target for angiogenesis-directed
diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(10), C-X-C chemokines
(11, 12) such as IL-8 or epithelial neutrophil-activating
peptide (ENA-78), fractalkine (13), and adhesion molecules
such as soluble E-selectin or soluble VCAM-1 (14).
IL-18 is a cytokine with many proinflammatory functions. IL-18 was
initially described as IFN-
-inducing factor in 1989
(15). Thus, IL-18 stimulates Th1 cytokines by T cells and
NK cells and promotes Th1 cell differentiation and immune responses
(16, 17). IL-18 can act in synergy with IL-12 in
regulating IFN- (18). However, IL-18 also up-regulates
other cytokines, such as TNF-, IL-1, and IL-8 in
non-CD14+ PBMCs, resting T cells, and NK cells
(19). IL-18 up-regulates GM-CSF in PBMCs (20)
and also in synovial membrane cultures, directly affecting macrophages
to stimulate cytokine production (21).
IL-18 has structural homology with IL-1, shares some common signaling
pathways (22), and also requires the cleavage at its
aspartic acid residue by IL-1-converting enzyme to become the active,
mature protein (23, 24). Thus, IL-1 and IL-18 share many
biologically similar inflammatory functions. Previous work has
implicated IL-18 in RA, as higher levels are present in RA vs
osteoarthritic synovial fluid (SF) and sera (21). Also,
IL-18 enhanced erosive, inflammatory arthritis in a murine model of
systemic arthritis (21). In the study by Leung et al.
(25) in which IL-18 promoted collagen-induced inflammatory
arthritis through a mechanism shown to be distinct from IL-12, cytokine
treatment enhanced synovial hyperplasia, inflammatory infiltrate, and
cartilage erosion. Interestingly, the IL-18-treated mice produced
significant amounts of TNF-, and splenic macrophages from mice
cultured with IL-18 also stimulated high levels of TNF-. The
influential role of IL-18 in articular inflammation was confirmed in
mice lacking the IL-18 gene that had reduced incidence and severity of
collagen-induced arthritis, which was reversed by treatment with rIL-18
(26). One important source of IL-18 is the macrophage,
which is a critical synovial tissue producer of IL-12
(27), and responsible for the production of many other
cytokines (28). However, various other sources of IL-18
have been identified, including Kupffer cells, dendritic cells,
keratinocytes, articular chondrocytes, osteoblasts, and synovial
fibroblasts (18, 21, 29, 30, 31, 32, 33).
In regard to arthritis, if IL-18 functions in an autocrine or paracrine
fashion, the increased expression of IL-18 in the synovium may play a
critical role in the development of synovial inflammation, synovial
hyperplasia, and articular degradation, to which angiogenesis may
contribute. Given the importance of angiogenesis in the pathophysiology
of RA, we hypothesized a role for IL-18 as an angiogenic mediator.
Supportive of this function is the finding that IL-18 has been shown to
stimulate production of angiogenic TNF- (19). The
establishment of the role of IL-18 in inflammation and RA led to the
question of its angiogenic potential.
In this study, we examined the capacity of IL-18 to mediate
angiogenesis in in vitro and in vivo models. We further compared the
angiogenic activity of IL-18 with that of known mediators, such as
bFGF. One potential mechanism of the angiogenic activity of IL-18 that
we examined is through the involvement of
v3 integrin
(34). This role of
v3 was examined in
the IL-18-induced migration of endothelial cells. IL-18 induced tube
formation in Matrigel matrix in vitro. To examine the role of IL-18 in
angiogenesis in vivo, we also implanted IL-18 in Matrigel plugs in
mice, and found a significant increase in blood vessel formation over
control, as measured by hemoglobin concentration. This effect was not
decreased by implanting neutralizing anti-TNF- Ab in the
Matrigel plug. Finally, in an alternative model of angiogenesis, a
wound granuloma model, we implanted sponge discs embedded with IL-18
into mice and showed a 4-fold increase in angiogenesis compared with
controls. These findings support a novel role for IL-18 as an
angiogenic mediator in RA and may elucidate a potential therapeutic
target for angiogenesis-directed disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human (rh) IL-1 and TNF-, with sp. act. of
2 x 107 and 1.3 x 107 U/mg,
respectively, were gifts from Upjohn-Pharmacia (Kalamazoo, MI). Some
TNF- was from R&D Systems (Minneapolis, MN). rhIL-18 was purchased
from R&D Systems, with sp. act. measured by ability to induce IFN-
produced by KG-1 cells in the presence of 10 ng/ml rhTNF- (100 ng/ml
rhIL-18 induces 0.5–2 ng/ml IFN- produced by 105/ml
KG-1 cells), and was used for the majority of the assays. An additional
source of rhIL-18 was PeproTech (Rocky Hill, NJ), with IL-18 sp. act.
determined by the dose-dependent stimulation of IFN- production by
human PBMC costimulated with human IL-12 (ED50 of 5 ng/ml);
and the experiments in which this IL-18 was used included the
checkerboard assay, the chemotaxis assays comparing the activity of
IL-18 with IL-8 or epithelial neutrophil-activating peptide-78
(ENA-78), and the Matrigel in vivo assays with IL-18 and
anti-TNF- Ab. PeproTech was also the source for the rhIL-8 and
ENA-78, with biological activities determined by significant ability to
chemoattract human peripheral blood neutrophils using a concentration
of 10–100 ng/ml and 5–10 ng/ml, respectively. Neutralizing monoclonal
mouse anti-human IL-8, mouse anti-human ENA-78, and polyclonal
goat anti-human IL-18 Ab were obtained from R&D Systems.
