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Interleukin-15 inhibits sodium nitroprussideinduced apoptosis of synovial fibroblasts and vascular endothelial cells.

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Vol. 46, No. 11, November 2002, pp 3010–3014
DOI 10.1002/art.10610
© 2002, American College of Rheumatology
Interleukin-15 Inhibits Sodium Nitroprusside–Induced
Apoptosis of Synovial Fibroblasts and
Vascular Endothelial Cells
Lin Yang, Sherry Thornton, and Alexei A. Grom
Objective. One of the pathologic hallmarks of
juvenile rheumatoid arthritis (JRA) is a tumor-like
expansion of inflamed synovial tissue, or pannus, which
causes much of the joint damage in this disease. The
expansion of pannus is supported by extensive formation of new blood vessels. We have previously shown that
revascularization of minced JRA synovial tissues engrafted into SCID mice correlated with the intensity of
inflammatory activity in the tissues and with
interleukin-15 (IL-15) expression. Since synovial vascular endothelial cells (VECs) expressed IL-15 receptors,
the present study was undertaken to investigate the
hypothesis that IL-15 might play a role in neovascularization of the pannus.
Methods. To evaluate IL-15 for possible angiogenic activity, we assessed the ability of recombinant
human IL-15 (rHuIL-15) to induce VEC growth directly
and to stimulate synovial cells to produce endothelial
growth factors. Since IL-15 had been shown to inhibit
apoptosis of certain immune cells, we were also interested in whether it might have similar effects on VECs.
Apoptosis was induced by addition of sodium nitroprusside (SNP) at 1–2 mM to >80% confluent primary
VECs, and numbers of apoptotic cells were determined
by annexin V assay.
Results. Addition of rHuIL-15 at 10–100 ng/ml to
primary synovial fibroblast cultures failed to upregulate expression of vascular endothelial growth fac-
tor and angiopoietin 1 by these cells. Although rHuIL-15
failed to induce a mitogenic response of VECs, it
promoted survival of these cells on Matrigel. Preincubation of VECs with rHuIL-15 at 50 ng/ml significantly
reduced the proportion of VECs undergoing apoptosis.
Conclusion. IL-15 promotes survival of VECs on
Matrigel and inhibits SNP-induced apoptosis of endothelial cells. We hypothesize that this mechanism may
be relevant to the stabilization of newly formed vascular
structures in JRA synovium.
Expansion of inflamed synovial tissue, or pannus,
is a characteristic pathologic feature of both rheumatoid
arthritis (RA) and juvenile rheumatoid arthritis (JRA).
With the progression of the diseases, the pannus invades
and destroys intraarticular cartilage. The expansion of
the pannus is supported by extensive formation of new
blood vessels (1). Suppression of such neovascularization in animal models of arthritis leads to a significant
reduction in the severity of synovial inflammation (2).
Although in healthy individuals the existing vasculature is extremely stable, under inflammatory conditions the volume of blood vessels may rapidly change (3).
Such changes are related to the ability of many inflammatory cytokines to modulate the expression of the
direct-acting vascular-specific growth factors, leading to
the changes in the overall angiogenic activity. For instance, in adult RA synovium, interleukin-1 (IL-1),
tumor necrosis factor ␣ (TNF␣), and transforming
growth factor ␤ have emerged as major mediators of
vascular endothelial growth factor (VEGF) release (4).
More recently, IL-15, another proinflammatory cytokine, has been associated with vascular endothelial cell
(VEC) functions in rheumatoid synovium. (5). The
expression of IL-15 in rheumatoid synovium and the
presence of IL-15 receptors on VECs have been reported by several groups (5–7). Furthermore, Angiolillo
Supported in part by NIH grants P30-AR-4736, P30-HD28827, and MAMDC P60-AR-44059-01, by Children’s Hospital Research Foundation of Cincinnati, and by the Schmidlapp Foundation.
Lin Yang, MD, Sherry Thornton, PhD, Alexei A. Grom, MD:
Children’s Hospital Medical Center, Cincinnati, Ohio.
Address correspondence and reprint requests to Alexei A.
Grom, MD, Division of Rheumatology, Pavilion Building 2-129,
Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati,
OH 45229. E-mail:
Submitted for publication November 8, 2001; accepted in
revised form July 26, 2002.
et al showed that IL-15, injected subcutaneously into
nude mice, consistently induced neovascularization of
Matrigel plugs (8), suggesting direct angiogenic activity
of the cytokine.
