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Double-Stranded RNA Induces Sickle Erythrocyte Adherence to
Endothelium: A Potential Role for Viral Infection in Vaso-Occlusive Pain
Episodes in Sickle Cell Anemia
By Paula A. Smolinski, Margaret K. Offermann, James
R. Eckman, and Timothy M. Wick
Vaso-occlusive pain episodes in sickle cell anemia are hypothesizedt o be precipitated by adherence of sickle
erythrocytes t o vascular endothelium in the microcirculation. Febrile episodes, thought t o be viral in etiology, are frequently
associated with vaso-occlusion; however, a direct link between viral infection and vascular occlusion has
not yet been
established. Many pathogenic viruses contain doublestranded RNA or replicate through double-stranded RNA
intermediates. Double-stranded RNA has beenshown to induce vascularcell adhesion molecule-l (VCAM-1)protein expression on endothelial cells. Recently,a new adhesionpathway hasbeendescribed between VCAM-1expressed on
cytokine stimulated endothelium and the a.& integrin complex expressed on sickle reticulocytes.Based on these observations, the hypothesis was developed that viral infection,
through double-stranded RNA intermediates, increases endothelial VCAM-1expression leading t o sickle erythro-
cyte adhesiont o endothelium via an a&-VCAM-l "dependent mechanism. In support of this hypothesis, endothelial
cells exposedt o the synthetic double-stranded RNA poly(l:C)
or the RNA virus parainfluenza 1 (Sendai virus) express increased levels of VCAM-1 andsupport increased sickleerythrocyte adherence under continuous flow at 1.0 dynelcm'
shear stress as compared with unstimulated endothelium.
Blocking antibodies directed against either VCAM-l on the
endothelium or a& on sickle erythrocytes inhibit nearly all
of the increased sickle cell adherence caused by poly(l:CI
or Sendai virus. These results support the hypothesis that
viruses, through double-stranded RNA elements, caninduce
sickle erythrocyte adherence t o endothelium through a&VCAM-l-mediated adhesion and provide a potential link
between viral infection and microvascular occlusion
precipitating sickle cell pain episodes.
0 1995 by The American Societyof Hematology.
S
in endothelial cells independent of cytokine
Based on these observations, we hypothesized that exposure
of endothelial cells to synthetic double-stranded RNA or to
parainfluenza 1 virus leads to increased sickle erythrocyte
adherence via a4PI-VCAM-1 -mediated
interactions.
These studies, coupled with previous observations supporting the role of endothelial adherence in initiating vaso-occlusion, provide a mechanism by which certain viruses could
induce pain episodes by initiating endothelial VCAM- 1 protein expression and promoting adherence of a,P,-positive
sickle erythrocytes.
ICKLE CELL ANEMIA is characterized by episodic
periods of painful microvascular occlusion with tissue
necrosis. Vaso-occlusion is hypothesized to occur when
sickle erythrocyte transit through the microcirculation is delayed such that hemoglobin polymerization and erythrocyte
sickling occur in the microcirculation and cause occlu~ i o n . "Adherence
~
of sickle erythrocytes to microvascular
endothelium is posited as a major determinant of intracapillary transit time and a modulator of vaso-occlusive complications!-6 In recent years, specific adhesion pathways between
both microvascular and large vein endothelium and sickle
erythrocytes have been elucidated in a number of independent studies?"'
Recent studies from our laboratory show that up to 25%
of sickle reticulocytes express the a4PIintegrin complex
and bind to vascular cell adhesion molecule-l (VCAM-1)
expressed by cytokine activated endothelium." Because reticulocyte counts are elevated in sickle blood in response to
shortened erythrocyte survival, this observation suggests that
VCAM-1 expression induced by proinflammatory factors in
vivo may cause significant adhesion of sickle erythrocytes
to endothelium during immune activation.