Neutralizing mouse anti-TNF- was obtained from R&D Systems.
Monoclonal mouse anti-human integrin
v3 (vitronectin receptor) was obtained
from BioWhittaker Clonetics (Temecula, CA). Nonimmune rabbit serum (R&D
Systems) and mouse serum IgG (PharMingen, San Diego, CA) were negative
controls. Nonspecific goat IgG control from R&D Systems was also used.
Recombinant bovine acidic FGF (aFGF) and recombinant human bFGF with
sp. act. defined as ED50 of 0.1–0.3 ng/ml and 0.1–0.25
ng/ml were purchased from R&D Systems. Recombinant VEGF was obtained
from Endogen (Woburn, MA). Growth factor-reduced Matrigel Matrix,
derived from Engelbreth-Holm-Swarm mouse tumor, was purchased from
Becton Dickinson (Bedford, MA). Human microvascular endothelial cells
(HMVECs) were obtained from Clonetics (San Diego, CA). Diff-Quik was
purchased from Baxter (North Chicago, IL). A Kontes’ homogenizer was
obtained from Kontes Glass (Vineland, NJ); tetramethylbenzidine and
methemoglobin were obtained from Sigma (St. Louis, MO); and ethylene
vinyl acetate copolymer was obtained from Elvax (Dupont,
Wilmington, DE).
Patient population
SFs were isolated from patients with RA during therapeutic arthrocentesis. RA SFs were used for HMVEC migration assays. All specimens were obtained with Institutional Review Board approval.
Bioassay for HMVEC migration activity
Subconfluent HMVECs (passage 3–12) were fed the night before the assay, to optimize the cellular conditions, with endothelial cell basal medium-2 (EBM-2; Clonetics) and 0.1% FBS. HMVECs (3.75 x 104 cells/25 µl EBM and 0.1% FBS) were placed in the bottom wells of a 48-well Boyden chemotaxis chamber (NeuroProbe, Cabin John, MD) with gelatin-coated polycarbonate membranes (8 µm pore size; Nucleopore, Pleasant, CA) (14). The chamber was inverted and incubated in a humidified incubator with 5% CO2/95% air at 37°C for 2 h, allowing endothelial cell attachment to the membrane. The chamber was reinverted, and the test substances, PBS, positive control bFGF (60 nM) in PBS, or RA SF were added to the wells in the top of the chamber, and the chamber was further incubated for 2 h at 37°C. IL-18 (R&D Systems) was used in varying concentrations (11.25 ng/ml or 0.625 nM to 180 ng/ml or 10 nM). PBS served as the negative control throughout these experiments, and testing PBS with 2% BSA, the diluent used to reconstitute the lyophilized cytokines, resulted in similar baseline levels of HMVEC migration. Additionally, this assay was also performed with a representative RA SF sample after incubation for 1 h at 37°C with combinations of neutralizing Ab (25 µg/ml) to human IL-18 and ENA-78 or IL-8 before adding samples to the bottom of the wells. Comparison was made to control nonspecific Abs, goat IgG for anti-IL-18 control, and mouse IgG for anti-IL-8 and anti-ENA-78. A confirmatory assay was performed with rhIL-18 and the addition of neutralizing anti-human IL-18 Ab or nonspecific isotype-matched control Ab (25 µg/ml of each). A comparative dose response to the cytokines IL-18, IL-8, and ENA-78 (PeproTech) was also examined. The membranes were then removed, fixed in methanol for 1 min, and stained with Diff-Quik. Readings represent the number of cells migrating through the membrane (the sum of three high power x40 fields/well, averaged for each quadruplicate well).
Checkerboard analysis
Checkerboard analyses were performed as previously described (14, 35) using the Boyden chamber. Briefly, increasing concentrations of IL-18 (0, 1, 10, and 100 nM) were added to the cell suspensions in the bottom wells in addition to the top wells. The effect of an alteration or decrease in the resultant gradient was examined. The assay was performed three times and analyzed in a similar manner as above.
The role of v3 integrin in
IL-18-mediated migration
To determine whether IL-18 induces endothelial migration via
v3 integrin, we
employed the modified Boyden chamber migration assay. HMVECs
were preincubated for 1 h at 37°C with Ab to integrin
v3 (25 µg/ml), in
an attempt to inhibit its function in the migration process, or
nonspecific isotype-matched control Ab. The cell suspensions were then
incubated again with Ab to
v3 or nonspecific Ab
and used in the cell migration assay, as described above. The test
substances included IL-18 (10 nM), bFGF (60 nM) as positive control,
and PBS as negative control. This assay was performed three times, with
additional positive control TNF- (15 ng/ml) or negative control VEGF
(10 ng/ml).
