[CANCER RESEARCH56, 5150-5155. November 15. 19961
Cell Adhesion Molecules Mediate Radiation-induced Leukocyte Adhesion to the
Vascular Endothelium'
Dennis Hallahan,2 Jaya Kuchibhotla, and Charles Wyble
Departments ofRadiation
and Cellular Oncology ID. H., J. K.J and Surgery [C. WI. Pri:zker School of Medicine. University of Chicago. Chicago. Illinois 60637
ABSTRACT
Examples of radiation-induced
stem cell factor
The predominant early histological changes in irradiated tissues are
edema
and leukocyte
infiltration.
Cell adhesion
molecules
(CAMs)
are
required for the extravasation ofleukocytes from the circulation. To study
the role of CAMSin the pathogenesis of radiation-mediated inflammation,
we quantified the expression of P-selectin, E-selectin, intercellular adhe
sion molecule-i (ICAM-1), and vascular cell adhesion molecule-i glyco
proteins on the surface of irradiated human endothelial cells. We found
that E-selectin and ICAM-1 expression increased after irradiation,
whereas there was no increased expression of other cytokine-inducible
adhesion molecules (P-selectin or vascular cell adhesion molecule-i). We
found a dose- and time-dependent increase in radiation-induced expres
sion of both E.selectin
and ICAM-1. Furthermore,
the threshold
dose for
cytokines include TNF3-a, IL-l, and
(1 1—16). These
cytokines
mediate
inflammation
by
inducing synthesis of CAM within the vascular endothelium (re
viewed in Ref. 17). IL-l and TNF bind to receptors on leukocytes and
endothelial cells to activate the inflammatory response (18). For
example, TNF stimulates the expression of cell adhesion molecules on
endothelial cells, which mediate emigration of leukocytes from the
circulation. CAMs expressed on the surface of vascular endothelial
cells include the selectin family (E-selectin and P-selectin) and the
immunoglobulin superfamily (e.g., ICAM-l). These CAMs interact
with their respective counter-receptors on leukocytes to initiate in
flammatory cell extravasation (reviewed in Ref. 17). E-selectin and
P-selectin are not expressed on the surface of unstimulated endothelial
E-selectin expression was 1 Gy, whereas the threshold dose for ICAM-1
cells. These
synthesis
irradiated
thelium after stimulation by cytokines. Following this primary adhe
was 5 Gy of X-rays. Northern blot analysis of RNA from
endothelial cells demonstrated that ICAM-1 is expressed at 3-6
sion of neutrophils
h following irradiation. No de novo protein synthesis was required for
increased ICAM-1 mRNA expression. The 1.1-kb segment of the 5' un
translated
region
chloramphenicol
ofthe
JCAM-1
gene was sufficient
acetyltransferase
reporter
gene
for X-ray
induction
expression.
We
of
meas
induction.
Radiation-mediated
leukocyte
adhesion
was pre.
vented by anti-ICAM-1 blocking antibodies. These data indicate that
ICAM-1 participates in the inflammatory response to ionizing radiation.
Moreover, radiation induction of these CAMs occurs in the absence of
tumor
necrosis
factor
and
interleukin
1 production.
An inflammatory-like response, in part, contributes to the acute and
subacute sequelae of radiation therapy. For example, histological
in radiation-mediated
pneumonitis
and pericarditis
have in
flammatory components. Studies of radiation-induced
tissue pathol
ogy noted
as the initial
neutrophil
infiltration
of irradiated
tissues
histological change prior to organ damage (1—4),and neutrophils are
known to bind to irradiated endothelial cells in vitro (5, 6). Normal
tissues respond to irradiation with increased adhesion of leukocytes to
the endothelium (7). For example, increased adherence of neutrophils
to endothelial cells occurs during acute pulmonary radiation injury
(3). Margination of neutrophils in the vasculature and infiltration of
the perivascular region are observed after irradiation or reactive
oxygen species (reviewed in Refs. 4, 6, 8, and 9). Our objective of this
study was to determine the mechanisms by which ionizing radiation
mediates leukocyte adhesion to the vascular endothelium.
Cytokines are proteins released by irradiated tissues and are impli
cated in the acute phase response to ionizing radiation (10, 11).
Received 4/5/96; accepted 9/17/96.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was
funded
by
NIH
Grant
CA58505,
the
Chicago
Tumor
Institute,
and
whom
Oncology,
requests
for
reprints
be
addressed,
at Department
of
inflammatory
cells
of endothelial
cells, and that E-selectin
gene expression
ated
mice
( 1 1, 25). In the present
study,
we found
that
ICAM- 1
protein is expressed on irradiated vascular endothelial cells. There was
no increase in the other cytokine-inducible CAMs, VCAM-1, or
P-selectin. Furthermore, radiation induction of E-selectin and
ICAM-l occurs immediately and without de novo protein synthesis.
These findings indicate that radiation-induced expression of E-selec
tin and ICAM-l is independent of radiation-induced cytokine produc
tion. We found that adherence of leukocytes to these irradiated endo
thelial cells was prevented by anti-ICAM-l antibody. The importance
of these findings is that ICAM-l mediates emigration of neutrophils
from the vasculature and migration of these inflammatory cells into
tissue. ICAM- 1 blocking
agents,
therefore,
preventing the inflammatory-like
hum toradiation
therapy.
MATERIALS
represent
a novel means of
response of the vascular endothe
AND METHODS
Endothelial Cell Cultures. HUVEC cultures were preparedfrom fresh
(<24
h of age) human
umbilical
veins transported
to the laboratory
in sterile
buffer at 4°C,as we have described (24). The veins were cannulated, filled
the
Radiation
MC 1105, 5841 S. Maryland, University of Chicago, Chicago, IL 60637.