Pain episodes are often associated with febrile events precipitated by viral or bacterial infection." Many different cell
types, including endothelial, have the capacity to recognize
and respond to double-stranded FWA.I3-l5Pathogenic viruses
with a double-stranded RNA genome or which replicate
through double-stranded RNA intermediates, including parainfluenza 1 virus, Coxsackie virus, flavivirus, rhinovirus, and
influenza virus, may directly induce proinflammatory cellular responses.'6 In animal studies, synthetic double-stranded
RNA such as poly(1:C) induces many of the same pathogenic
responses, as do intact viruses, including the development
of a proinflammatory ~tate.'"'~
Among the genes induced by double-stranded RNA are
class I interferons (a and @),l5 interleukin-6 (IL-6),l3 and
IL-1." Recent studies show that double-stranded RNA also
induces cellular adhesion molecules, including VCAM-l,
Blood, Vol 85,
No 10 (May 15). 1995: pp 2945-2950
MATERIALS AND METHODS
Endothelial cell culture. Human umbilical vein endothelial cells
(HUVECs) were harvested from umbilical cords by collagenase digestion" and grown to confluence in 0.1% gelatin-coated T-flasks
in primary medium consisting of M199 (Sigma Chemical CO, St
Louis, MO) with 20% heat-inactivated fetal calf serum (FCS; Intergen, Purchase, NY), 2 mmol/L L-glutamine (Sigma), and 100 U/
mL penicillin, 1 0 0 pg/rnL streptomycin, and 0.25 pg/mL amphotericin B (GIBCO Laboratories, Grand Island, NY). Confluent endothe-
From the School of Chemical Engineering, Georgia Institute of
Technology, Atlanta; the Division of Hematology/Oncology, Department of Medicine, Winship Cancer Center, Atlanta;and the Georgia
Comprehensive Sickle Cell Center, Emory University School of Medicine, Atlanta, GA.
Submitted April 15, 1994; accepted December 28, 1994.
Supported in part b.y Grants No. HL-44960 and HL-48482 from
the Heart, Lung, and Blood Institute ofthe National Institutes of
Health
Address reprint requests to Timothy M . Wick, PhD,Associate
Professor, School of Chemical Engineering, Georgia Institute of
Technology, 778 Atlantic Dr, Atlanta, GA 30332-0100.
The publication costsof 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.
0 1995 by The American Society of Hematology.
0006~4971/95/8510-0033$3.00/0
2945
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2946
SMOLlNSKl ET AL
lial monolayers
were
trypsinized
using 0.001% trypsin-EDTA
Cell Wells plate (Corning Glass Works, Corning,
N Y ) i n passage
(GIBCO)andsplit
1:3. Endothelialcells of passagetwo to four
medium. After reaching confluence, each well was incubated for 20
were grown to confluence
in individual Nunc Permanox Lab Tek
hours with 200 pL control medium or medium supplemented with
(Naperville, [L) wells coated with 0.1% gelatin. Passaged endothelial
either IO0 pg/mL poly(I:C), 100 pg/mL poly(I), o r S00 U/mL TNFcells were culturedin passage medium consistingof primary medium
LY or infectedwithparainfluenza
I virus as describedabove.The
supplemented with 100 pg/mL heparin (Sigma) and 25 pg/mL endo- remainder of the ELISA analysis was performed as previously dethelial cell growth factor(ECGF Boehringer Mannheim, Indianaposcribed." Endothelial VCAM-I protein expression was
read on an
lis, IN).
ELISABio-TekMicroplateReader(Winooski,VT)at450nm.
Background values were subtracted and all measurements were perErythrocyte suspensions. Wholebloodsampleswereobtained
formed in quadruplicate and reported as mean values.