Our group’s previous studies of JRA synovial
tissue demonstrated increased vascularization and VEC
activation. These features remained prominent in JRA
synovial tissue fragments engrafted into SCID mice, and
correlated with the intensity of inflammatory activity
and IL-15 expression (9). Based on these observations,
we hypothesized that IL-15 may play a major role in
neovascularization of pannus tissue in JRA. To evaluate
this cytokine for possible angiogenic activity, we assessed
the ability of recombinant human IL-15 (rHuIL-15) to
promote VEC growth directly and to stimulate other
synovial cells to produce endothelial growth factors.
Synovial tissue samples. Synovial tissue specimens
were obtained from JRA patients undergoing joint replacement surgery as a normal part of their clinical care (the tissues
would otherwise have been discarded). All patients satisfied
the American College of Rheumatology criteria for the diagnosis of JRA (10).
Recombinant proteins and cell lines. Recombinant
human IL-15 and recombinant human basic fibroblast growth
factor (bFGF) were purchased from R&D Systems (Minneapolis, MN). To establish primary synovial fibroblast cultures,
synovial tissue samples were minced, resuspended in serumfree RPMI 1640 with type I collagenase (Worthington, Lakewood, NJ) at a final concentration of 4 mg/ml, and incubated
at 37oC with 10% CO2 with gentle stirring for 12 hours. Cell
suspensions were then filtered through sterile mesh, washed,
and transferred to fresh RPMI 1640 with 10% fetal calf serum
(FCS). Primary human umbilical vein endothelial cells
(HUVECs) were purchased from BioWhittaker (San Diego,
CA). Cells were expanded in basal media supplemented with
2% FCS and endothelial growth factors (EGM-2 BulletKit
System; BioWhittaker).
Western blotting. Cell pellets (106 cells/sample) were
lysed in 1% Triton X-100 lysis buffer, subjected to sodium
dodecyl sulfate–12.5% polyacrylamide gel electrophoresis, and
transferred to polyvinylidene difluoride membranes. The
membranes were immunoblotted with goat IgG polyclonal
anti-human IL-15 receptor ␣ chain (IL-15R␣) antibodies
(R&D Systems) at 0.1 ␮g/ml and washed. Biotinylated antigoat IgG (Jackson ImmunoResearch, West Grove, PA) was
used as secondary antibody. The membranes were then incubated with streptavidin–alkaline phosphatase, washed, and
developed in BCIP/nitroblue tetrazolium. Human peripheral
blood lymphocytes were used as a positive control.
Cell proliferation assay. HUVECs or primary fibroblasts from the second and third passages were plated in
triplicate at 5 ⫻ 103 in 0.2 ml of complete media with or
without additives in 96-well flat-bottomed plates. The plates
were incubated for 72 hours. DNA synthesis was determined
by 3H-thymidine deoxyribose incorporation (1 ␮Ci/well; ICN
Biomedicals, Costa Mesa, CA) during the last 18 hours of
Endothelial cell tube formation on Matrigel. HUVECs
plated on Matrigel-coated wells rearrange themselves to form
tube-like structures. The advantage of using growth factor–
reduced Matrigel (GFR-Matrigel) as opposed to standard
Matrigel is that differences in tube density in the presence of
exogenous stimulants are more readily recognized (11). GFRMatrigel was purchased from Collaborative Biomedical Products (Bedford, MA). Tube formation was evaluated as described previously (11). Briefly, 300 ␮l of GFR-Matrigel used
at 4oC to coat a 24-well plate was allowed to polymerize at
37oC for 2 hours. HUVECs (2 ⫻ 105/well) in a final volume of
1.0 ml of EGM-2–2% FCS (with no endothelial cell growth
factor supplementation) were added to the wells and incubated
overnight at 37oC with or without rHuIL-15 (100 ng/ml). Basic
FGF at 25 ng/ml was used as a positive control. Three
independent experiments were performed for each culture
condition. Tube formation was examined visually at 10⫻
magnification after 24 hours of incubation. Photographs of the
central part of each well were taken, and the overall length of
tube-like structures was measured.
Induction of apoptosis by sodium nitroprusside
(SNP). The nitric oxide (NO) donor SNP (Sigma-Aldrich, St.
Louis, MO) was chosen as a proapoptotic agent because the
overproduction of NO has been demonstrated in RA synovium
(12,13). Primary synovial fibroblasts or HUVECs were cultured in appropriate media until ⬎80% confluence was
achieved. HUVECs were grown in EGM-2 supplemented with
endothelial growth factors and 2% FCS. Primary synovial
fibroblasts were grown in RPMI supplemented with 10% FCS.