IL-18 induced HMVEC proliferation
HMVEC proliferation in response to rhIL-18 was determined using a modified method from previously described procedures (14, 36). Briefly, HMVECs fed 1 day before use (with a fresh media exchange to obtain optimal growth conditions) were trypsinized and resuspended at 5 x 104 cells/ml in EBM (Clonetics) + 2% FBS + gentamicin. An equivalent number of cells (2.5 x 103) in 50 µl cell suspension was plated nonconfluently onto each well of a 96-well plate and allowed to adhere for 4 h at 37°C. Serial IL-18 dilutions were prepared in this media at 2x concentration, of which 50 µl was added to the 50 µl cell suspension already in the wells, resulting in 100 µl 1x dilutions. In a similar manner, serial dilutions of bFGF were also prepared for a positive control; media alone were added for the negative control. All dilutions were performed in quadruplicate wells. The plate was incubated for up to 72 h at 37°C. The Cell Titer 96 Aqueous nonradioactive cell proliferation assay kit (Promega, Madison, WI), which contains a tetrazolium compound and phenazine methosulfate solution, was diluted according to the recommended protocol and added to each well (20 µl/well). The plates were incubated at 37°C for 2–12 h until color development occurred. Absorbance was read at 490 nm on an ELISA plate reader with Microplate Manager (Bio-Rad, Richmond, CA). The absorbance readings were performed when the maximal response (plateau y-axis value) by bFGF concentrations represented at least a doubling from baseline control (no stimulation), and the absorbance at 490 nm (y-axis) vs concentration of stimulant (x-axis) was plotted and examined. Proliferation curves for HMVECs were determined, and measurements were obtained and compared in the log proliferation phase, which was 3 days (t = 72 h) for the conditions described. Serial dilutions of IL-18, ranging from 1 fM to 100 nM, or bFGF (positive control), ranging from 10 fM to 100 nM, in EBM and 2% FBS in a 100-µl vol were assayed.
Matrigel in vitro HMVEC tube formation assay for in vitro angiogenesis
Matrigel was used to examine HMVEC tube formation in response to IL-18. Matrigel was plated in eight-well chamber slides after thawing on ice, and the Matrigel was allowed to polymerize at 37°C for 30–60 min. HMVECs were removed from culture, trypsinized, and resuspended at 4 x 104 cells/ml in Medium 199 (Invitrogen, Carlsbad, CA) containing 2% FBS and 200 µg/ml endothelial cell growth supplement (Becton Dickinson, Bedford, MA). Four hundred microliters of cell suspension were added to each chamber, followed by 1, 5, or 10 nM IL-18, 50 nM PMA, or vehicle control (PBS for IL-18, and DMSO for PMA) placed directly into the suspension in the chambers. The chamber slides were then incubated for 16–18 h at 37°C in 5% CO2 humidified atmosphere. Culture media were aspirated off the Matrigel surface, and the cells were fixed with methanol and stained with Diff-Quick Solution II. Each chamber was photographed using Polaroid Microcam camera at x22 magnification. The number of tubes formed was quantitated blindly, as previously described (36). Briefly, a connecting branch between two discrete endothelial cells was counted as one tube and required a consistent intensity, thickness, and minimum length (>1 mm on a 4x enlarged copy of the photomicrograph) to be counted. This tube analysis was determined from one focal plane generated from a photomicrograph of the center of the well, focusing on the surface of the Matrigel, which was also the most prominent portion of the Matrigel (<0.5 cm deep after pipetting 400 µl into the chamber). Each concentration of the test substance and vehicle control was performed in triplicate within the same chamber, while PMA and its vehicle DMSO were tested in the chamber’s remaining two wells. This assay was performed four times for each IL-18 concentration.
Matrigel plug angiogenesis in vivo assay
To examine the effect of IL-18 on angiogenesis in vivo, we used a Matrigel plug assay (37, 38). C57BL/6 mice were anesthetized by Metofane inhalation. Each mouse was shaved on its ventral aspect and given an s.c. injection of sterile Matrigel (500 µl/injection) with a 27-gauge needle. Matrigel plus PBS served as the negative control, Matrigel containing bovine aFGF (1 ng/ml) served as the positive control, and Matrigel with IL-18 (10 nM) was the test substance. After 7–10 days, the mice were sacrificed by Metofane inhalation. The Matrigel plugs were then carefully dissected out, with removal of any surrounding connective tissue, and then analyzed by hemoglobin measurement or by histology. The Drabkin’s reagent method of determining hemoglobin content was employed. For histological analysis, we also embedded some of these plugs in paraffin for tissue sectioning and staining.
Additionally, we examined the effect of IL-18 in the presence of
neutralizing anti-TNF- Ab in the Matigel plug. Although the aim
of this experiment was not to block endogenous TNF- completely with
the dose employed, the effect of IL-18 while inhibiting the local
activity of TNF- on angiogenesis at the site of the Matrigel was
assessed. Matrigel plugs containing 10 nM IL-18 (PeproTech) and 25
µg/ml anti-TNF- Ab or isotype-matched control IgG were
injected into mice (n = 18 per group). Matrigel with
PBS also served as a negative control.