by venipuncture into sodium citrate ( l :9 vol/vol) or sodium heparin
(14.3 USP Units/mL) Vacutainer tubes (Becton Dickinson, RutherAdhesion assuys. The flow assay was performed using a parallel
plate flow chamber at 37"C, as previously described." The treated
ford, NJ) from normal donors or asymptomatic homozygous sickle
endothelial monolayer was exposed to continuous flow at I dyne/
patients at the Georgia Comprehensive Sickle Cell Center at Grady
cm' consisting of an initial 5-minute serum-free medium rinse, folMemorial Hospital (Atlanta, CA). Informed consent was obtained
lowed by a 10-minute perfusion with 1 % hematocrit sickle cells in
from all participants, and protocols were approved
by the Human
Investigations Committees of Emory University School
serum-freemedium,andconcluding
with a20-minuteserum-free
of Medicine,
medium rinse to remove nonadherent cells. The endothelial monoGeorgia Institute of Technology, and Grady Memorial Hospital. Patients showed no signs of pregnancy, obvious infection, thromboen- layer was then scanned at 20 X magnification, and adherent erythrobolic disease, or liver disease and werenot receiving anticoagulation
cytesper field werecountedfor
20 randomfields.These
values
as adherenterythrocytes per square
were averagedandreported
therapy. Within 4 hours, the whole
blood was centrifuged at l00g
millimeter.
for 10 minutes at 25°C and used in the experimental protocol within
24 hours. The erythrocyte-rich fraction was washed as previously
Datu unalysis und .statistical met1zod.c.. Eachexperimentconsisted of aseriesofadherencemeasurementsusingendothelium
described" and suspended to 1% hematocrit in serum-free medium
from the same harvest and erythrocytes from the same patient (eg.
I3 I (GIBCO)plus
consistingofendothelialbasalmediaMCDB
Fig l ) . For eachexperiment,theaveragenormalizederythrocyte
0.292 mg/mLL-glutamine(Sigma),0.227mg/mLcyclicAMP
adherence obtained from counting the 20 microscopic
fields is treated
(Sigma), 0.1 mg/mLstreptomycin,neomycin,andamphotericin
as a single observation. Each experiment was repeated several times
(GIBCO), 0.001 mg/mL hydrocortisone (Sigma), 0.01 pg/mL epiusing sickle cells from different patients and grouped data for the
dermalgrowthfactor(Clonetics,SanDiego,CA),0.2%(wt/vol)
experiments are reported as mean -t standard error of mean. Percent
human albumin (Sigma), 5.0 pg/mLhuman transferrin (Sigma), and
blocking was calculated using the following equation: ([poly(I:C)5.0 pg/mL bovine insulin (Sigma).
mediated adhesion] - [poly(l:C)-mediated adhesion with specified
Endothelial and erythrocyte treatments. In preparation for flow
antibody present])/([poly(I:C)-mediated adhesion]
[basaladheassays, confluent endothelial cell monolayers were preincubated for
sion]) X 100, andthecalculatedvaluesareindicatedabovethe
20 hours before flow with either control medium or control medium
bars. Paired Student's t-tests were used to determine the statistical
supplemented with either 500 U/mL tumor necrosis factor-a (TNFsignificance of differences."
a;generously supplied by Dr Robert Swerlick, Emory University
100 pg/mLpolyinosinicacid:
SchoolofMedicine,Atlanta,CA),
polycytidilicacid[poly(I:C);PharmaciaLKBBiotechnologyInc,
RESULTS
Piscataway, NJ], 100 pg/mL polyinosinic acid [poly(I); Pharmacia],
Effects of poly(I:C) on erythrocyte adherence. Toexor 100 pg/mL polycytidilic acid [poly(C); Pharmacia]. Control meplore the possible involvement of viruses through doubledium consisted of serum-free medium supplemented with 30% (voU
stranded RNA intermediates in sickle cell vaso-occlusion,
vol) human AB serum (GIBCO). A solution of 20 p L parainfluenza
the adherence of sickle erythrocytes to poly(1:C)-stimulated
I virus (Sendai virus; 10,000 HAU/mL; Spafas, Stoors, CN) per 3
mL M199 (Sigma) wasused when infecting endothelial cells. Where
endothelial cells was compared with the adherence to unindicated,thevirussolutionwasaddedtoawashedendothelial
stimulated endothelium. Sickle erythrocyte adherence was
monolayerfor 1 hour of incubation.The virus solution was then
increased more than fourfold when endothelial cells were
removed, the monolayer was washed, and complete passage medium stimulated with poly(1:C). In contrast, normal erythrocyte
was replaced. The endothelial cells were then used in a flow assay
adherence did not increase when endothelial cells were stimor enzyme-linked immunosorbent assay (ELISA)20 hours postinfeculated with poly(1:C) (Fig 1). The level of sickle erythrocyte
tion.
