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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1612-1621
Contribution of Natural Killer Cells to Inhibition of
Angiogenesis by Interleukin-12
By
Lei Yao,
Cecilia Sgadari,
Keizo Furuke,
Eda T. Bloom,
Julie Teruya-Feldstein, and
Giovanna Tosato
From the Divisions of Hematologic Products and of Cellular and Gene
Therapies, Center for Biologics Evaluation and Research, Food and Drug
Administration; and the Hematopathology Section, Laboratory of
Pathology, National Cancer Institute, Bethesda, MD.
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ABSTRACT |
Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing
interferon- (IFN-) and other downstream mediators. Here, we
report that neutralization of natural killer (NK) cell function with
antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of
basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic
mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10-positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK
cells were locally activated. Experimental Burkitt lymphomas treated
locally with IL-12 displayed tumor tissue necrosis, vascular damage,
and NK-cell infiltration surrounding small vessels. After activation in
vitro with IL-12, NK cells from nude mice became strongly cytotoxic for
primary cultures of syngeneic aortic endothelial cells. Cytotoxicity
was neutralized by antibodies to IFN-. These results document that
NK cells are required mediators of angiogenesis inhibition by IL-12,
and provide evidence that NK-cell cytotoxicity of endothelial cells is
a potential mechanism by which IL-12 can suppress neovascularization.
This is a US government work. There are no restrictions on its use.
| |
INTRODUCTION |
INTERLEUKIN-12 (IL-12), a
disulfide-linked heterodimer composed of two subunits, p35 and p40,
stimulates T and natural killer (NK) cells to secrete interferon-gamma
(IFN-) and augments T- and NK-cell proliferation and cytolytic
activity.1-5 Through these functions, IL-12 plays a
critical role in the regulation of early inflammatory responses and
promotes the development of Th1-type T-cell responses that favor
cell-mediated immunity.6,7 Preclinical testing has
demonstrated that IL-12 can be effective against certain microbial and
fungal agents, can serve as an immune adjuvant, and can exert antitumor
activity against several experimental malignancies.8-13
Recently, IL-12 was reported to inhibit new vessel formation in an in
vivo assay that detects murine corneal vascularization induced by basic
fibroblast growth factor (bFGF), a potent inducer of
angiogenesis.14,15 This property of IL-12 was also
demonstrated in another in vivo angiogenesis assay that measures
bFGF-induced neovascularization of subcutaneous Matrigel
(Becton-Dickinson Labware, Bedford, MA) plugs in nude
mice.16 Neutralizing antibodies to IFN- removed most of
the IL-12 inhibitory effect on neovascularization in both mouse models,
and experiments in vitro failed to demonstrate a direct effect of IL-12
on endothelial-cell growth.16 Furthermore, a neutralizing
antibody to IP-10, a CXC chemokine induced by IFN-, substantially
reduced the inhibition of angiogenesis induced by IL-12.16
Previous studies had documented that IP-10 can act as an inhibitor of
neovascularization induced by bFGF and other stimuli.17
These findings indicated that IL-12 inhibits angiogenesis indirectly
through the stimulation of IFN- and, at least in part, IP-10.
The mechanisms by which IP-10 inhibits neovascularization are presently
unknown, and it is unclear whether the chemokine can act directly on
endothelial cells.18 In one study, a recombinant IP-10
alkaline phosphatase fusion protein was found to bind to endothelial
cells and other cells through heparan sulfate
proteoglycan.19 This binding was specific and saturable,
and was reversed by heparin, but signaling was not reported.
Endothelial-cell proliferation in vitro was inhibited by IP-10, albeit
at substantially higher concentrations than those required for an
angiostatic effect in vivo.19 Recently, a signaling
receptor for human IP-10, CXCR3, was identified on activated T cells,
NK cells, and other cells.20 Using reverse-transcriptase
polymerase chain reaction (RT-PCR), we found no evidence of CXCR3
expression in primary cultures of human umbilical cord-derived
endothelial cells (unpublished results, November 1996 to January
1997). While others have also reported that human
umbilical cord-derived endothelial cells do not express CXCR3,21 a recent study found evidence of CXCR3 expression
in murine endothelial cells.22 Thus, it is currently
unclear whether endothelial cells can be direct targets of IP-10 effects.
In the present study, we examined further the mechanisms that mediate
angiogenesis inhibition by IL-12. Our results provide evidence that NK
cells can play a critical role in the regulation of angiogenesis.
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MATERIALS AND METHODS |
Mice, reagents, cytokine, and antibodies.
Five- to 6-week-old female BALB/c nude mice (National Cancer Institute,
Bethesda, MD, Frederick Cancer Research Center [FCRC]) maintained in pathogen-limited conditions were used throughout, except
for one experiment that used 4- to 6-week-old female C57BL/6 nude mice
(Taconic, Germantown, NY). Matrigel was purchased from Becton Dickinson
Labware. Recombinant murine IL-12 was a gift of Genetics Institute, Inc
(Cambridge, MA). Recombinant murine IFN- was a gift of Genentech,
Inc (South San Francisco, CA). Rabbit antimurine IP-10 antiserum was a
gift of Dr J.M. Farber (Laboratory of Clinical Investigation, National
Institute of Allergy and Infectious Diseases, National Institutes of
Health [NIH], Bethesda, MD). Rabbit antiasialo GM1 antibody was
purchased from WAKO (Dallas, TX). Murine antiasialo GM1 monoclonal
antibody (clone SH34) was a gift of Dr R. Stout (James H. Quillen
College of Medicine, Johnson City, TN).23,24 Murine
antimouse NK1.1 (clone PK136) and rat antimouse B220 (clone RA3-6B2)
monoclonal antibodies were purchased from PharMingen (San Diego, CA).