Phone: (312) 702-2526; Fax: (312) 702-0610.
endothelium,
to the endo
is regulated through the activation of NFkB (24). Furthermore, the
ICAM-l mRNA level is increased in the brains and dermis of irradi
3 The
should
to the vascular
of leukocytes
of leukocytes (23). Neutrophils then migrate into the tissue region of
highest adhesiveness, which is regulated by ICAM-l expression (17).
Monoclonal antibodies directed against ICAM-l inhibit transendothe
hal migration of neutrophils (21, 22), indicating that ICAM-l is
required for this migration.
We studied the expression of CAMs in irradiated human endothe
hal cells. We have shown previously that E-selectin is induced by
Center for Radiation Therapy.
2 To
the adhesion
extravasate and migrate into the inflammed tissue (reviewed in Refs.
17 and 19). Extravasation of leukocytes from the vasculature requires
ICAM-l, which is expressed on the surface of endothelial cells in
X-irradiation
INTRODUCTION
changes
initiate
response to inflammatory
cytokines (20—22). ICAM-l
interacts with
counter-receptors
on the surface of leukocytes to mediate emigration
ured whether ICAM-1 mediates adhesion of leukocyte to the irradiated
endothelium and found that leukocyte adhesion occurred concurrently
with ICAM-1
selectins
abbreviations
used
are:
TNF,
tumor
necrosis
factor;
IL,
interleukin;
CAM,
cell
adhesion molecule; ICAM, intercellular adhesion molecule; NFkB, nuclear factor-KB;
VCAM, vascular cell adhesion molecule; HUVEC, human umbilical vein endothelial cell;
CAT, chloramphenicol acetyltransferase.
S 150
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
X-RAY-INDUCEDCELL ADHESION MOLECULES
with 0.2% collagenase, and incubated at 37°Cfor 15 mm. Cells were flushed,
HL-60 cells were labeled with 51Cr according to methods described previously
and complete medium was added, followed by centrifugation at 2000 rpm for
5 mm. The cell pellet was resuspended and maintained in M199 with 10%
FCS, 10% human serum, and penicillin/streptomycin/AmphotericinB solution
(5, 33, 34). 51Cr-labeled HL-60 cells (106) were added 4 h after irradiation and
(Sigma Chemical Co.) on gelatin-coated
in 5% CO2. The purity of endothelial
@
(0.2%) tissue culture dishes at 37°C
cell cultures
was verified
by staining
for
incubated
with rocking
for 30 mm. Cultures
whereas
cells exposed
to identical
von Willebrand factor. Confluent cells were harvested with 0. 1% collagenase,
0.01% EDTA, and subcultured at a ratio of 1:3. HUVECs were used at third
served as negative control.
passage;
above and treated
this reduced
the number
of passenger
cells and allowed
for uniform
were then washed
three times, and
adherent HL-60 cells were quantified by scraping and scintillation counting for
5tCr. IL-l (10 ng/ml)-stimulated HUVECs served as a positive control,
conditions
without
irradiation
or cytokines
During antibody-blocking studies, HUVECs were irradiated as described
with ICAM-l
blocking
antibody
(Becton
Dickinson:
1:50) at
expression of CAMs.
20h afterirradiation.Cultureswerethenwashed,
andHL-60cellswereadded
The 3B-I I endothelial cell line was derived from a solid tumor in a nude
mouse injected with SVET4-l0. These murine endothelial cells were cloned by
limited dilution (26, 27). This cell line expresses the cell surface MHC class 1
51Crquantification, and light microscopy. Experiments were repeated three to
four times, and the mean and SE were determined. Statistical significance was
antigen,
determined
H2K,
maintained
VCAM-l,
ICAM-l,
in 90% DMEM
and SP4O-T
with high glucose
antigen.
3B-l 1 cells
were
(4.5 gIl), 10% heat-inactivated
Endothelial
collagenase, 0.01% EDTA, and 0.25% BSA and pelleted in polystyrene tubes.
Cells were then incubated with primary IgGI antibody (mouse antihuman
P-selectin,
VCAM-l,
ohs, MN, and Becton
and ICAM-l;
Dickinson,
R&D Systems,
Inc., Minneap
San Jose, CA) for 20 mm at 4°C. The cells
conjugated secondary antibody (goat antimouse IgGl ) for 20 mm at 4°C.The
cells
were
rinsed
in PBS
and fixed
in PBS
containing
0.01% paraformaldehyde. Nonspecific binding was evaluated with the use of
FITC-conjugated
secondary
antibody
alone
and with
a lymphocyte-specific
cell sorting
analysis
1) was
was used for quantification
cloned upstream
(Promega)
tion, which
created
cotransfected
after removal
the pBS-CAT
with a plasmid
of the CAT
coding
of the SV4O promoter
plasmid
containing
region of the pCAT
by BglII/StuI
(35). The pBS-CAT
a cytomegalovirus
diges
plasmid
promoter
was
linked to
the LacZ gene (1 @.tg)
and 12 @gof carrier DNA into HUVECs by use of
lipofection.
The medium
with Lipofectin
was changed
reagent
to optiMEM,
(Life Technologies,
and cells were transfected
Inc.) for 8 h, followed
medium, and incubation
overnight.
by rinsing,
Transfectants
were incubated for 16 h after transfection, followed by treatment with 10 Gy
(I Gy/min; GE Maxitron) of ionizing radiation or with IL-I (10 ng/ml). The
cells were harvested
quantified
by scraping
to normalized
was measured,
at 36 h and lysed. f3-Galactosidase
transfection
as we have described
efficiencies.
previously
Reporter
levels were
gene expression
(24, 3 1, 36). Experiments
were
repeated three to four times, and the mean and SE were determined. Statistical
first-step antibody, anti-CD10,which does not bind to endothelial cells.
Fluorescence-activated
analysis.
the addition of complete
were then rinsed with isotonic PBS, pelleted, and incubated with FITC
fluorescein-labeled
(—.