Hemoglobin determination of angiogenesis in Matrigel plugs
The Matrigel plugs dissected from the mice were carefully stripped of any remaining peritoneum. The plugs were weighed by placing them into preweighed 1-ml tube of ddH20 and homogenized for 5–10 min on ice. The samples were spun at 10,000 rpm on a microcentrifuge for 6 min, and the supernatants collected for hemoglobin measurement. Fifty microliters of supernatant were mixed with 950 µl Drabkin’s reagent and allowed to sit at room temperature for 15–30 min, and then 100 µl of this mixture was placed in a 96-well plate. Absorbance was read with a Microplate Manager ELISA reader at 540 nm. Hemoglobin concentration was determined by comparison with a standard curve in g/dl. These values were normalized by dividing the hemoglobin percentage by the plug weight. Hemoglobin concentration is a reflection of the number of blood vessels in the plugs.
For the Matrigel in vivo experiments comparing plugs containing IL-18
and anti-TNF- or IL-18 and control Ab, the hemoglobin
concentrations were determined by the tetramethylbenzidine method
(39, 40). In brief, methemoglobin (17.5 mg) in 25 ml
double-distilled water was brought to room temperature, and glycerol
30% was added. Serial dilutions were prepared to generate a standard
curve. Fifty microliters of supernatant or standard were added to a
96-well plate in duplicate, 50 µl tetramethylbenzidine was added to
each sample, the plate was allowed to develop at room temperature for
15–20 min with gentle shaking, and the reaction was terminated with
150 µl 2 N H2SO4 after
3–5 min. Absorbance was read with an ELISA plate reader at 450 nm. To
calculate hemoglobin concentrations, the values were normalized by the
weight of the plugs.
Masson’s trichrome staining of Matrigel plugs
To examine the Matrigel plugs histologically, some of the plugs were paraffin embedded. Sections (5 µm) were deparaffinized and stained using Masson’s trichrome straining. In brief, sections were hydrated in distilled water, dipped in Bouin’s solution for 1 h at 56°C, washed in water, dipped in Weigert’s iron solution for 7 min, washed, and dipped in Biebrich scarlet-acid fuchsin solution for 2 min. Sections were then rinsed, incubated in phosphomolybdic-phosphotungstic acid solution for 10 min, dipped in aniline blue solution for 5 min, rinsed, dipped in glacial acetic acid solution for 3–5 min, and dehydrated in two changes of 95% alcohol, 100% alcohol, and xylene. The slides were mounted with Cytoseal 60 (Stephens Scientific, Kalamazoo, MI).
Sponge granuloma in vivo angiogenesis assay
The animal model of inflammatory angiogenesis using a sponge granuloma has been described by Fajardo and colleagues (41). C57BL/6 mice were divided into several groups: negative control PBS; positive control, aFGF; and test substance, IL-18. One-centimeter sponge discs cut from sheets of 2-mm-thick polyvinyl alcohol foam sponges obtained from sterile packs (M-pact, Eudora, KS) were prepared, and a 2-mm hole was cut into the disc center to serve as a depot for control PBS or test substances, and then closed back with the cut plug. After adding the stimulant to the center hole, the sponge discs were coated with an inert slow release ethylene vinyl acetate copolymer, Elvax (DuPont Packages), and both disc surfaces were sealed with Millipore filters (0.45 µm) using Millipore glue number 1 (Millipore, Bedford, MA). The animals were anesthetized with pentobarbital 40 mg/kg of body weight, the hair on their left back area was shaved and sprayed with 70% ethanol, and the disc inserted into the s.c. layer at a site 2 cm distant from the incision, which was then sutured to prevent disc dispersion. After 9–12 days, the animals were sacrificed, and the sponge discs were harvested. The sponges were then analyzed by hemoglobin quantitation.
Hemoglobin quantitation of sponge granulomas by tetramethylbenzidine
To quantitate angiogenesis in the sponge model, the sponges were placed in 2 ml double-distilled water in 24-well plates. The sponges were cut into small pieces with scissors and then homogenized with a Kontes’ homgenizer. The samples were then spun at 8000 rpm for 6 min, and the resultant supernatants were filtered through a 0.22-µm filter. The hemoglobin percentage was determined by tetramethylbenzidine (39, 40). The protocol as described for the hemoglobin concentration determination in the Matrigel plug assay above was then followed, in this case using 50 µl of the filtered supernatant. To calculate hemoglobin concentrations, the values were normalized by the weight of the sponges.
Statistical analysis
Statistical analysis was performed using the nonparametric Wilcoxon rank-sum test, with statistical significance defined by p < 0.05. Values were expressed as means ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the effect of rhIL-18 on HMVEC migration, we tested
varying concentrations of IL-18 in a modified Boyden chamber assay. A
representative assay of three is shown (Fig. 1
). IL-18 induced HMVEC migration in a
concentration-dependent manner, reaching significance at 1 nM
(p < 0.05). Cell migration measured by the
number of migrating cells/well was determined by three blinded readings
per each quadruplicate well. The mean cell migration induced by 1 and
10 nM IL-18 was 18 ± 0.8 and 20 ± 0.4 cells/well,
respectively, while 100 nM IL-18 (29 ± 3.3 cells/well) exceeded
cell migration by control stimulant 60 nM bFGF (24.7 ± 1
cells/well).