adherence to poly(1:C)-stimulated endothelial cells was simiMonoclonal antibodies (MoAbs) were used in adhesion blocking
l a r to that induced by the potent endothelial activator TNFexperiments. To block endothelial receptors, cells were incubated
a (Fig 2). There was no increase in sickle erythrocyte adherwith 10 pg/mL anti-VCAM-I clone 1 G1 1 (AMAC Inc, Westbrook,
ence to endothelial cells stimulated with the single-stranded
ME) or 10 or 50 pg/mL anti-ICAM clone 84H10 (AMAC)" for I
RNA molecules poly(1) (Fig 2) or poly(C) (data not shown),
hour between the 20 hours
of stimulation and the flow assay. To
blockerythrocytereceptors,
washed 1% erythrocytesuspensions
indicating the specificity of poly(1:C)-induced sickle erythrowere centrifuged and the erythrocyte pellet was incubated
with 10
cyte adherence.
or 50 pg erythrocyte MoAbs, anti-a, clone HP2/1 (AMAC), antiPnly(I:C)-induced adherence is via a&,--VCAM-I interDl clone MoAbl3* (Collaborative Biomedicals, Bedford, MA), or
actions. The increased sickle erythrocyte adherence was
anti-glycophorinAcloneD2.10(AMAC)for
15 minutes."After
associated with increased endothelial VCAM-l protein exincubation with the MoAbs, erythrocytes were suspended in serumpression (Fig 2, inset), suggesting that VCAM-I is involved
free medium, centrifuged to remove unbound antibody, and resusin
the observed adherence. MoAb blocking assays were perpended in fresh serum-free medium to I % hematocrit
formed to further elucidate the mechanism of poly(1:C)-inMeasuring cell adhesion molecule protein expression. VCAM- I
protein expression was quantified using an ELISA. Briefly, HUVECs duced sickle erythrocyte adherence. Poly(1:C)-mediated
sickle erythrocyte adherence was blocked 98%by preincubawere grown to confluence in a 0.1% gelatin-coated 96-well Corning
~
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SICKLECELLADHERENCE
2941
ENHANCED BY VIRUS
L
Unstimulated
Poly(l:C) stimulated
v)
0
Fig 1. Sickle erythrocytes adhere t o poly(l:C)-stimulated endothelial cells. Erythrocyte adherence t o unstimulated or poly(l:C)-stimulated endothelium was
quantified under continuous flowat 1 dynelcm2.
Sickle erythrocyte adherence t o polyll:C)-stimulated
endothelium was significantly greater than adherence t o unstimulated endothelium ( P < .0005, n =
11). Data are reported as mean 2 standard error for
five normal donors and 11 asymptomatic sickle patients.
l
100
tion of poly(1:C)-stimulated endothelial cells with antiVCAM-I antibody (Fig 3). Similarly, preincubation of sickle
erythrocytes with anti-a4 antibody inhibited 80% of the observed sickle cell adhesion (Fig 3).In addition, preincubation
of sickle erythrocytes with an anti-Dl antibody blocked 68%
of poly(1:C)-induced adhesion (data not shown), further
demonstrating the involvement of a4PI.
To exclude nonspecific antibody interactions as a cause
of the observed inhibition, the blocking abilities of antibodies against adhesion molecules not believed to be involved in sickle cell adhesion were determined. Preincu-
bation of endothelialcells with 10 pglmL endothelial
anti-ICAM-l antibody did not inhibit poly(1:C)-mediated
adhesion (37.8 t 10.1 17 32.2 -t 9. I cellslmm’. n = 4).