1162/+
promoter
Cells. Endothelialcells were irradiatedwith a GE MaxitronX-raygenerator,
as described previously (14). Cells were removed from flasks with 0.1%
E-selectin,
by
Analysis of Transcriptional Regulation. The ICAM-l promoter fragment
fetal bovine serum, and penicillin/streptomycin/Amphotericin B solution.
3B-I 1 cells were grown to 90% confluence for all experiments.
Quantification
of Cell Adhesion Molecules in Irradiated
30 mm after drug or antibodies.Cell adhesionwas quantified by cell counting,
of
significance
was determined
by y@analysis.
receptor expression of E-selectin on HUVECs. The Becton Dickinson FAC
Scan was used with Lysis II software. Forward and side scatter fluorescence
data identified 10,000 viable endothelial cells in each experimental group for
unlabeled cells, nonspecific antibody-labeled cells, and anti-CAM antibody
labeled cells. Fluorescence data were then accumulated on each group of
10,000 cells at 530 nm; the wavelength was emitted by FITC after treatment
with X-rays
or IL-I (10 ng/ml;
R&D Systems).
These
fluorescence
data were
expressed as histograms of events versus log fluorescence and analyzed in
comparison
to the autofluorescence
of unlabeled
cells as well as the fluores
RESULTS
Adhesion Molecule Expression in Irradiated Endothelial Cells.
To examine the effects of ionizing radiation on the expression of
CAMs, we exposed 3B-l 1 cells and HUVECs to doses of 1, 2, 5, 10,
and 20 Gy. Cells were then incubated with antibodies to E-selectin,
ICAM-l, and VCAM-l at 1, 2, 4, 8, 16, and 24 h. ICAM-l was
cence of baseline or anti-CAM-labeled cells as appropriate. The percentage of
expressed
cells expressing adhesion molecules was determined by quantification
25% of untreated 3B-ll cells. Increased expression of ICAM-l was
not observed before 20 h following treatment with X-rays or cyto
of the
number of cells demonstrating an increase in log fluorescence beyond that of
untreated control cells. Experiments were performed three to four times and the
mean and SE were calculated. Endothelial cell viability was determined at the
time of antibody incubation (4 h) by use of trypan blue dye exclusion.
Northern
Blot Analysis
of Irradiated
HUVECs.
HUVECs
were exposed
to 10 Gy (GE Maxitron X-ray generator) as described previously (14). RNA
was extracted with the single-step guanidinium thiocyanate-phenol-chloroform
method (28) following irradiation. RNA from nonirradiated cells and treated
cells were size-fractionated by 1% agarose formaldehyde electrophoresis.
Cycloheximide,
S @.tg/ml,and actinomycin
D, S
@g/ml,were added to HU
equal loading
of each lane. RNA gels were then transferred
(29, 30). We quantified
75 RNA (3 1) to verify equal loading
into lanes followed by autoradiography
screens.
RNA
levels
were quantified
to a nylon
of RNA
for 3 days at —85°C
with intensifying
by densitometry
HUVECs
of cells expressing
increased
treatment
2—3-fold (P < 0.05) following
and
ICAM-l
with S to 20 Gy at
24 h afterirradiation.Moreover,the percentageof cellsdemonstrating
E-selectin synthesis increased 20-fold at 4 h following exposure of
HUVEC to 2 Gy (P < 0.001). Expression of VCAM-l and P-selectin
proteins
was not increased
by ionizing
radiation
(Fig.
2), whereas
expression was induced by IL-I and TNF.
expression
expression
began to increase
at 2 h, peaked
at 4—6 h, and
remained
at baseline
levels until 16 h after irradiation,
and
peak expression occurred at 24 to 36 h following irradiation. HUVECs
were then irradiated with doses ranging from 0.5 to 50 Gy and assayed
at 4 or 24 h for study of the dose dependence of the X-ray-mediated
expression
as we have described
(32).
of cell adhesion
molecules.
E-selectin
expression
increased
at 4 h after exposure to 0.5 Gy and increased in a dose-dependent
Leukocyte Adhesion Studies. HL-60 and U937 cells express the counter
manner
receptor for ICAM- 1 binding and, therefore, serve as standards for assaying
Ieukocyte adhesion to stimulated endotheliai cells (5, 33, 34). 3B-l 1 cells were
grown to 90% confluence and irradiated with 10 Gy. Twenty-four h later,
U937 cells were added at a density
of 106/dish with rocking
for 30 mm at 36°C.
Cultures were then rinsed three times with PBS and rocking. Cell cultures were
scraped,
control
gradually returned to baseline at 20 h (Fig. 2). In contrast, ICAM-l
membrane (Genescreen Plus; DuPont NEN). Northern blots were hybridized to
a 32P-labeled 830-bp segment from the NarI and Asp-cut human ICAM-l
cDNA
of 30% of untreated
kines. As shown in Fig. 1, the percentage
E-selectin
VECs 1 h before irradiation. Ethidium bromide staining of the RNA demon
strated
on the surface
and leukocytes
were counted
by hemocytometer.
Cell counts
were
performed in three separate experiments, and the mean and SE were deter
mined. HUVECs were grown to 90% confluence and irradiated with 2—10
Gy.
up to 20 Gy, where a plateau
sion returned
to baseline
within
was reached.
20 h after
E-selectin
irradiation.
expres
In contrast,
ICAM- 1 expression was not increased at X-ray doses below S Gy, but
demonstrable
increases
occurred
at 24 h after treatment
with higher
doses. ICAM- 1 expression persisted at 48 h after irradiation (Fig. 2).
These data indicate that E-selectin is induced transiently after low
doses of irradiation, whereas
ation doses and is sustained.