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To confirm the specificity of rhIL-18-induced HMVEC migration, we
preincubated the IL-18 stimulant with either neutralizing goat
anti-human IL-18 IgG Ab or nonspecific isotype-matched control IgG
Ab, at a concentration of 25 µg/ml for 1 h before and during the
2 h of the cell migration assay. The response to the stimulant
with or without Ab was determined by the number of migrating cells/well
based on three blinded readings per each quadruplicate well (Fig. 2A). Neutralization of IL-18
resulted in inhibition of cell migration to basal values (9 ±
1.32 cells/well) similar to that seen upon incubation of HMVECs with
control PBS (7 ± 0.71 cells/well). Results are representative of
three similar assays. In addition, 10 nM IL-18 alone stimulated HMVEC
migration (27.25 ± 1.44 cells/well) comparable to that by 60 nM
bFGF (28.75 ± 0.65 cells/well).
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The checkerboard analysis was performed with the chemotaxis assay
using increasing concentrations of IL-18 in the cell suspensions at the
bottom of the chamber as well as in the top of the chamber. A
representative assay of three is shown (Fig. 2
B). The
concentrations were chosen for their known induction of HMVEC
migration, starting at 1 nM, although the effect of 100 nM may not have
been greatly dampened by the addition of 1 or 10 nM on the opposide
side of the membrane. There was evidence of some HMVEC migration
induced by the presence of IL-18 within the cell suspension, which
exceeded that by PBS, reflecting a chemokinetic contribution. However,
the presence of equivalent concentrations of IL-18 on either side of
the membrane reduced the resultant migration to levels closer to PBS
control. These data suggest a primarily chemotactic effect of IL-18 on
HMVEC migration.
IL-18 contributes to RA SF-induced HMVEC migration
To determine the relative contribution of IL-18 to RA SF-induced
HMVEC migration, we preincubated RA SF samples with neutralizing goat
anti-human IL-18 Ab or nonspecific isotype-matched control goat IgG
Ab, at a concentration of 25 µg/ml for 1 h before and during
2 h of the assay. We found significant inhibition of RA SF-induced
HMVEC migration with anti-IL-18 Ab, compared with nonspecific Ab
(Fig. 3). The inhibition of HMVEC
migration after neutralization of the RA SF samples (n
= 4) with the anti-IL-18 Ab (13.5 ± 2.9 cells/well) was
68 ± 5% compared with sham neutralization with the control IgG
Ab (41.5 ± 4.3 cells/well). HMVEC migration with control IgG was
equivalent to RA SF alone (40.6 ± 1.4 cells/well)
(p < 0.05). These findings suggest a prominent
role for IL-18 in RA SF-induced HMVEC migration.
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To compare the relative contribution to RA SF-induced HMVEC
migration, we preincubated four different RA SF samples with
neutralizing Abs to IL-18, IL-8, or ENA-78 (Fig. 4). Immunodepletion of IL-18 resulted in
a similar percentage of HMVEC migration inhibition (68 ± 5%) as
immunodepletion of IL-8 (64 ± 7%). However, immunodepletion of
IL-18 resulted in a 1.6-fold greater percent inhibition than
immunodepletion of ENA-78 (43 ± 6%). Values shown are means
generated from four different RA SF patient samples and are significant
compared with isotype-matched control Ab (*, p <
0.05). Inhibition was not further enhanced with combinations of
neutralizing Abs to IL-18 and either IL-8 or ENA-78 (62 ± 3% and
70 ± 3%, respectively).
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The neutralization of IL-18 present in RA SF reduced SF-induced
HMVEC migration to a similar or greater degree as neutralization of
IL-8 or ENA-78, respectively. We compared the bioactivities of these
recombinant human cytokines in the HMVEC migration assay. The dose
responses were generated from three different experiments, and a
representative assay is shown (Fig. 5).
IL-18 and ENA-78 induced HMVEC migration to a similar degree at the
lower concentrations 0.1–10 nM. IL-18 exceeded the effect of ENA-78 at
higher concentrations (10–100 nM). In comparison, IL-8 showed greater
effect on HMVEC migration at the lower and higher doses compared with
IL-18 and ENA-78, which had more comparable bioactivities. Overall,
assaying a wide range of concentrations, it appears that IL-8 is a more
potent angiogenic agent than IL-18, which is of greater potency than
ENA-78.
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v3
integrin
To determine the mechanism by which IL-18 mediates angiogenesis,
we hypothesized that IL-18 may act through integrins present on HMVECs.
TNF- is angiogenic in part via its action on integrin
v3 (34).
Thus, we examined whether blocking
v3 would inhibit
IL-18-induced angiogenesis in the HMVEC migration assay. HMVECs were
first incubated with
anti-v3 Ab or
nonspecific control IgG at 25 µg/ml for 1 h before and during
the chemotaxis assay. IL-18 (10 nM) was then used to stimulate HMVEC
migration, and comparison was made with the control stimulants bFGF and
TNF-. To illustrate a contrasting mechanism, VEGF was also used as
another stimulant, which would not be expected to be inhibited by
blocking v3, since
the angiogenic ability of VEGF is thought to occur via the integrin
v5 (42).