After preincubation of cells with or without rHuIL-15 for 48
hours, they were treated for an additional 24 hours with SNP
at various concentrations. Cells were then harvested and
analyzed for numbers of apoptotic cells. All experiments were
performed in triplicate.
Detection of apoptosis. The annexin V assay was used
to quantify numbers of apoptotic cells (Annexin Apoptosis
Detection Kit; BD PharMingen, San Diego, CA). After exposure to rHuIL-15 for 48 hours and incubation with SNP for an
additional 24 hours, cells were analyzed by flow cytometry on
a FACScan (Becton Dickinson, Mountain View, CA). Results
were integrated with CellQuest software (Becton Dickinson)
and expressed as the percentage of annexin-positive, propidium iodide–negative cells. The concentration of SNP was
titrated to determine conditions in which considerable numbers of VECs were in the early apoptotic stage but the numbers
of dead cells were still low.
Ribonuclease protection assay (RPA). Total cellular
RNA was isolated from either fresh HUVECs or primary
fibroblasts, utilizing RNA Stat-60 according to the instructions
of the manufacturer (Tel-Test, Friendswood, TX). The expression of angiogenic factors and their receptors was measured by
the Riboquant Multi-Probe RPA (BD PharMingen), as previously described (9). The template set hAngio1, which includes
the key angiogenic factors VEGF, angiopoietin 1, and their
receptors, was selected for this study.
Statistical analysis. Normal distribution of samples
was confirmed using the Wilk-Shapiro test, and the data were
Figure 1. Human interleukin-15 receptor ␣ chain (IL-15R␣) gene
expression in primary synovial fibroblasts and endothelial cells. Cell
lysates obtained from 106 cells were subjected to sodium dodecyl
sulfate–12.5% polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted with anti–IL15R␣ antibodies. Lane 1, Negative control. Lane 2, Human peripheral
blood lymphocytes (positive control). Lane 3, Primary synovial fibroblasts. Lane 4, Human umbilical vein endothelial cells. Molecular mass
standards are shown on the right.
analyzed using Student’s t-test. P values less than or equal to
0.05 were considered significant.
IL-15R␣ expression by primary synovial fibroblasts and VECs. In previous studies, investigators at
our institution demonstrated high levels of IL-15 in the
majority of JRA synovial tissue samples (7). In this
study, the expression of IL-15R␣ in primary synovial
fibroblast and VEC cultures was initially demonstrated
by reverse transcriptase–polymerase chain reaction analysis (results not shown) and then confirmed by Western
blot analysis (Figure 1). IL-15R␣ is detectable by Western blotting as a protein of 60–65 kd (14).
IL-15 promotion of human VEC survival on
GFR-Matrigel. Most biologic effects of IL-15 have been
demonstrated with concentrations in the range of 10–
100 ng/ml. This range is also comparable with the levels
of IL-15 protein found in synovial fluid of patients with
RA (6). In our initial experiments, addition of IL-15 to
the culture media at final concentrations of 10 ng/ml and
100 ng/ml did not induce proliferation of the cells
(results not shown). Since proliferation of VECs also
depends on their adhesive interactions with extracellular
matrix proteins, we assessed the ability of rHuIL-15 to
stimulate VEC growth on GFR-Matrigel. Previous studies have shown that VECs grown on Matrigel in the
presence of appropriate growth factors form tube-like
structures (11). HUVECs from the second or third
passage were plated on GFR-Matrigel (2 ⫻ 105/well)
with or without IL-15 at 100 ng/ml. Basic FGF (at 25
ng/ml), a known angiogenic agent, was used as a positive
Although no proliferative response to IL-15 was
noted (results not shown), we observed a higher density
of surviving tube-like structures when cells were grown
in the presence of rHuIL-15. As shown in Figure 2, no
tube formation was seen in the wells in which HUVECs
were grown on GFR-Matrigel alone, and the vast majority of the cells in these wells were dead. In contrast,
addition of rHuIL-15 was associated with formation of
tube-like structures similar to those observed in the wells
containing bFGF. This observation, combined with the
known antiapoptotic effects of IL-15 on human immune
cells and fibroblasts, led us to hypothesize that IL-15
might protect VECs against apoptotic death.