Murine anti-alpha-smooth muscle actin monoclonal antibody (clone 1A4)
and a rabbit antihuman von Willebrand factor (factor VIII-related
antigen) polyclonal antibody were purchased from DAKO (Carpinteria,
CA). Mouse antirat IFN- (clone DB-1, cross-reactive to mouse)
monoclonal antibody was a gift of Dr D.S. Finbloom (Division of
Cytokine Biology, Center for Biologics Evaluation and Research,
Food and Drug Administration, Bethesda, MD). bFGF was purchased from
R&D Systems (Minneapolis, MN).
Treatment with antiasialo GM1 antibody and in vivo Matrigel assay.
BALB/c nude mice were injected intraperitoneally (IP) with rabbit
antiasialo GM1 antibody (1 mg/mouse; WAKO) and C57BL/6 nude mice were
injected IP with purified monoclonal anti-NK1.1 antibody (clone PK136,
100 µg/mouse) 1 day before Matrigel injection, and the same treatment
was repeated on day 3 after Matrigel injection. Antiasialo GM1 or anti
NK1.1 antibodies were also mixed with Matrigel and injected
subcutaneously. The Matrigel assay was performed as described
previously.16,17 In brief, Matrigel (liquid at 4°C) was
mixed with medium alone; with 150 ng/mL bFGF (R&D system); with bFGF
(150 ng/mL) plus 100 ng/mL IL-12; with bFGF (150 ng/mL) plus 250 µg/mL antiasialo GM1 (BALB/c mice) or 50 µg/mL anti-NK 1.1 (C57BL/6 mice) antibodies; or with bFGF (150 ng/mL) plus IL-12 (100 ng/mL) plus antiasialo GM1 (BALB/c mice, 250 µg/mL) or anti-NK 1.1 (C57BL/6 mice, 50 µg/mL). A 0.5-mL quantity of the Matrigel mixture
was injected subcutaneously into the midabdominal region of BALB/c or
C57BL/6 nude mice. Five mice were injected with each mixture. At body
temperature, Matrigel polymerizes to form a plug. After 7 days, the
animals were killed, Matrigel plugs were removed together with the
abstract epidermis and subepidermis, fixed in 10% neutral buffered
formalin solution (Sigma Chemical Co, St Louis, MO), and then embedded
in paraffin. All tissues were sectioned (5-µm thickness) and mounted
onto slides for further staining.
Histologic sections from Matrigel plugs were stained with Masson's
trichrome or esterase by standard techniques. Quantitative analysis of
angiogenesis in Matrigel plugs used a computerized semiautomated
digital analyzer (Optomax, 40-10 System, Hollis, NH) essentially as
described.17 In brief, the instrument is adjusted to
measure the total area occupied by cells within a circular area
measuring 1.26 × 105 µm2 of the Matrigel
plug. For each plug, 12 to 15 separate fields (each field reflecting a
circular area of 1.25 × 105 µm2) are
evaluated; the total plug area measures 12 to 18 × 106
µm2. The fields are randomly selected from each plug, and
the operator is blind to the experimental design. The average area
occupied by infiltrating cells/1.26 × 105
µm2 Matrigel field is calculated. Results are expressed
as the mean area occupied by cells per Matrigel field (±SD) of five
replicate plugs from five distinct mice.25
Isolation of RNA and RT-PCR.
Total cellular RNA was isolated form Matrigel plugs by Trizol
(GIBCO-BRL, Gaithersburg, MD). RNA 4 µg was reverse-transcribed using
Superscript Preamplification System for First Strand cDNA Synthesis
(GIBCO-BRL), according to the manufacturer's instructions. The
resultant cDNA was diluted with H2O to a final volume of
200 µL. For RT-PCR analysis, cDNA equivalents to 80 ng of total RNA were subjected to PCR amplification in a 50-µL reaction mixture containing 20 mmol/L Tris-HCl (pH 8.5), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 200 µmol/L of each dNTP, 2.5 U Taq DNA polymerase
(GIBCO-BRL), and 0.2 µmol/L of each primer (Table
1).
Immunohistochemistry.