Blocking HMVEC v3
resulted in significant inhibition of IL-18-induced cell migration
comparable with that seen with control (Fig. 6). The same effect was seen with 25 or
10 µg/ml blocking Ab concentration. Significant inhibition of cell
migration by blocking HMVEC
v3 before stimulation
with bFGF and TNF- was also demonstrated, while no change in cell
migration occurred after stimulation with VEGF. Shown are the
representative data from three separate assays.
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The effect of rhIL-18 on endothelial cell proliferation was
assessed using varying concentrations of IL-18
(10-6 to 100 nM) to stimulate HMVEC growth.
Control stimulation with bFGF (10-5 to 10 nM)
demonstrated an expected 2-fold increase in HMVEC proliferation.
However, IL-18 did not directly stimulate or inhibit HMVEC
proliferation on repeated assays (n = 4), but behaved
similarly to PBS control. The time point of 72 h at which cell
growth was measured was selected when control bFGF-induced
proliferation was optimal. Additionally, proliferation curves for
HMVECs were determined for bFGF, media alone, and IL-18; and
measurements were compared in the log proliferation phase (Fig. 7).
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Endothelial cell tube formation in Matrigel is one measure of
angiogenesis in vitro. The role of IL-18 in angiogenesis was assessed
by determining the induction of HMVEC tube formation on Matrigel matrix
plated onto eight-well chamber slides. Tube-like structures formed
after a 16-h incubation in the presence of IL-18, whereas grossly fewer
tubes formed with PBS control (Fig. 8).
Shown are representative wells (magnified x88): A shows
tube formation with IL-18 (1 nM), compared with its PBS control
(B), and C shows tube formation with IL-18 (10
nM), compared with its PBS control (D). IL-18 at 1 and 10 nM
induced significant HMVEC tube formation with an increase of 77% and
87% compared with PBS control (p < 0.05).
Results represent the average of three wells per four similar assays at
two different IL-18 concentrations, in which the number of tubes formed
per each well were blindly counted from photomicrographs taken by
Polaroid at x22 magnification of the center of the well focused on the
Matrigel surface. Shown are the data for 1 nM IL-18
stimulation (Fig. 8E). Comparison with the stimulant PMA and
its vehicle control DMSO is also shown.
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The angiogenic role of IL-18 in vivo was assessed by examining the
effect of IL-18 on blood vessel growth in the Matrigel plugs in mice.
The Matrigel plugs containing IL-18 (10 nM) induced significantly
greater angiogenesis than control PBS. Histology illustrates the
difference qualitatively in the representative photomicrographs of
Masson trichrome-stained sections (x50 magnification) (Fig. 9A). Marked new blood vessel
growth can be seen in the IL-18-containing plugs (left
panel) compared with the control (right panel). Five
Matrigel plugs were embedded with stimulus for each test group. The
hemoglobin content of the 10 nM and 1 nM IL-18-containing plugs was
52-fold and 64-fold higher, respectively, than that for PBS control
(Fig. 9B): 1.65 ± 0.45 g/dl/(mg) for 10 nM IL-18,
2.05 ± 1.3 g/dl/(mg) for 1 nM IL-18, and 0.032 ± 0.02
g/dl/(mg) for PBS (p < 0.05). IL-18-stimulated
angiogenesis is comparable with positive control aFGF in the Matrigel
plugs.
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To examine the angiogenic effect of IL-18 in the presence of
neutralizing anti-TNF- Ab, Matrigel plugs containing 10 nM IL-18
and 25 µg/ml anti-TNF- Ab or isotype-matched control IgG were
injected into mice (n = 18 per group). After 7 days
incubation, the mean normalized hemoglobin contents were determined,
and there was no significant difference between the two groups:
2.67 ± 0.62 g/dl/(mg) for the IL-18 plus IgG-containing plugs vs
2 ± 0.31 g/dl/(mg) for the IL-18 plus anti-TNF--containing
plugs (Fig. 10). The hemoglobin content
of the 10 nM IL-18 plus IgG-containing plugs was 3.7 times greater than
that of PBS control.
|
To examine whether IL-18 induced inflammatory angiogenesis in
vivo, the sponge granuloma model was used. In this model, angiogenesis
was measured as increased blood vessel growth in discs made from
polyvinyl alcohol foam sponges containing either IL-18 or control
stimulant aFGF and implanted into mice. After 11 days, there was a
significant increase in blood vessel growth with IL-18 stimulation vs
PBS control (Fig. 11). A 5-fold
increase in angiogenesis with IL-18 (10 nM) was demonstrated by greater
hemoglobin concentrations measured with tetramethylbenzidine, and the
hemoglobin value induced by IL-18 was even greater than that for aFGF,
which resulted in a 4-fold increase: 5.77 ± 1.21 g/dl/(mg) for
IL-18, 4.45 ± 1.81 g/dl/(mg) for aFGF, and 1.13 ± 0.29
g/dl/(mg) for PBS (p < 0.05).
|
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Integral to the inflammation in RA is the process of angiogenesis. We found that IL-18 induced endothelial migration in the HMVEC migration assay, which is one aspect of angiogenesis in vitro. IL-18 induced HMVEC migration in the nanomolar range in a concentration-dependent manner, between 1 and 100 nM, with 100 nM IL-18 achieving a comparable degree of cell migration to 60 nM bFGF, a potent chemotactic stimulus. We were able to verify this finding by neutralizing the rhIL-18 with specific anti-IL-18 Ab, thereby blocking its chemotactic function compared with blocking with control-nonspecific IgG. Checkerboard analysis demonstrated a primarily chemotactic property of IL-18, whose effect is dependent on a concentration gradient rather than random chemokinesis. With the knowledge that RA SF is highly chemotactic and that IL-18 is elevated in RA SF, we examined the contribution of RA SF IL-18 to endothelial cell migration. Four different RA SFs immunodepleted of IL-18 resulted in a 68 ± 5% decrease in endothelial migration. This finding supports a potent role of IL-18 in endothelial cell migration in RA.