ICAM-l
induction
requires
higher
5151
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
radi
@
@
@
@
.Do@
@5t@
I
@T.
@H ‘@
j
@.
X-RAY-INDUCED CELL ADHESION MOLECULES
HUVECs
L@
IL-i
x-ray
Control
Cl)@
0)
-
-
@
j
@
E1.@
=@
@
zi
;
r@,
,@,F;'
R@-k@
-@- i;I@
@,,
Fluorescence
3B-11 Cells
L@Control
x-ray
C@
ILl
Co
a)
‘@
@
@
11
w
—@
—@
1@
—
_@__
‘1
.
@!f
-@
____________
Fluorescence
Fig. 1. ICAM-l expression in irradiated endothelial cells. Endothelial cells were incubated with primary IgGl antibody (mouse antihuman ICAM-l) and FITC-conjugated secondary
antibody (goat antimouse IgGI) at 24 h after treatment. Shown are histograms of the number of endothelial cells expressing [CAM- I (M2 region) following no treatment (Control),
treatment with 10 Gy (x-ray), or IL-l (10 ng/ml).
TNF and IL-i Are Not Induced
in Irradiated
Endothelial
Cells.
ICAM-l induction by TNF and IL-l occurs in a time-dependent
manner that is similar to that observed in Fig. 2 (17). Because TNF
and IL-l are known radiation-inducible cytokines, we quantified these
proteins in the medium and in endothelial cells following irradiation.
We found no detectable levels of TNF or IL-i in cells before or after
irradiation. IL-l did induce TNF expression in endothelial cells.
Therefore, endothelial cells have the capacity to synthesize TNF but
not in response to X-rays. These findings are supported by the
observation that TNF and IL-I induce VCAM-l, whereas radiation
does not activate
expression
Radiation-mediated
mechanism
of this adhesion
molecule.
ICAM-1 mRNA Expression.
of radiation-mediated
ICAM- 1 expression,
HUVECs with 10 Gy, and RNA was extracted at 3, 6, 8, 10, and 12 h.
Northern blot hybridization to human ICAM-l cDNA probe revealed
a 2.5-fold increased ICAM-l mRNA expression at 3—8h (Fig. 3).
This increased
mine whether
expression
protein
returned
synthesis
to baseline
is required
at 10—12 h. To deter
before
radiation-mediated
ICAM-l mRNA expression, we treated HUVECs with the protein
synthesis inhibitor cycloheximide for 1 h before irradiation. Total
60-
cellular RNA was then extracted
was used for quantifying
mRNA
40-
Percent
Shiftin log
Fluorescence
at 6 h, and Northern blot analysis
expression
(Fig. 3). When endothe
hal cells were treated with cycloheximide,
increased.
When
cells were treated
expression of ICAM-l
with cycloheximide
followed
by
irradiation, superinduction of ICAM- 1 expression was observed. This
20
indicates
duction
I
that protein-synthesis
of ICAM-l
mRNA
inhibition
expression.
did not prevent
Inhibition
X-ray
of transcription
in
by
actinomycin D prevented X-ray induction of ICAM.
The promoter segment of the JCAM-1 gene was linked to the CAT
hours
Fig. 2. Time-dependent
To study the
we irradiated
increase in adhesion molecule synthesis after x-irradiation.
HUVECs were irradiated and fixed at the indicated times. Cells were then incubated with
reporter
gene and transfected
reagent;
Life Technologies,
into HUVECs
Inc.).
by lipofection
We found
a 3.1-fold
(Lipofectin
increase
in
primary IgGl antibody (mouse antihuman E-selectin, P-selectin, VCAM-l, or ICAM) and
FITC-conjugated secondary antibody (goat antimouse IgGl). Fluorescence-activated cell
sorting analysis was used for quantification of fluorescence intensity. Shown are the mean
(bars, SE) of the increase in the number of cells expressing P-selectin, VCAM-l,
CAT expression (P < 0.05) following irradiation of transfected en
dothelial cells (Fig. 4). These data, taken together with the inhibition
of mRNA expression by actinomycin D, suggest that ICAM-1 tran
ICAM-l, and E-selectin at the indicated time intervals after irradiation.
scription
is induced
by ionizing
radiation.
5152
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
X-RAY-INDUCEDCELL ADHESION MOLECULES
>1
Cu
_3
@
@
00
@
x-ray
.C
.C
C')
CO
.C
(5
)@c x
+
I-
C
>1
.C
(‘@1
0
I-
0
+
0
>-
<
0
,-
@4 I
Fig. 3. Northern blot analysis of JCAM-l gene expression in irradiated
HUVEC. HUVECs were irradiated, and total cellular RNA was isolated at
the indicatedtimesafterirradiation.Northemblotswerehybridizedto the
@
ICAM-l cDNA and 75. Cycloheximide (CYC) and actinomycin D (ACT)
were added 1 h before irradiation. Positive controls are endothelial cells
treated with TNF or IL-l.
28S—@@W!MØI@@
@
ICAM-I-
@
18S —
@
7S—
pBS-CAT(ICAM)
•@@*@—;
••
@.
. .@
*. @‘
--•.-‘w 5@t@w
@J)
@.
-
5..@ ..@
incubated with the anti-ICAM-l antibody demonstrated a reduction in
HL-60 cell binding that reflected baseline adhesion (P < 0.005).
However, the anti-ELAM-l blocking antibody did not prevent radia
tion-mediated leukocyte adhesion at 24 h. IL-l-treated HUVECs
incubated with the anti-ICAM-l antibody demonstrated an attenuation
of HL-60 cell binding.
Fold Induction
0123456
control
HUVEC
x-ray
5454 @g5*@
IL-I
DISCUSSION
Fig. 4. Transcriptional
activation of the ICAM-l promoter by ionizing radiation. The
Inflammatory
1.1-kb segment of the 5' untranslated region of the JCAM-I gene was cloned to the CAT
reporter gene and transfected into HUVECs by use of liposomes. Transfectants were
irradiated, and CAT enzyme activity is shown as the means (bars, SE) of three experi
ments.