Paraffin-embedded tissue sections were deparaffinized twice in xylene
and twice in 100% ethanol. Tissue sections were further incubated with
3% hydrogen peroxide in methanol, and then rehydrated through graded
ethanol washes (100%, 90%, 70%, and 50%) followed by Tris-buffered
saline (TBS). Sections were either treated with 1×
trypsin (Sigma) for 10 minutes, or immersed in 10 mmol/L citrate buffer
and heated in a microwave pressure cooker (Nordic Ware, Minneapolis,
MN) at maximum power (800 W) for 25 minutes. Sections were then blocked
with 3% goat serum in TBS, followed by incubation (overnight at 4°C)
with primary antibody (undiluted SH34 hybridoma culture supernatant for
antiasialo GM1; 1:200 dilution of rabbit antimurine IP-10 antiserum;
1:50 dilution of purified rabbit anti-von Willebrand factor [factor
VIII-related antigen]; 1:200 dilution of rat antimouse B220
monoclonal antibody; and 0.25 µg/mL mouse anti-alpha smooth muscle
actin monoclonal antibody). After washing, biotinylated goat
antirabbit, goat antirat, or horse antimouse secondary antibody (2 µmol/L/mL; Vector Labs, Burlingame, CA) was applied followed by
VECTASTAIN ABC peroxidase complex (Elite ABC-kit; Vector
Labs). The sections were developed using peroxidase 3,3'-diaminobenzidine (DAB) substrate and then counterstained with
hematoxylin. Double staining for asialo GM1 and von Willebrand factor
was performed by first staining with antiasialo GM1 monoclonal antibody
as described earlier using DAB substrate (color brown) followed by
staining with anti-von Willebrand factor as described earlier using
Vector VIP substrate (color purple). No counterstaining was done after
double staining. All sections were mounted, dehydrated, and examined by
light microscopy.
Purification and culture of murine cells.
Mouse aortic endothelial cells were obtained from aorta of C57BL/6 nude
mice using 1 mg/mL collagenase (Worthington, Biochemical Corp,
Freehold, NJ). The cells were cultured in endothelial cells' growth
medium (GIBCO) supplemented with 15 µg/mL of endothelial cell growth
supplement (ECGS) (Sigma). Cells from passages 4 or less were used for
cytotoxicity assay. By immunocytochemical staining, greater than 90%
of the cells were positive for the endothelial cells' marker von
Willebrand factor (factor VIII-related antigen).
Mouse splenocytes were prepared from spleens of littermate C57BL/6 nude
mice. For NK-cell enrichment, the cells were cultured in RPMI 1640 at 5 × 106/mL in the presence of 500 IU/mL of IL-2 (Chiron,
Emeryville, CA) for 6 days.26 At completion of cultures
with IL-2, more than 95% of the cells were positive for asialo GM1 by
flow cytometry. One day before cytotoxicity was tested,
NK-cell-enriched splenocytes were washed free of IL-2 and incubated
with medium alone, medium plus murine IFN- (200 ng/mL), murine IL-12
(10 ng/mL), or murine IL-12 plus antibody to rat IFN- (BD-1, 5 µg/mL) for 24 hours. Cells were collected, washed, and suspended at
the desired concentrations for cytotoxicity assay.
Cytotoxicity assay.
Cytotoxicity assays were performed in 96-well flat-bottom plates
(Becton Dickinson, Lincoln Park, NJ). Mouse aortic
endothelial cells (5 × 103) were added to each well
and incubated overnight. Subsequently, the cells were labeled with 2 µci of Na2 51CrO4 (New England
Nuclear, Boston, MA) per well for 16 hours. After the labeled cells
were washed, NK-cell-enriched splenocytes were added to each well at
effector-to-target (E:T) ratios of 120:1 in a final volume of 100 µL/well. After a 4-hour incubation at 37°C in a humidified 5%
CO2 atmosphere, the plates were centrifuged for 4 minutes
at 100 × g. The supernatants, harvested using a Skatron-Titertek harvester (Skatron, Sterling, VA), were counted in an
automated gamma counter (Pharmacia, Piscataway, NJ).
Maximum release of 51Cr was obtained by lysis of the cells
in 2% sodium dodecyl sulfate (SDS). Spontaneous 51Cr
release was determined in wells containing labeled target cells incubated with medium alone. Spontaneous release was less than 15% of
maximum release in all experiments. Each condition was tested in
quadruplicate wells. Percent specific 51Cr release was
calculated by using the following equation: Percent specific release: = ([Experimental Release  Spontaneous Release] ×100)/Maximum
Release Spontaneous Release.
Mouse tumor model.
BALB/c nude mice, 6 to 8 weeks of age, received 400 rad (1 rad = 0.01
Gy) total-body irradiation and 24 hours later were injected subcutaneously in the right abdominal quadrant with 107
exponentially growing human Burkitt lymphoma cells (CA49 cell line)27 in 0.2 mL RPMI medium 1640. Tumor size was
estimated (in square centimeters) twice weekly as the product of
two-dimensional caliper measurements (longest perpendicular length and
width). Test mice bearing Burkitt tumors ( 0.2 cm2 in
size) were injected subcutaneously next to the tumor daily for 34 days
with either murine IL-12 (200 ng/mL) or formulation buffer (saline
containing 50 mg/mL human serum albumin and 5 mg/mL mannitol);
the total injection volume was 100 µL. The care and use of mice was
in accordance with NIH guidelines.
| |
RESULTS |
NK-cell infiltration at sites of IL-12-inhibited vascularization.
When Matrigel, a basement membrane extract that is liquid at 4°C but
rapidly solidifies at body temperature, is inoculated with bFGF
subcutaneously into athymic mice, an angiogenic response is induced
within 5 to 7 days.17 Histologically, bFGF-impregnated Matrigel plugs, but not Matrigel-alone plugs, reveal the presence of
capillary vessels containing red blood cells, in conjunction with
tubular structures resembling primordial vessels, and many isolated
cells. When IL-12 is present in the Matrigel plugs in conjunction with
bFGF, the angiogenic response induced by bFGF is markedly
reduced.16 Since IL-12 is known to directly activate T and
NK cells and to indirectly promote their migration, through stimulation
of IFN- and the IFN--inducible chemokine IP-10,18 we
looked for the presence of NK cells in Matrigel plugs containing IL-12.