To compare the contribution of RA SF IL-18 with other mediators of RA SF that also induce endothelial cell migration, we further immunodepleted RA SFs of IL-18 alone and in combination with the chemokines IL-8 or ENA-78 (12, 43). IL-18 appeared to account for a significant portion of chemotactic activity for HMVECs in the SFs comparable with that by IL-8. The contribution by IL-18 was 1.6-fold greater than that by ENA-78. Combining immunodepletions of IL-18 and IL-8 or ENA-78 did not augment suppression of HMVEC migration. The lack of further suppression suggests that inhibiting the effect of one of these cytokines may be blocking common pathways involved in HMVEC migration. IL-18 accounted for approximately two-thirds of the migrating activity of HMVECs in RA SFs. These results indicate that IL-18 is one of the main mediators of RA SF-induced HMVEC migration.
A direct comparison of the HMVEC-migrating activity of the rh IL-18 with that of the chemokines rhIL-8 and rhENA-78 in the HMVEC migration assay revealed that IL-8 was a more potent agent than IL-18, which was more potent than or comparable with ENA-78.
IL-18 is also ascribed many inflammatory functions that would be
consistent with a role in angiogenesis. For instance, IL-18 stimulates
the production of other proangiogenic cytokines, such as TNF-,
IL-1 and IL-8, from human PBMCs, the responding cells being
predominantly resting T cells and NK cells. IL-18’s stimulation of the
chemokine IL-8 has also been shown in the macrophage cell line derived
from histiocytic lymphoma cell, U937, and the myelomonocytic leukemia
cell line, KG-1 (44, 45), to produce IL-8 and other C-X-C
chemokines. Besides its direct angiogenic role, other potential
mechanisms of the angiogenic function of IL-18 may be through IL-8 and
other angiogenic cytokines that it up-regulates. Indeed, IL-18 exerts
an inflammatory cytokine cascade in the mixed PBMC population by
stimulating the constitutive expression of IL-18R on NK cells and
lymphocytes, which leads to TNF- production, ultimately stimulating
macrophage IL-1 and IL-8 production (19). In turn,
treatment with IL-1 and TNF- of RA synovial fibroblast cultures,
not constitutively expressing IL-18, induced IL-18 gene and protein
production, suggesting a feedback interaction to promote Th1 cytokine
and cell development in RA (21).
IL-18 promotes inflammation, not only via other cytokine actions as above, but also through the promotion of other inflammatory mediators, some of which are themselves implicated in angiogenesis. IL-18 stimulates the expression of the soluble adhesion molecule ICAM-1 on monocytic cell lines (46), and we have found that IL-18 up-regulates the expression of VCAM-1 on RA synovial fibroblasts (47). Soluble VCAM-1 is potently angiogenic (14), and it may be that IL-18 also acts to stimulate RA fibroblast VCAM-1 expression, hence amplifying the angiogenic response.
To elucidate a mechanism by which IL-18 mediates angiogenesis, we
examined the potential role of the integrin
v3 in HMVEC
migration. Endothelial cell invasion, migration, and proliferation are
regulated in part by the integrin family of cell adhesion molecules,
and we have shown that
v3 is up-regulated on
RA compared with osteoarthritic or normal synovial blood vessels
(48). We therefore hypothesized that like bFGF and TNF-
(34), IL-18 may also act via this integrin in
angiogenesis. After blocking
v3 on HMVECs with
specific anti-v3
Ab, the degree of IL-18-induced cell migration was inhibited by 34%
compared with blocking with isotype-matched control Ab.
v3 antagonists have
successfully modulated angiogenesis in a rabbit model of synovitis
(49). Targeting IL-18, which appears to function in part
through this integrin, could be an alternative therapeutic approach
for RA.
Endothelial cell proliferation is another aspect of angiogenesis. We did not demonstrate direct inhibition or enhancement of basal endothelial cell (HMVEC) proliferation, which is in contrast to previous reports of the antimitogenic activity of IL-18. IL-18 at 1–10 nM concentration has been reported to inhibit FGF-2-induced capillary endothelial cell proliferation (50). However, the effect of IL-18 on basal proliferation, as examined in our study, was not explored. This group also found that murine, not human, IL-18 suppressed FGF-2-induced corneal neovascularization in mice, although the IL-18 was administered by i.p. injections, and it is unclear whether local rather than systemic treatment would have induced divergent results. Interestingly, they also found that IL-18 suppressed angiogenesis in the chick chorioallantoic membrane model. Our finding that IL-18 did not induce HMVEC proliferation is consistent with results obtained for other angiogenic mediators, such as soluble VCAM-1 or soluble E-selectin, which also do not induce endothelial proliferation (14).