Leukocyte Adhesion to Irradiated Human Endothelial Cells.
HL-60 cells and U937 cells serve as standards for assaying leukocyte
adhesion to stimulated endothelial cells (5, 33, 34). To determine
whether leukocytes adhered to irradiated endothelial cells, we added
U937 cells to 3B-1 1 cultures at 24 h after x-irradiation or treatment
with IL-l. U937 cell counts demonstrated a 4-fold increase in cell
adhesion to x.irradiated 3B-l 1 endothelial cells as compared to un
treated 3B- 11 cells (Fig. 5). HUVEC cells were grown to 90%
confluence and irradiated with 10 Gy. 51Cr-labeled HL-60 cells (106)
added 20 h after irradiation were quantified by scraping and scintil
lation counting. A 3.5-fold increase in HL-60 binding occurred
(P < 0.01) after irradiation (Fig. 5). IL-i stimulated HUVECs served
as a positive control and demonstrated a 5-fold increase in HL.60 cell
binding. HUVECs exposed to identical conditions, but without irra
diation
or cytokine
stimulation,
served
as negative
include
cytokines,
leukocytes,
and
the
sion of cytokines
such as TNF and IL- 1. These
cytokines
stimulate
endothelial cells, resulting in leukocyte adhesion and extravasation.
Adhesion molecules must be expressed on the surface of the vascular
endothelium before leukocyte extravasation can occur (20—22).Our
purpose in the present study was to determine whether adhesion
molecules are induced by x-irradiation of human endothelial cells. We
FoldIncrease
in leukocyte
adhesion
control.
Effects of antl-ICAM-1 Blocking Antibody on Leukocyte Ad
hesion to Irradiated Endothelial Cells. HUVECs were irradiated as
described above and treated with the anti-ICAM-l blocking antibody
(Becton Dickinson; 1:50) at 20 h after irradiation. Cultures were then
washed, and HL-60 cells were added 30 mm after antibodies. We
found that the anti-ICAM-l blocking antibody eliminated radiation
mediated leukocyte adhesion to the irradiated endothelial cells (Fig.
6). UntreatedHUVECsincubatedwith anti-ICAM-l antibodydem
onstrated no decrease in HL-60 cell binding. X-irradiated HUVECs
mediators
adhesion molecules that mediate leukocyte infiltration of the in
flammed tissue (reviewed in Ref. 17). We and others have studied the
role of cytokines in the pathogenesis of radiation injury ( 13, 14, 37).
These studies have shown that ionizing radiation induces the expres
Fig. 5. Increased adhesion of leukocyte cells to endothelial cells exposed to x
irradiation. 3B- 11 cells (A) and HUVECs (B) were treated with X-rays ( 10 Gy), IL- 1 (10
ng/ml), or sham irradiation (control). Twenty-four h after treatment, U937 (A) or HL-60
(B) cells were added with rocking for 30 mm. Cultures were then washed three times, and
adherent cells were counted. Shown are the means (bars, SE) of three experiments.
5153
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
X-RAY-INDUCED CELL ADHESION MOLECULES
interleukin-1
extent, IL- 1-a and glial fibrillary acidic protein mRNA were increased
in the brain after irradiation to doses below 7 Gy (1 1). ICAM-l
expression was increased by doses as low as 2 Gy. Pretreatment of
mice
Fold Increase
in leukocyte
adhesion at
24 hours
Fig. 6. Effects of anti-ICAM-l
blocking antibody on cell adhesion to irradiated
endothelial cells. HUVECs were treated with X-rays (10 Gy), IL-I (5 @zg/ml),
or sham
irradiation
@
(control). Twenty-four
h after treatment,
HUVECs were treated with no
with
dexamethasone
or pentoxiphyllin
suppressed
radiation
induced acute-phase gene expression either partially or completely
(1 1). Pharmaceuticals that block leukocyte adhesion to E-selectin or
ICAM-l may prevent radiation-mediated inflammation in normal
tissues. These findings may lead to more effective pharmaceuticals for
use in the treatment of acute and subacute sequelae of radiation
therapy.
Several findings support the hypothesis that CAM induction by
ionizing radiation is independent of cytokine production: (a) CAM
expression occurs in vitro in the absence of leukocytes or cytokines;
(b) CAM induction is more rapid (2—3h) than cytokine induction (6
h; Refs. 14, 32, and 41); (c) cytokine-inducible CAMs are not induced
antibody (U), anti-ICAM-I blocking antibody (0), or anti-ELAM-I antibody (@) for 30
by X-rays,
mm with rocking at 24 h after irradiation. HL-60 cells labeled with ‘Crwere added to
treated HUVECs. Cultures were then washed, and adherent cells were counted by
scintillation counting. Shown are the means (bars, SE) of three experiments.
detectable TNF or IL- I in the irradiated endothelial cells; (e) inhibi
tion of protein synthesis does not prevent the increase in expression of
either E-selectin or ICAM-l genes after irradiation. Taken together,
these findings indicate that CAM expression in the irradiated vascular
endothelium does not require the induction of cytokines.
The mechanisms by which X-rays induce adhesion molecules on
the endothelium have been studied previously (24). We have found
that the transcription factor NFkB is activated following irradiation of
endothelial cells. It has been reported that ICAM-l mRNA is depend
ent on NFkB activation (42, 43). NFkB is also activated by inhibition
of protein synthesis (44). We found that inhibition of protein synthesis
whereas
they are induced
by TNF or IL-l ; (d) we found no
found no IL-i or TNF produced by endothelial cells following irra
diation. These results are supported by the finding that TNF and IL-l
induced P-selectin and VCAM-l expression on the cell surface,
whereas radiation did not increase the expression of these CAMs.