Plugs of Matrigel alone, Matrigel impregnated with bFGF (150 ng/mL)
showing evidence of neovascularization, and Matrigel impregnated with
bFGF (150 ng/mL) plus murine IL-12 (100 ng/mL) showing evidence of
profound (>80%) inhibition of bFGF-induced neovascularization by
IL-12, were stained with a monoclonal antibody to asialo GM1. In a
representative experiment (Fig 1), asialo GM1-positive cells were identified in sections from Matrigel plugs impregnated with bFGF plus IL-12, but generally not from Matrigel plugs
impregnated with bFGF or from Matrigel-alone plugs. Most of the
strongly positive cells localized at the margins of the plug and at the
interface between Matrigel and mouse tissues (epidermis and
subepidermis). Since NK cells, and to a lesser extent monocytes, express asialo GM1,28,29 these results suggested that NK
cells may localize in proximity and within IL-12-impregnated Matrigel plugs displaying histologic evidence for IL-12-inhibited
neovascularization.
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| Fig 1.
Immunohistochemical analysis of asialo
GM1-positive cells infiltrating and surrounding Matrigel plugs.
Paraffin-embedded sections from Matrigel plugs alone or Matrigel plugs
impregnated with bFGF, or bFGF + IL-12 were stained with a monoclonal
antimouse asialo GM1 antibody, counterstained with hematoxylin, and
examined by light microscopy. Original magnification ×40.
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We wanted to define further the cellular composition of Matrigel plugs
stimulated with bFGF alone or in conjunction with IL-12. Using an
antibody against von Willebrand factor (factor VIII-related antigen)
that identifies endothelial cells, bone marrow megakaryocytes, and
platelets (due to their storage of von Willebrand factor), we found
most of the nucleated cells to be immunopositive in both control and
IL-12-induced plugs (Fig 2). This result
provided evidence that most cells in bFGF-stimulated Matrigel plugs
with or without IL-12 are of endothelial-cell origin. It should be noted that the total number of immunopositive cells infiltrating the
bFGF plus IL-12-induced plugs was lower than in bFGF-induced plugs,
reflecting inhibition of angiogenesis by the cytokine. Several cells in
control (bFGF) and occasional cells in IL-12-induced plugs (bFGF plus
IL-12) displayed staining for the alpha-smooth muscle isoform of actin
that identifies smooth muscle cells of vessels and other parenchymas
(Fig 2). In addition, rare cells in control and IL-12-induced plugs
were esterase-positive, displaying a red staining that identifies cells
of monocyte/macrophage lineage (Fig 2). Staining for B220, an antigen
expressed on B cells and subsets of activated NK cells,30
identified several positive cells at the margins of IL-12 plus
bFGF-induced plugs and at the interface between the plug and the
surrounding mouse tissues, but only rare B220-positive cells in control
Matrigel plugs (bFGF, Fig 2). Because the pattern of B220 staining was
similar to the pattern with antiasialo GM1 antibody, and because IL-12
is not known to directly stimulate B cells, we concluded that activated NK cells are likely to contribute most of the B220-positive cells in
IL-12 plus bFGF-induced plugs.
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| Fig 2.
Histochemical analysis of cells infiltrating Matrigel
plugs. Paraffin-embedded sections from Matrigel plugs induced by either
bFGF alone or in conjunction with IL-12 were stained with rabbit
anti-von Willebrand factor, mouse anti-alpha smooth
muscle actin monoclonal antibody, or rat antimouse B220
monoclonal antibody and counterstained with hematoxylin. Staining for
esterase used conventional methods. Original magnification ×40.
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Spatial relationships between endothelial cells and NK cells
infiltrating Matrigel plugs were examined. Using double staining for
asialo GM1 (dark brown) and von Willebrand factor (purple, Fig
3), NK cells were identified
at the periphery of the plug in close proximity to endothelial cells
present as isolated cells or less frequently in tubular structures.
Together, these results provided evidence that IL-12 stimulates NK-cell
migration to the margins of bFGF-induced Matrigel plugs, where
endothelial cells are also recognized.
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| Fig 3.
Cell identification and localization by double staining
for asialo GM1 and von Willebrand factor. Immunohistochemical analysis
of a paraffin-embedded section from a Matrigel plug stimulated with
bFGF + IL-12 using mouse monoclonal antibody to asialo GM1 and a
rabbit anti-von Willebrand factor antiserum. Asialo GM1-positive cells
(dark brown) are indicated by arrows; von Willebrand factor positive
cells are purple. Original magnification ×40.
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NK-cell requirement for angiogenesis inhibition by IL-12.