Preincubation of sickle erythrocytes with 10 pg anti-glycophorin A antibody also did not inhibit poly(1:C)-induced
adhesion (37.8 2 10.1 v 35.8 2 11.8 cellslmm’, n = 4).
Increasing the control antibody levels to S0 pg still did
not initiate blocking (data not shown). These studies illustrate that poly(1:C)-induced sickle erythrocyte adherence
occurs through interactions between erythrocyte a4Dl and
endothelial VCAM- I .
70
60
50
-
-
--
--
20
-
--1---
10
-
-1
40
30
... .
\~\~\~\~.~\‘\’\~\~\~.‘.’
............
T
\’\’\’\’\‘\,
\
0
Unstimulated
TNFa
Poly(l:C)
HUVEC Treatment
pOlY(l)
Fig 2. Sickle erythrocyte adherence levels parallel endothelial VCAM-1 expression. Sickle
erythrocyte adherence t o unstimulated endothelial cells or
endothelial cells stimulated with
either 500 UlmL TNFa, l00 p g l
mL poly(l:C), or 100 p g l m L
polyll) was quantified under continuous flow at1 dynelcm2.Data
are reported as mean ? standard
error
for
five
asymptomatic
sickle patients [except n = 4 for
poly (l)]. (Inset1 Endothelial
VCAM-1 expression was quantifiedusing ELlSA forunstimulated endothelial cells or endothelia1 cells treated for 20 hours
with the indicatedagonist. Data
are reported as average OD reading for four identical wells and
represent one data set.
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SMOLlNSKl ET AL
2948
Fig 3. Poly(l:C)-induced sickle erythrocyte adherence occurs via ancx&,-VCAM-l -mediated mechanism. Sickle erythrocyte adherence t o endothelial
cells stimulatedfor
20 hours with 100 pg/mL
poly(l:C) was quantified under continuous flow of 1
dyne/cm2. Either 50 p g anti-cr4 antibody on sickle
erythrocytes ( P c ,021 or 10 pg/mL anti-VCAM-l
antibody on endothelium ( P < .02) inhibited
poly(l:C)-induced sickle cell adherence. Data are reported as mean 2 standard error for four asymptomatic sickle patients.
Enthrocyte adherence to endothelirml ir!fected w i t h pnroir!fllcen:a I virus. To determine whether an intactvirus
would lead to similar increases in adherence as that observed
with synthetic double-stranded RNA, endothelial cells were
incubated with parainfluenza I virus (Sendai virus). The increased level of sickle erythrocyte adherence was comparable to that observed with poly(1:C)(Fig 4).and parainfluenza
1 led to increased levels of endothelial VCAM-I protein
Fig 4. Adherence of sickle
erythrocytes t o endothelial cells
infected with parainfluenza 1 virus (Sendai virus) occurs via an
cr&-VCAM-l-mediated
mechanism. Sickle erythrocyte adherence t o endothelial cells infected
with parainfluenza 1 virus (Sendai virus) was quantified under
continuous flow at 1 dyne/cm2
as described. Where indicated,
erythrocytes were incubated with
50 p g anti-cr, antibody or endothelial cells were incubatedwith
10 pg/mLanti-VCAM-lantibody. Data are reported as mean
2 standard error for five
asymptomatic sickle patients (except n
= 4 for antibody blocking). (Inset) Endothelial VCAM-1 expression was quantified usingELISA
for unstimulated endothelial cells
or endothelialcells treated for20
hours withthe indicated agonist. Data are reported as average OD reading for four identical
wellsand represent one data
set.
10
l5
5
0
expression (Fig 4, inset). Blocking antibodies directed
against endothelial VCAM- 1 inhibited the virus-induced adhesion by 92% (Fig 4).Similarly, anti-a, or anti-P, antibodies on sickle erythrocytes reduced the adhesion by 63% (Fig
4)and 45% (data not shown), respectively. In addition, neither anti-ICAM-I (50 pg/mL) nor anti-glycophorin A (S0
pg) blocked the virus-induced adherence (data not shown).