IL-15 inhibition of SNP-induced apoptosis of
VECs in vitro. To test the above hypothesis, we examined the effects of IL-15 on apoptosis of VECs induced
Figure 2. Effects of recombinant human interleukin-15 (hrIL-15) on endothelial cell survival on growth factor–reduced
Matrigel (GFR-Matrigel). Human umbilical vein endothelial cells (2 ⫻ 105/well) in a final volume of 1.0 ml of growth
factor–free EGM-2–2% fetal calf serum with (A) or without (B) hrIL-15 (at 100 ng/ml) were placed on GFR-Matrigel in a
24-well plate and incubated overnight at 37oC. Basic fibroblast growth factor (bFGF) at 25 ng/ml was used as a positive control
(C). Three independent experiments were performed for each culture condition. Tube formation was examined visually at 10⫻
magnification after 24 hours of incubation. Photographs of the central portion of each well were taken, and the overall length
of tube-like structures was measured.
Figure 3. Effects of recombinant human interleukin-15 (rHuIL-15)
on sodium nitroprusside (SNP)–induced apoptosis of endothelial cells.
Human umbilical vein endothelial cells from the second or third
passage were grown in EGM-2–2% fetal calf serum with or without
rHuIL-15 at 50 ng/ml for 48 hours prior to addition of SNP at various
concentrations. Cells were then incubated for 24 hours with or without
SNP and harvested. The proportion of apoptotic cells was determined
using the annexin V assay as described in Patients and Methods.
Apoptotic cells were distinguished from dead cells based on propidium
iodide (PI) uptake. A, Mean and SEM proportion of apoptotic cells as
determined in 3 independent experiments for each culture condition.
Light grey–shaded bars represent cells preincubated with rHuIL-15;
dark grey–shaded bars represent cells not preincubated with rHuIL-15.
With SNP concentrations of 1.5 mM and 2 mM, respectively, P ⫽ 0.01
and P ⫽ 0.004 for the proportion of apoptotic cells with rHuIL-15
preincubation versus without rHuIL-15 preincubation. B, Typical flow
cytometry results in a representative experiment (1.5 mM SNP).
by SNP, a donor of NO. The overproduction of NO in
rheumatoid synovium and the ability of NO to induce
apoptosis of human synoviocytes have been demonstrated by several groups (12,13). The effects of NO on
VECs, however, are more complex. Physiologic levels of
NO regulate vascular tone and protect microvasculature
from injury, whereas higher NO concentrations are
pathologic and promote VEC apoptosis both in vivo and
in vitro (13). Therefore, in initial experiments, we determined the length of the exposure and the concentration
of SNP at which a considerable proportion of cells
become apoptotic but the numbers of dead cells are still
low. Twenty-four–hour exposure to SNP at concentrations in the 1.5–2.0 mM range was found to be optimal
for analysis of the antiapoptotic activities of IL-15. At
these concentrations, after 24 hours of incubation, up to
25% of VECs showed staining characteristic of early
apoptosis. We then tested rHuIL-15 for its ability to
inhibit VEC apoptosis induced by 1–2 mM SNP. As
shown in Figure 3, preincubation of HUVECs with
rHuIL-15 at 50 ng/ml for 48 hours prior to addition of
SNP resulted in a statistically significant reduction in the
proportion of apoptotic cells.
IL-15 inhibition of SNP-induced apoptosis of
primary synovial fibroblasts in vitro. Similar results
were obtained with primary fibroblast cultures derived
from fresh JRA synovial tissue samples (data not
shown). Preincubation of primary fibroblasts with
rHuIL-15 at 50 ng/ml for 48 hours prior to addition of
SNP resulted in a statistically significant reduction in the
proportion of apoptotic primary synovial fibroblasts.
Failure of IL-15 to up-regulate expression of
VEGF in primary fibroblasts in vitro. Since proinflammatory cytokines such as TNF␣ and IL-1 exert their
angiogenic effects through stimulation of VEGF production by primary fibroblasts (4), we were interested in
whether IL-15 might have similar properties. Primary
fibroblast cultures were established from a fresh synovial
tissue sample, and the synovial cells were stimulated with
rHuIL-15 at a final concentration 50 ng/ml for 48 hours.
Three independent experiments were performed for
each culture condition, and levels of expression of
VEGF were assessed by RPA. No differences were
noted between stimulated and unstimulated fibroblasts
(data not shown).
In our evaluation of the angiogenic activity of
IL-15, we showed that addition of IL-15 to the media
promoted VEC survival and tube formation on Matrigel.
This observation combined with the known antiapo-
ptotic effects of IL-15 on immune cells and fibroblasts
led us to hypothesize that IL-15 might protect VECs
from apoptotic death. Indeed, we found that preincubation of VECs with rHuIL-15 made them more resistant
to SNP-induced apoptosis. We also demonstrated that
rHuIL-15 had similar effects on synovial fibroblasts.