To dissect a potential role for NK cells as mediators of angiogenesis
inhibition by IL-12, we used two reagents that can suppress NK-cell
function: antiasialo GM1 antibody31,32 and anti-NK1.1 antibody.33 Groups of five athymic mice (BALB/c or C57BL/6) were first inoculated IP with antiasialo GM1 antibody (BALB/c, 1 mg/mouse) or anti-NK1.1 antibody (C57BL/6, 100 µg/mouse), and 24 hours later were injected subcutaneously (0.5 mL total injection volume) with Matrigel plus bFGF (150 ng/mL) plus antiasialo GM1 antibody (BALB/c, 250 µg/mL) or anti-NK 1.1 antibody (C57BL/6, 50 µg/mL), or with Matrigel plus bFGF (150 ng/mL) plus IL-12 (100 ng/mL)
plus antiasialo GM1 antibody (BALB/c, 250 µg/mL) or anti-NK1.1 antibody (C57BL/6, 50 µg/mL). On day 3 after the injection of Matrigel, the mice were injected again IP with antiasialo GM1 antibody
(BALB/c, 1 mg/mouse) or anti-NK1.1 antibody (C57BL/6, 100 µg/mouse).
Groups of five control mice received subcutaneous injections (0.5 mL
total injection volume) or Matrigel alone, Matrigel plus bFGF (150 ng/mL), or Matrigel plus bFGF (150 ng/mL) plus IL-12 (100 ng/mL). All
plugs were removed on day 7, processed for histology, and angiogenesis
quantified by a semiautomated digital analyzer set to measure areas
occupied by cells on Matrigel sections. The results of four experiments
using antiasialo GM1 antibodies and of one experiment using NK 1.1 antibody are listed in Table 2. In both
sets of experiments, bFGF stimulated angiogenesis as documented by a
19- and 23-fold increase in the area occupied by cells on Matrigel
sections, and IL-12 inhibited this bFGF-induced angiogenesis by 67.9%
and 76.9%. Antiasialo GM1 and anti-NK 1.1 antibody treatment reversed
by 92.6% and 76.9%, respectively, the inhibition of angiogenesis by
IL-12, but had little effect on the stimulation of angiogenesis by
bFGF. These results demonstrated that neutralization of NK-cell
function reduces significantly the ability of IL-12 to act as an
inhibitor of bFGF-induced angiogenesis in vivo, and strongly suggested
that NK cells can mediate angiogenesis inhibition by IL-12.
Histologic sections depicting representative Matrigel plugs derived
from the five experimental groups are depicted in Fig 4. As shown,
plugs with Matrigel alone (Fig
4A) characteristically contained only few infiltrating cells at the margin of the plug, while
bFGF-impregnated plugs (Fig 4B) contained abundant infiltrating cells,
often organized to form tubular structures or capillaries containing
red blood cells. Plugs containing bFGF and IL-12 (Fig 4C) usually
displayed cells confined to the margin of the plug, but not reaching
deeper in the plug. bFGF-impregnated plugs from mice treated
systemically and locally with antibody to asialo GM1 (Fig 4D) were
similar in morphology to bFGF-impregnated plugs from animals not
treated with the antibody (Fig 4B). In addition, bFGF plus IL-12
impregnated plugs from mice treated with antibody to asialo GM1 (Fig
4E) contained numerous cells, often organized in tubular structures and
capillaries, and thus differed substantially from bFGF plus
IL-12-treated plugs in mice not treated with the antibody (compare Fig
4E and C), resembling instead plugs containing bFGF alone (compare Fig
4E and B). Thus, the angiogenic process induced by bFGF appeared
to be only modestly reduced by IL-12 when NK cells were
functionally impaired.
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| Fig 4.
Representative photomicrographs depicting the
effects of antiasialo GM1 antibody on inhibition of bFGF-induced
neovascularization by IL-12. Female BALB/c mice were injected
subcutaneously with Matrigel alone (A), Matrigel impregnated with bFGF
alone (B), bFGF + IL-12 (C), bFGF + antiasialo GM1 antibody (D), or
bFGF + antiasialo GM1 antibody + IL-12 (E). Matrigel plugs were
removed 7 days after injection and were processed for histology. The
sections were stained with Masson's trichrome. Original magnification
×20.
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Pathway of NK-cell recruitment and activation.
Since the data above demonstrated that IL-12 requires NK cells to act
as an inhibitor of angiogenesis, and that NK cells are present at sites
of inhibited neovascularization by IL-12, we examined potential
mechanisms by which NK cells might mediate the angiostatic effect of
IL-12. It is known that IL-12 is a potent inducer of
IFN-4 and IP-10, an IFN--inducible chemokine that acts as a potent chemotactic factor for activated T and NK
cells.18,34,35 It was thus possible that in the nude mouse
that is T-cell-immunodeficient, IFN- and IP-10 stimulation by IL-12
might account for the presence of activated NK cells at the sites of
inhibited vascularization by IL-12. We therefore looked for evidence of
IFN- and IP-10 expression in Matrigel plugs removed from nude mice
displaying evidence of an angiostatic effect by IL-12. Using RT-PCR
analysis applied to total RNA extracted from bFGF-induced Matrigel
plugs, we found the PCR products of IFN- and IP-10 amplifications to be more abundant from Matrigel plugs impregnated with IL-12 than without it (representative results from duplicate plugs shown in Fig
4). By contrast, in antiasialo GM1-treated animals, the amplification
products of IFN- and IP-10 from plugs induced or not induced with
IL-12 were similar in magnitude, and less abundant than the
corresponding PCR products from Matrigel plugs impregnated with IL-12
in the absence of antibody (representative results shown in Fig
5). These results suggested that IL-12
induces expression of the IFN- and IP-10 genes at the Matrigel plug
site, and that this effect of IL-12 requires the presence of functional
NK cells.