Furthermore, we found that X-ray-mediated E-selectin gene induction
occurs without protein (e.g., cytokine) synthesis. Therefore, inhibition
of radiation-induced TNF is not sufficient to prevent X-ray-mediated
increases ICAM- 1 expression and results in superinduction
by X-rays.
leukocyte adhesion to the vascular endothelium, whereas blocking of
Superinduction also results from protein synthesis inhibition com
leukocyte binding to ICAM-i may be more effective.
bined with cytokine or phorbol ester treatment (45). Furthermore,
The present study revealed that the threshold dose for increased
expression of ICAM-l was 5 Gy. This differed from the 1-Gy thresh
others have shown that glucocorticoids attenuate ICAM- I expression
old dose for E-selectin synthesis in human endothelial cells (24). The
in the irradiated mouse brain (1 1). The mechanism
of steroid inhibi
importance of the difference in these threshold doses for ICAM-l and
tion of adhesion molecules involves the ligand-activated glucocorti
coid receptor, which represses RelA-mediated activation of the
E-selectin is that radiation-mediated pneumonitis occurs at a higher
frequency when large rather than small fractions of radiation are used
ICAM- 1 NFkB site (46). Transcriptional repression of NFkB, medi
(38, 39). For example, patients treated with total-body irradiation have
ated by glucocorticoid, is not caused by binding of glucocorticoid
a higher risk of pneumonitis when they are treated with single large
receptor to the ICAM-l NFkB element but by a physical interaction
doses of radiation as compared to multiple small doses (38). Similarly,
between the glucocorticoid receptor and RelA proteins (46). Several
lung cancer patients have a higher incidence of pneumonitis when
studies have shown that NFkB participates in X-ray-mediated tran
treated with larger daily fractions of radiotherapy. An alternative view
scriptional regulation (24, 44, 47, 48). Taken together, these findings
of these threshold doses is that ICAM- I induction is less likely to
indicate that radiation-activated NFkB may regulate CAM expression
through a cytokine-independent pathway.
participate in radiation-mediated inflammation than is E-selectin in
duction, because ICAM-l is not induced by conventional radiation
therapy doses. Previous studies have shown increased ICAM- 1
ACKNOWLEDGMENTS
mRNA levels after exposure of mouse brains to 2 Gy. However, 5 Gy
was required for ICAM. 1 mRNA expression in skin cultures ( 11, 25).
We thank Dr. S. W. Caughman for the ICAM-l promoter and Dr. Brian
Seed for the ICAM-l cDNA.
E-selectin and ICAM-l participate in leukocyte adhesion to the
irradiated endothelium. We found that leukocyte adhesion to the
irradiated endothelium increases at 24 h following irradiation. This
REFERENCES
time interval coincides with the onset of ICAM- 1 expression on the
I . Fajardo, L. F., and Stewart, J. R. Pathogenesis of radiation-induced myocardial
surface of endothelial cells. We have previously shown that adhesion
fibrosis. Lab. Invest., 29: 244—257, 1973.
of leukocytes to the irradiated endothelium occurs through the induc
2. Narayan, K., and Cliff, W. J. Morphology of irradiated microvasculature: a combined
in vivo and electron-microscopic study. Am. J. Pathol., 106: 47—62,1982.
tion of E-selectin, and that E-selectin blocking antibodies prevent this
3. Slauson, D. 0., Hahn, F. F., Benjamin, S. A., Chiffelle, T. L., and Jones, R. K.
adhesion (24). These findings are supported by the observation that
Inflammatory sequences in acute pulmonary radiation injury. Am. J. Pathol., 82:
549-572, 1976.
leukocytes adhere to the irradiated vascular endothelium in vivo (7). In
4. Hopewell, J. W., Calvo, W., Jaenke, R., Reinhold, H. S., Robbins, M. E., and
addition to the role of ICAM-l and E-selectin in leukocyte adhesion,
Whitehouse, E. M. Microvasculature and radiation damage. In: W. Hinkelbein, 0.
platelet/endothelial cell adhesion molecule has been shown to play a
Bruggmoser, H. Frommhold, and M. Wannenmacher (eds.), Recent Results in Cancer
Research, Vol. 130, pp. 1—16.Berlin: Springer-Verlag, 1993.
role in radiation-mediated leukocyte extravasation (40). There are,
5. Colden-Stanfield, M., Kalinich, J. F., and Gallin, E. K. Ionizing radiation increases
therefore, multiple mechanisms by which leukocytes adhere to the
endothelial and epithelial cell production of influenza virus and leukocyte adherence.
irradiated vascular endothelium.
J. Immunol.,153:5222—5229,
1994.
6. Dunn, M. M., Drab, E. A., and Rubin, D. B. Effects of irradiation on endothelial
The correlation between cytokine induction by radiation and the
cell-polymorphonuclear
leukocyte interactions. J. AppI. Physiol., 60: 1932—1937,
acute phase response has been exemplified best in the lung and brain.
1986.
For example, levels of TNF-a, IL-lO-/3, ICAM-l, and, to a lesser
7. Wu, N. Z., Ross, B. A., Gulledge, C., Klitzman, B., Dodge, R., and Dewhirst, M. W.
5154
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
X-RAY-INDUCEDCELL ADHESION MOLECULES
Differences in leucocyte-endothelium interactions between normal and adenocarci
noma bearing tissues in response to radiation. Br. J. Cancer, 69: 883—889, 1994.
8. Reinhold, H. S., Fajardo, L. F., and Hopewell, J. W. The vascular system. Adv.
Radiol. Biol.. 14: 177—226,1990.