Consistent with the chemotactic properties of IL-18, we also showed
IL-18 induction of HMVEC tube formation in Matrigel matrix in vitro.
The degree of tube formation by IL-18 at 1 nM was comparable with the
stimulant PMA (50 mM), a potent inducer of HMVEC differentiation and
tube formation (51). In the Matrigel in vivo mouse
angiogenesis model, IL-18 also stimulated actual blood vessel
formation, which was visibly in excess of control. The potency of IL-18
relative to other angiogenic stimulants in this assay can be assessed,
since 1 nM (or 18 ng/ml) IL-18 produced results similar to that induced
by 1 ng/ml aFGF, which is clearly one of the strongest angiogenic
stimuli. By comparison, an excess of 100-fold more bFGF would be
required to produce the same effect in this assay as 1 ng/ml aFGF (0.86
g/dl vs 1.30 g/dl hemoglobin), and 10-fold more TNF- (10 ng/ml
hemoglobin) would be expected to produce twice the effect as 1 ng/ml
aFGF (2.3 g/dl vs 1.3 g/dl hemoglobin) (37).
The Matrigel in vivo assay also allowed for another examination of a
potential mechanism behind the angiogenic activity of IL-18. The
angiogenic effect of IL-18 in the presence of anti-TNF- Ab was
evaluated by adding IL-18 and neutralizing anti-TNF- Ab to the
Matrigel before injection. Although the endothelial cells were the
target of the effect of IL-18 in cell migration and tube formation
observed in the earlier assays, the new blood vessel formation in the
Matrigel plug could involve cellular targets other than endothelial
cells, at least indirectly. In our hands, blocking the local TNF-
did not significantly alter the effect of IL-18 on Matrigel plug
hemoglobin content. However, it is not clear whether systemic
inhibition of TNF- would have diminished the effect of IL-18. In
addition, the downstream effect of IL-18 on PBMCs, lymphocytes, and
other cells via up-regulation of other cytokines cannot be overlooked.
The various mediators known to orchestrate the angiogenic process may
function in part through common pathways. Although redundancy in the
system exists and serves to maintain the angiogenic state
characteristic of certain physiologic or pathologic conditions,
evidence from clinical trials clearly demonstrates that blocking one
key element through treatment targeting TNF- alone reduced RA
disease activity (52). Further studies examining potential
mechanisms of angiogenesis, including common signaling pathways, cell
adhesion, and up-regulation of cytokines, integrins, and other
mediators, may shed light on the significance of the current
findings.
The sponge angiogenesis system allows testing of the effect of various antagonists and agonists on the proliferation of blood vessels and stroma, provides histological and quantitative information, and is easily reproducible. This system applied to mice was suitable to test the independent effect of IL-18 on angiogenesis, and specifically, new blood vessel formation, which this assay measures by virtue of the constant sample area of the disc and the planar growth of the vessels (41). IL-18 at 10 nM strongly induced angiogenesis, as measured by a 5-fold increase in the amount of hemoglobin over control, similar to the 4-fold increase seen with the positive control stimulant aFGF (1 nM). This finding not only confirms our results from the Matrigel plug in vivo assay, but it establishes IL-18 as a direct angiogenic stimulus.
In summary, we found that IL-18 induces endothelial cell chemotaxis,
and that IL-18 accounts for a significant portion of the vascular
migration characteristic of RA SF. This contribution is independent of
that made by the chemokines IL-8 and ENA-78 also found in RA SF. One
mechanism for IL-18-induced HMVEC migration is through the integrin
v3, placing IL-18 in
company with TNF- and bFGF, which also act in part through this
specific integrin. In our hands, IL-18 did not affect basal endothelial
cell proliferation. We demonstrated the ability of IL-18 to induce tube
formation in Matrigel in vitro as well as significant blood vessel
formation in the Matrigel plug in vivo. An attempt to block the effect
of TNF- present at the angiogenesis site with neutralizing Abs in
the Matrigel plug did not significantly diminish the angiogenic effect
of IL-18 within the plug. Finally, IL-18 induced neovascularization in
the sponge disc angiogenesis model in mice. These findings support the
notion of IL-18 as a potent angiogenic stimulus, with the capacity to
mediate vascular migration and new blood vessel formation in the
development of inflammatory and angiogenesis-driven disease, such as
RA. Ultimately, this evidence for a novel angiogenic function of IL-18
may lend itself to potential therapeutic targets in the treatment
of RA.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 C.C.P. and J.C.M.M. contributed equally to the work reported in this study.
3 Address correspondence and reprint requests to Dr. Alisa E. Koch, Gallagher Research Professor of Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Ward 3-315, Chicago, IL 60611. E-mail address: ae-koch{at}northwestern.edu
4 Abbreviations used in this paper: RA, rheumatoid arthritis; aFGF, acidic FGF; bFGF, basic FGF; EBM, endothelial cell basal medium; ENA-78, epithelial neutrophil-activating peptide; FGF, fibroblast growth factor; HMVEC, human microvascular endothelial cell; rh, recombinant human; SF, synovial fluid; VEGF, vascular endothelial growth factor.
Received for publication November 22, 2000. Accepted for publication May 25, 2001.
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