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| Fig 5.
RT-PCR analysis of IP-10 and IFN- expression in
Matrigel plugs. mRNA was prepared from duplicate sets of Matrigel plugs
impregnated with bFGF alone, bFGF + IL-12, bFGF + antiasialo GM1
antibody, or bFGF + IL-12 + antiasialo GM1 antibody,
reverse-transcribed, and subjected to PCR amplification using specific
primers for murine IP-10 and IFN-. The amplified products were
electrophoresed through a 1.5% agarose gel.
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We wished to confirm these results and looked for the presence of IP-10
protein by immunohistochemistry (Fig 6, see page
1616). Using an antiserum to murine IP-10,
essentially no immunopositive cells were detected in Matrigel plugs
alone (Fig 6A), Matrigel plugs induced by bFGF (Fig 6B), or Matrigel
plugs induced by bFGF in mice treated with anti-NK 1.1 antibody (Fig
6D). By contrast, strong positive staining was detected in Matrigel
plugs impregnated with bFGF and IL-12, mostly localized at the
periphery of the plug (Fig 6C). Since most of the cells in these plugs
were identified as endothelial cells due to their staining for von
Willebrand factor (Fig 2), we concluded that endothelial cells
infiltrating the plugs are a likely source of IP-10 in the presence of
IL-12. After treatment with anti-NK 1.1 antibody, Matrigel plugs
impregnated with bFGF and IL-12 displayed occasional IP-10 faintly
immunopositive cells (Fig 6E). These results are consistent with those
from RT-PCR analysis, and demonstrate the presence of IP-10 at those
sites where angiogenesis is inhibited by IL-12.
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| Fig 6.
Immunohistochemical detection of murine IP-10 in Matrigel
plugs from C57BL/6 mice treated with or without anti-NK 1.1 monoclonal
antibody treatment. Paraffin-embedded sections from Matrigel plugs
alone (A); plugs impregnated with bFGF (B); plugs impregnated with
bFGF + IL-12 (C); plugs impregnated with bFGF + monoclonal anti-NK 1.1 antibody (D); and plugs impregnated with bFGF + IL-12 + monoclonal anti-NK 1.1 antibody (E) were stained with a
rabbit antimouse IP-10 antiserum and counterstained with hematoxylin.
Some IP-10-positive cells (brown) are indicated by arrows. Original
magnification ×40.
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To examine whether IL-12-induced NK cells at the Matrigel plug site
are activated, we looked for evidence of increased expression of
perforin and granzyme B, molecules that are involved in
NK-cell-mediated killing, and expression of which is increased with
NK-cell activation.36,37 As shown in representative results
(Fig 7), the RT-PCR products of perforin
and granzyme B amplifications were more abundant in RNA samples from
Matrigel plugs impregnated with IL-12 plus bFGF as opposed to plugs
containing bFGF alone, suggesting that IL-12 promotes NK-cell
activation in this system. As expected, Matrigel plugs from antiasialo
GM1-treated animals did not display evidence of IL-12-induced
expression of perforin and granzyme B genes (Fig 7).
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| Fig 7.
RT-PCR analysis of murine perforin and granzyme B
expression in Matrigel plugs. mRNA was prepared from Matrigel plugs
impregnated with bFGF alone, bFGF + IL-12, bFGF + antiasialo GM1
antibody, or bFGF + IL-12 + antiasialo GM1 antibody,
reverse-transcribed, and subjected to PCR amplification using specific
primers for murine perforin and granzyme B. The amplified products were
electrophoresed through a 1.5% agarose gel.
|
|
NK-cell accumulation and vascular damage in experimental Burkitt
tumors treated locally with IL-12.
The results presented above demonstrated that NK cells are required for
inhibition of angiogenesis by IL-12, and that NK cells expressing the
activation markers perforin and granzyme B accumulate at sites where
angiogenesis is inhibited by IL-12. We asked whether NK cells might
contribute to angiogenesis inhibition by IL-12 more directly than by
providing for IFN- and IP-10. Particularly, we looked for evidence
that activated NK cells might kill endothelial cells and disrupt
vascularization by this mechanism. To this end, we examined the effects
of IL-12 on a preexisting and yet rapidly evolving tumor vascular bed,
and selected a model in which human Burkitt tumors are reproducibly
established subcutaneously in athymic mice.27,38 As
expected,39 local inoculations of IL-12 (200 ng/d, 7 d/wk),
but not buffer alone, reduced Burkitt tumor growth (Fig 8A, see page
1616). Burkitt tumors treated locally with
IL-12 for 34 days displayed the characteristic central necrosis, extending to the epidermis (Fig 8B). Histologically, there was diffuse
evidence of vascular injury, characterized by intraluminal thrombosis
(Fig 8C) and intimal thickening in IL-12-treated, but not
buffer-treated tumors. Immunocytochemical staining for activated NK
cells using either anti-B220 monoclonal antibody (Fig 8D and E) or
staining with antiasialo GM1 antibody (not shown) showed intense focal
staining in IL-12-treated, but not buffer-treated Burkitt tumors. NK
cells localized almost exclusively within the boundary area that
separates live from necrotic tumor tissue. Here, NK cells were detected
surrounding capillaries (Fig 8D) and small arterioles (Fig 8E). RT-PCR
analysis demonstrated expression of perforin and granzyme genes
in IL-12-treated, but not control-treated tumors, suggesting the
presence of activated NK cells (not shown). These experiments
demonstrate that, when applied to a tumor vascular bed, IL-12 can
promote extensive vascular damage and tumor tissue necrosis. In
addition, IL-12 can induce the accumulation of activated NK cells at
those sites where tumor tissue necrosis usually progresses, particularly surrounding blood vessels.