9. Matzner,Y.,Cohn,M.,Hyam,E.,Razin,E.,Fuks,Z.,Buchanan,M.R.,Hans,T. A.,
Vlodavsky, I., and Eldor, A. Generation of lipid neutrophil chemoattractant by
irradiated bovine aortic endothelial cells. J. Immunol., 140: 2681—2685, 1988.
10. Rubin, P., Johnston, C. J., Williams, J. P., McDonald, S., and Finkelstein, J. N. A
perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis (see Corn
ments). Int. J. Radiat. Oncol. Biol. Phys., 33: 99—109, 1995.
11. Hong, J. H., Chiang, C. S.. Campbell, I. L., Sun, J. R., Withers, H. R., and McBride,
W. H. Inductionof acutephasegeneexpressionby brainirradiation.Int.J. Radiat.
Oncol. Biol. Phys., 33: 619—626, 1995.
12. Sherman, M. L., Weber, B. L., Datta, R., and Kufe, D. W. Transcriptional and
posttranscriptional regulation of macrophage-specific colony stimulating factor gene
expression by tumor necrosis factor. Involvement of arachidonic acid metabolites.
J. Clin.Invest.,85: 442—447,
1990.
13. Woloschak, G. E., Chang-Liu, C. M., Jones, P. S., and Jones, C. A. Modulation of
gene expression in Syrian hamster embryo cells following ionizing radiation. Cancer
Res., 50: 339—344, 1990.
14. Hallahan, D. E., Spriggs. D. R., Beckett, M. A., Kufe, D. W., and Weichselbaum,
R. R. Increased tumor necrosis factor a mRNA after cellular exposure to ionizing
radiation. Proc. NatI. Acad. Sci. USA, 86: 10104—10107, 1989.
15. Baker, W. H., Limanni, A.. Chang, C. M., Jackson, W. E., Seemann, R., and Patchen,
M.L.Comparisonof interleukin-la geneexpressionandproteinlevelsinthemurine
16.
17.
18.
19.
20.
spleen after lethal and sublethal total-body irradiation. Radiat. Res., /43: 320—326,
1995.
Ishihara, H., Tsuneoka, K., Dirnchev, A. B., and Shikita, M. Induction of the
expression of the interleukin-l j3 gene in mouse spleen by ionizing radiation. Radiat.
Res.,133:321—326,
1993.
Springer, T. A. Traffic signals for lymphocyte recirculation and leukocyte emigration:
the multistep paradigm. Cell, 76: 301—314,1994.
Fraker, D. L., Alexander, H. R., and Pass, H. I. Biologic therapy with TNF. System
administration and isolation perfusion. In: V. T. DeVita, S. Hellrnan, and S. A.
Rosenberg (eds.), Biologic Therapy of Cancer, pp. 329—346. Philadelphia: J. B.
Lippincott Co., 1995.
Fantone, J. C., and Ward, P. A. Polymorphonuclear leukocyte-mediated cell and
tissue injury: oxygen metabolites and their relations to human disease. Hum. Pathol.,
16: 973—978,1985.
Sligh, J., Jr., Ballantyne. C. M., Rich, S. S., Hawkins, H. K.. Smith, C. W., Bradley,
A., and Beaudet,A. L. Inflammatoryand immuneresponsesare impairedin mice
deficient in intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. USA, 90:
8529—8533,
1993.
21. Rothlein, R., Mainolfi. E. A.. and Kishimoto, T. K. Treatment of inflammation with
anti-ICAM-l. Res. Immunol., 144: 735—739,1993.
22. Luscinskas, F. W., Cybulsky, M. I., Kiely, J. M., Peckins, C. S., Davis, V. M., and
Gimbrone, M., Jr. Cytokine-activated human endothelial monolayers support en
hanced neutrophil transmigration via a mechanism involving both endothelial-leuko
cyte adhesion molecule-l and intercellular adhesion molecule- 1. J. Immunol., 146:
1617—1625,
1991.
23. Smith, C. W., Rothlein, R., Hughes, B. J., Mariscalco, M. M., Rudloff, H. E.,
Schrnalstieg, F. C., and Anderson, D. C. Recognition of an endothelial determinant
for CD18-dependent human neutrophil adherence and transendothelial migration.
J. Clin.Invest.,82: 1746—1756,
1988.
24. Hallahan, D. E., Clark, E. T., Kuchibhotla, J., Gewertz, B., and Collins, T. E-Selectin
induction by ionizing radiation. Biochem. Biophys. Res. Commun., 217: 784—795,
1995.
25. Behrends, U., Peter, R. U., Hintermeier-Knabe, R., Eissner, G., Holler, E., Bomkamm,
G.W.,Caughman,S.W.,andDegitz,K.Ionizingradiationinduceshumanintercellular
adhesion molecule-l in vitro. J. Invest. Dermatol., 103: 726—730,1994.
26. O'Connell, K. A., and Edidin, M. A mouselymphoid endothelialcell line immortal
ized by simian virus 40 binds lymphocytes and retains functional characteristics of
normal endothelial cells. J. Immunol., 144: 521—525,1990.
27. O'Connell, K., Landman, G., Farmer, E., and Edidin, M. Endothelial cells trans
formed by SV4O T antigen cause Kaposi's sarcomalike tumors in nude mice. Am. J.
Pathol., 139: 743—749,1991.
28. Chornczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem., 162: 156—
159, 1987.
29. Bevilacqua, M. P., Stengelin, S., Gimbrone, M. A. J., and Seed, B. Endothelial
leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to
complement regulatory proteins and lectins. Science (Washington DC). 243: 1160—
1165,1989.
30. Simmons, D., Makgoba, M. W., and Seed, B. ICAM, an adhesion legand of LFA-l
is homologous to the neural cell adhesion molecule NCAM. Nature (Lond.), 33/:
624—627,
1988.