We used the NO donor SNP as a proapoptotic
agent for two reasons: first, NO has been shown to
induce apoptosis of synovial fibroblasts in vitro, and
second, overproduction of NO in RA synovium has been
documented in many studies (12,13). The levels of NO in
our experimental system are likely to be higher than the
physiologic levels of NO in inflamed synovium. This
certainly makes it difficult to extrapolate the findings to
JRA pathogenesis. However, combined with the ability
of IL-15 at physiologic concentrations to promote survival of VECs on Matrigel, our data suggest that the
observed effects may be relevant to the in vivo situation.
Fraser et al have recently demonstrated high levels of
VEC apoptosis in RA synovium, suggesting increased
turnover of these cells in vivo (15). Based on the present
findings, we hypothesize that IL-15 may promote survival of endothelial cells in JRA synovium and thus be
involved in the stabilization of newly formed blood
1. Firestein GS. Starving the synovium: angiogenesis and inflammation in rheumatoid arthritis. J Clin Invest 1999;103:3–4.
2. Peacock DJ, Banquerigo ML, Brahn E. Angiogenesis inhibition
suppresses collagen arthritis. J Exp Med 1992;175:1135–8.
3. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ,
Holash J. Vascular-specific growth factors and blood vessel formation. Nature 2000;407:242–8.
4. Paleolog EM, Young S, Stark AC, McCloskey RV, Feldmann M,
Maini RN. Modulation of angiogenic vascular endothelial growth
factor by tumor necrosis factor ␣ and interleukin-1 in rheumatoid
arthritis. Arthritis Rheum 1998;41:1258–65.
5. Oppenheimer-Marks N, Brezinschek RI, Mohamadzadeh M, Vita
R, Lipsky PE. IL-15 is produced by endothelial cells and increases
the transendothelial migration of T cells in vitro and in the SCID
mouse-human rheumatoid arthritis model. J Clin Invest 1998;101:
6. McInnes IB, al-Mughales J, Field M, Leung BP, Huang FP, Dixon
R, et al. The role of IL-15 in T-cell migration and activation in
rheumatoid arthritis. Nat Med 1996;2:175–82.
7. Scola MP, Thompson S, Brunner HI, Tsoras M, Witte D, van Dijk
MA, et al. IFN gamma:IL-4 ratios and associated type 1 cytokine
expression in JRA synovial tissue. J Rheumatol 2002;29:369–78.
8. Angiolillo AL, Kanegane H, Sgadari C, Reaman GH, Tosato G.
Interleukin-15 promotes angiogenesis in vivo. Biochem Biophys
Res Commun 1997;233:231–7.
9. Scola PM, Imagawa T, Boivin GP, Giannini EH, Glass DN, Hirsch
R, et al. Expression of angiogenic factors in juvenile rheumatoid
arthritis: correlation with revascularization of human synovium
engrafted into SCID mice. Arthritis Rheum 2001;44:794–801.
10. Cassidy JT, Levinson JE, Bass JC, Baum J, Brewer EJ Jr, Fink
CW, et al. A study of classification criteria for a diagnosis of
juvenile rheumatoid arthritis. Arthritis Rheum 1986;29:274–81.
11. Blair RJ, Meng H, Marchese MJ, Ren S, Schwartz LB, Tonnesen
MG, et al. Human mast cells stimulate vascular tube formation:
tryptase is a novel, potent angiogenic factor. J Clin Invest 1997;
12. Aupperle KR, Boyle DL, Hendrix M, Seftor EA, Zvaifler NJ,
Barbosa M, et al. Regulation of synoviocyte proliferation, apoptosis, and invasion by the p53 tumor suppressor gene. Am J Pathol
13. Shen YH, Wang XL, Wilcken DE. Nitric oxide induces and
inhibits apoptosis through different pathways. FEBS Lett 1998;
14. Bulanova E, Budagian V, Pohl T, Krause H, Dürkop H, Paus R, et
al. The IL-15R␣ chain signals through association with Syk in
human B cells. J Immunol 2001;167:6292–302.
15. Fraser A, Fearon U, Reece R, Emery P, Veale DJ. Matrix
metalloproteinase 9, apoptosis, and vascular morphology in early
arthritis. Arthritis Rheum 2001;44:2024–8.
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endothelial, apoptosis, inhibits, vascular, sodium, interleukin, nitroprussideinduced, synovial, cells, fibroblasts
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