View larger version (60K):
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[in a new window]
| Fig 8.
Vascular effects and NK-cell recruitment in experimental
Burkitt tumors treated locally with IL-12. (A) Effects of local diluent
(FB) or IL-12 inoculation (200 ng/mouse/d, 7 d/wk) on the growth of
Burkitt tumors established subcutaneously in nude mice. The results
reflect mean (±SD) tumor sizes of 4 animals/group. (B) Gross
morphology of a representative Burkitt tumor treated locally with IL-12
for 34 days (no magnification). (C) Microscopic morphology of a
representative Burkitt tumor treated locally with IL-12 for 34 days
depicting vascular pathology (original magnification ×40). (D, E)
Immunohistochemical detection of NK cells by the B220 monoclonal
antibody in representative Burkitt tumors treated locally with IL-12
depicting positive cell staining surrounding a capillary vessel with
discontinuous endothelium (D, original magnification ×40), and
positive cell staining surrounding a small arteriole (E, original
magnification ×40).
|
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NK-cell cytolysis of endothelial-cell targets.
We wished to test directly whether activated NK cells could kill
endothelial cells in an in vitro syngeneic system. For these experiments, we used NK-cell-enriched populations (95% positive for
asialo GM1) derived from spleens of C57BL/6 nude mice as effector cells, and activated them by 24-hour culture with IL-12 (10 ng/mL). As
targets, we used primary cultures of mouse aortic endothelial cells
(>90% positive for von Willebrand factor) obtained from C57BL/6 nude
littermates. In a representative 51Cr release experiment
(Fig 9), IL-12-treated NK-cell-enriched splenocytes exhibited strong cytolytic activity against aortic endothelial cells (63% killing above background). IFN- treatment alone increased NK-cell killing by 28% above background. A
neutralizing antibody to IFN- added in conjunction with IL-12 during
preculture reduced significantly (47% reduction) endothelial-cell
killing by NK cells stimulated with IL-12. These experiments
demonstrate that activated NK cells can kill endothelial-cell targets.
View larger version (13K):
[in this window]
[in a new window]
| Fig 9.
Endothelial-cell killing by IL-12-activated NK cells.
NK-cell-enriched splenocytes from C57BL/6 nude mice were first
incubated for 24 hours in medium alone, medium supplemented with IL-12
(10 ng/mL), IFN- (100 ng/mL) or a combination of IL-12 (10 ng/mL)
and an anti-IFN- antibody (5 µg/mL), and then tested for
cytotoxicity against 51Cr-labeled aortic endothelial cells
from littermate C57BL/6 nude mice at an E:T ratio of 120:1. Results are
expressed as percent specific cytotoxicity, and reflect the mean
(±SD) of four replicate cultures.
|
|
| |
DISCUSSION |
These studies demonstrate that NK-cell function is required for IL-12
to act as an inhibitor of angiogenesis. Using Matrigel plugs
impregnated with bFGF in an athymic mouse model, we found that
neutralization of NK-cell activity with antibodies to asialo GM1 or NK
1.1 antigens reduced by 77% to 93% the level of angiogenesis inhibition induced by IL-12. NK cells were readily demonstrable in
those sites where neovascularization was inhibited by IL-12, but not in
controls, and there was evidence that these NK cells were activated
because IFN-, as well as the lytic mediators perforin and granzyme
B, were locally expressed. The chemokine IP-10 was also expressed at
these sites, suggesting that it may serve to chemoattract NK cells at
the plug site. When applied to a preexisting, and yet rapidly evolving,
tumor vascular bed, IL-12 caused extensive vascular damage, tumor
tissue necrosis, and accumulation of activated NK cells surrounding
small tumor vessels. In vitro, IL-12-activated NK cells exerted
effective cytotoxicity against syngeneic endothelial cells. Together,
these results may explain how IL-12 can inhibit angiogenesis. In brief,
we believe that IL-12 stimulates the production of IFN- by the rare
NK cells present locally in the tissues, and IFN- produced by these
NK cells in turn stimulates IP-10 secretion by neighboring endothelial
cells, monocyte/macrophages, and other resident cells. IP-10
chemoattracts additional NK cells, and these cells contribute to the
secretion of additional IFN- and, secondarily, IP-10. In addition to
being locally recruited, the NK cells become activated by IFN-. Once
activated, NK cells can become more strongly cytolytic and kill
neighboring endothelial cells. As a result, neovascularization is
impaired. It should be noted that the current results derive from
experiments in athymic mice that are T-cell-immunodeficient. The
relative contribution of NK cells in the context of euthymic mice was
not examined here, and it is clear that CD8-positive T cells have been
found to mediate most of the antitumor effects of
IL-12.11-13
A number of reports have documented effective endothelial cells killing
in vitro by activated human allogeneic NK cells.40,41 We
have shown here that once activated in vitro by IL-12, murine NK cells
become strongly cytotoxic for primary cultures of syngeneic endothelial
cells. However, the extent to which NK-cell killing of endothelial
cells contributes to IL-12 inhibition of bFGF-induced angiogenesis in
vivo is presently unclear. The current experiments localized NK cells
at sites where angiogenesis was inhibited by IL-12, and detected
expression of the perforin and granzyme B genes, results that are
consistent with the presence of activated cytolytic NK cells. However,
we cannot exclude the involvement of soluble inhibitors of angiogenesis
produced by activated NK cells.