3 1. Hallahan, D. E., Gius, D., Kuchibhotla, J., Sukhatme, V., Kufe, D. W., and Weich
selbaum, R. R. Radiation signaling mediated by Jun activation following dissociation
from a cell type-specific repressor. J. Biol. Chem., 268: 4903—4907,1993.
32. Hallahan, D. E., Virudachalam, S., Sherman, M. L., Huberman, E., Kufe, D. W., and
Weichselbaum, R. R. Tumor necrosis factor gene expression is mediated by protein
kinase C following activation by ionizing radiation. Cancer Res.. 51: 4565—4569,
1991.
33. Phillips. M. L.. Nudelman, E., Gaeta, F. C., Perez, M., Singhal, A. K., Hakomori. S.,
and Paulson, J. C. ELAM-l mediates cell adhesion by recognition of a carbohydrate
ligand. sialyl-Lex. Science (Washington DC), 250: 1130—1132, 1990.
34. Berg. E. L., Fromrn, C., Melrose, J., and Tsurushita, N. Antibodies cross-reactive with
E- and P-selectinblockbothE- andP-selectinfunctions.Blood,85: 31—37,
1995.
35. Degitz, K., Li, L. J., and Caughman, S. W. Cloning and characterization of the
5-transcriptional regulatory region of the human intercellular adhesion molecule I
gene. J. Biol. Chem., 266: 14024—14030, 1991.
36. Weichselbaum, R. R., Hallahan, D. E., and Chen, G. T. Y. Biological and physical
basis of radiation oncology. in: J. Holland, E. Frei, R. C. Bast, D. Kufe, D. L. Morton,
and R. R. Weichselbaum (edt.), Cancer Medicine, 4th Ed., pp. 697—726.Baltimore,
MD: Williams & Wilkins, 1997.
37. Haimovitz-Friedman, A., Vlodavsky, I., Chaudhuri, A., Witte, L., and Fuks, Z.
Autocrine effects of fibroblast growth factor in repair of radiation damage in endo
thelial cells. Cancer Res., 5/: 2552—2558, 1991.
38. Cosset, J. M., Socie, G., Dubray, B.. Girinsky. T., Fourquet. A., and Gluckrnan, E.
Single dose versus fractionated total body irradiation before bone marrow transplan
tation: radiobiological and clinical considerations. Int. J. Radiat. Oncol. BioI. Phys.,
30: 477—492,1994.
39. Roach, M., Gandara. D. R., Yuo, H. S., Swift, P. S., Kroll, S., Shrieve, D. C., Wara,
W. M., Margolis, L., and Phillips, T. L. Radiation pneumonitis following combined
modality therapy for lung cancer: analysis of prognostic factors. J. Clin. Oncol., /3:
2606—2612,
1995.
40. Kimura, H., Wu, N. Z., Dodge, R., Spencer, D. P., Klitzman, B. M., McIntyre, T. M.,
and Dewhirst, M. W. Inhibition of radiation-induced up-regulation of leukocyte
adhesion to endothelial cells with the platelet-activating factor inhibitor, BN5202 1.
Int. J. Radiat. Oncol. Biol. Phys., 33: 627—633, 1995.
41 . Hallahan, D. E., Virudachalam, S., Kuchibhotla, J., Kufe, D. W., and Weichselbaum,
R. R. Membrane-derived
secondmessengerregulatesx-ray-mediated
tumornecrosis
factor a gene induction. Proc. NatI. Acad. Sci. USA, 9/: 4897—4901, 1994.
42. Muller, S., Kammerbauer, C., Simons, U., Shibagaki, N., Li, L. J., Caughman, S. W.,
and Degitz, K. Transcriptional regulation of intercellular adhesion molecule- 1: PMA
induction is mediated by NF KB. J. Invest. Dermatol., 104: 970—975, 1995.
43. Ledebur, H. C., and Parks, T. P. Transcriptional regulation of the intercellular
adhesion molecule- I gene by inflammatory cytokines in human endothelial cells.
Essential roles of a variant NF-kappa B site and p65 homodimers. J. Biol. Chem.,
270:933—943,
1995.
44. Sen. R., and Baltimore, D. Inducibility of K immunoglobulin enhancer-binding
protein NF-KB by a posttranslational mechanism. Cell, 47: 921—928,1986.
45. Bemasconi, S., Matteucci, C., Sironi, M., Conni, M., Colotta, F., Mosca, M.,
Colombo, N., Bonazzi, C., Landoni, F., Corbetta, G., et al. Effects of granulocyte
monocyte colony-stimulating factor (GM-CSF) on expression of adhesion molecules
and production of cytokines in blood monocytes and ovarian cancer-associated
macrophages. Int. J. Cancer, 60: 300—307, 1995.
46. Caldenhoven, E., Liden, J., Wissink, S., Van de Stolpe, A., Raaijmakers, J.,
Kocnderman, L., Okret, S., Gustafsson, J. A., and Van der Saag, P. T. Negative
cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for
the antiinflamrnatory action of glucocorticoids. Mol. Endocrinol., 9: 401—412, 1995.
47. Brach, M. A., Gruss, H. J., Kaisho, T., Asano, Y., Hirano, T., and Hemnann, F.
Ionizing radiation induces expression of interleukin 6 by human fibroblasts involving
activation of nuclear factor-KB. J. Biol. Chem., 268: 8466—8472, 1993.
48. Jung, M., Zhang, Y., Lee, S., and Dritschilo, A. Correction of radiation sensitivity in
ataxia telangiectasia cells by a truncated I K B-a. Science (Washington DC), 268:
1619—1621, 1995.
5155
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
Cell Adhesion Molecules Mediate Radiation-induced Leukocyte
Adhesion to the Vascular Endothelium
Dennis Hallahan, Jaya Kuchibhotla and Charles Wyble
Cancer Res 1996;56:5150-5155.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/56/22/5150
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.