In an athymic murine tumor model, treatment of established tumors with
IP-10 over a period of 30 to 40 days produced extensive damage to the
tumor vasculature, including capillary wall fragmentation, intimate
thickening, and intravascular thrombosis.27 Similar extensive destruction of the established tumor vasculature was noted
with prolonged local treatment with IL-12.39 Here, we found
that tumors treated in this manner also displayed evidence of NK-cell
recruitment and activation at the tumor site, particularly at the
interface between viable and necrotic tumor tissue, surrounding small
vessels. Since Burkitt cells are resistant to NK
cytotoxicity,42 these results further support the notion
that IL-12 can initiate an NK-cell-mediated process leading to
extensive damage of established or developing vasculature.
Once activated with IL-2, NK cells can adhere to and lyse human
endothelial cells cultured in vitro.40,43 The destruction of endothelial cells by IL-2-activated NK cells was proposed to contribute to the toxicity of high-dose IL-2 treatment, particularly to
the capillary leakage syndrome.43,44 One might speculate that NK-cell killing of endothelial cells at a tumor site might directly contribute to the antitumor effects of IL-2 treatment by
reducing tumor vascularization. However, when tested for their effects
on bFGF-induced Matrigel neovascularization, IL-2 exhibited little or
no effect.45 Unlike IL-12, IL-2 is not known to directly or
indirectly promote NK-cell migration to a particular site. In the
absence of sufficient accumulation of activated NK cells at a site of
neovascularization, endothelial-cell killing may be insufficient to
cause inhibition of neovascularization. It is of interest that IL-15, a
cytokine functionally related to IL-2, stimulated
angiogenesis.45 Stimulation by IL-15 was attributed to a
direct effect of this cytokine on endothelial cells that express the
 ,  , and subunits of the IL-15 receptor and undergo rapid
protein phosphorylation in response to IL-15.45
For many inhibitors of angiogenesis, including fumagillin, angiostatin,
endostatin, platelet factor 4, and thrombospondin, the mechanisms of
action are presently unknown, but for others mechanisms have been
proposed.46 Apoptosis in endothelial cells engaged in
neovascularization was believed to represent the mechanism by which
antagonists of the integrin v3 inhibit
angiogenesis.47 Neutralization of a growth factor required
by endothelial cells for growth was the reason why a monoclonal
antibody to vascular endothelial growth factor could inhibit
neovascularization.48,49 Intravascular thrombosis induced
by a monoclonal antibody to a cell-surface domain of human tissue
factor was the cause of reduced blood supply in an experimental tumor
model.50 Inactivation of enzymes that degrade extracellular
matrix proteins thereby favoring endothelial-cell spread may contribute
to the inhibition of angiogenesis by inhibitors of
metalloproteinase.51,52 However, NK-cell cytotoxicity of
angiogenic endothelial cells have not been proposed previously as
effective mechanisms for regulation of tissue vascularization.
IL-12 has shown promise as an anticancer agent in a number of
experimental tumor models. The anticancer properties of IL-12 have
generally been attributed to its ability to boost host immunity, particularly the activity of cytotoxic T and NK cells.11-13
The observation that IL-12 is also an effective inhibitor of
neovascularization raised the possibility that this mechanism of action
might account for some of the antitumor activities of the cytokine,
even in the context of T-cell immunodeficiency.11,13 The
findings, described in this report, that NK-cell activation,
chemotaxis, and ultimately lysis of endothelial cells engaged in
neovascularization is a mechanism by which IL-12 may act as an
inhibitor of angiogenesis, confirm the central role of T and NK cells
as mediators of the biologic activities of IL-12 through their
secretion of IFN-. They further stress the important role of the
interplay between host immunity and tumor-cell biology.
| |
ACKNOWLEDGMENT |
The authors thank Drs J.A. Farber, A. Rosenberg, and R. Yarchoan for
their suggestions and critical review of the manuscript.
| |
FOOTNOTES |
Submitted August 11, 1998; accepted October 20, 1998.
Supported in part by an appointment to the Postgraduate Research
Participation Program at the Center for Biologics Evaluation and
Research administered by Oak Ridge Institute for Science and Education
through an Interagency Agreement between the Department of Energy and
the Food and Drug Administration.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
This is a US government work. There are no restrictions on its use.
Address reprint requests to Lei Yao, PhD, Division of Hematologic
Products, Center for Biologics Evaluation and Research, FDA, Building
29A, Room 2D06, 8800 Rockville Pike, Bethesda, MD 20892; e-mail:
YAO{at}A1.CBER.FDA.GOV.
| |
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Top
Abstract
Introduction