These data suggest that live RNA virus, as well as synthetic
1
1
t
Media
Poly(l:C) Sendai
Sendai
Sendai
virus
virus
virus
X-VCAM-1
HUVEC Treatment
X-a4
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2949
SICKLECELL ADHERENCE ENHANCED BY VIRUS
purified double-stranded RNA, enhance sickle erythrocyte
adherence to vascular endothelium in large part through an
a4pI-VCAM- l -mediated mechanism.
DISCUSSION
The present data show that induction of endothelial
VCAM-l protein expression by synthetic double-stranded
RNA, or by an RNAvirus, increases sickle erythrocyte endothelial adherence under dynamic flow conditions. The sickle
erythrocyte adherence appears to occur through interactions
between endothelial VCAM-1 and the integrin complex
a4PI,known to be expressed on sickle reticulocytes." These
data suggest that endothelial cell activation in vivo via double-stranded RNA could initiate sickle erythrocyte adherence
and microvascular occlusion. These observations provide a
possible mechanism by which viral infection precipitates
pain episodes and other vaso-occlusive complications.
Double-stranded RNA has been used to model viral effects
on endothelial cells in this experimental work. Although
poly(1:C) is a synthetic double-stranded RNA that is not a
pathogen, it does provide a model for changes that could
result from viral infection of endothelial cells by many different RNA viruses with naked double-stranded RNA intermediates. The mechanism responsible for increased VCAM-1
expression in response to poly(1:C) is currently under investigation. Poly(1:C) activates NFKB,'~.*'a transcription factor
important in VCAM-1 transcription.26Activation through
this mechanism likely contributes to the observed increase
in VCAM-1 gene expression after poly(1:C) stimulation.
Data presented with parainfluenza 1 virus illustrate that
changes in VCAM-1 expression in response to synthetic
double-stranded RNA also occur in response to live RNA
virus. Other RNA viruses, including measles, also induce
VCAM-1 (B. Harcourt and M.K. Offermann, unpublished
observation). Studies with intact viruses are complicated by
multiple issues, whereas studies with purified viral or synthetic double-stranded RNA allow examination of the effects
of a specific macromolecular component present in a diverse
array of viruses. Endothelial cells express several proteins
that use double-stranded RNA as a cofactor, including the
double-stranded RNA-activated protein kinase and 2'-5'-oligoadenylate synthetase." Enzymes activated by doublestranded RNA allow cells to rapidly respond to a wide variety of viruses with change in gene expression. Genes shown
to be directly induced by double-stranded RNA are generally
proinflammatory, including interferon-a (IFN-a),
IFN-p,IL6, IL-l, and cellular adhesion molecules. Thus inflammatory
responses can be mounted before cells are able to generate
cytotoxic T lymphocytes or antibodies to specific viruses.
Endothelial infection by Herpes simplex virus type 1, a
DNA virus, has previously been reported to increase sickle
erythrocyte adherence in a static assay." The observed adherence appears to be mediated by interactions between endothelial Fc receptors induced by virus and surface-associated IgG on sickle erythrocytes. The erythrocyte-endothelial
interactions reported here appear to be mediated by a different mechanism than that reported for Herpes simplex virus.
Clinical association of febrile events"with acute pain
episodes in sickle cell patients suggests a role for infection in
the development of vaso-occlusive pain episodes. Although
research has focused on bacterial infection in sickle cell
ane~nia,'~.~'
viral infection also appears to play a role in
causing complications in sickle cell disease. During confirmed cases of infection by the RNA virus influenza B,
acute pain episodes have been reported31 andviral infections
are thought to precipitate such complications as acute chest
~yndrome.~'Infection is known to activate certain leukocytes, causing release of cytokines such as type I interferons
and TNF-a, leading to the development of an antiviral environment in the attacked h o ~ t . ' ~Many
, ~ ~ sickle cell patients
have been clinically observed to have consistently elevated
cytokine levels.35 Induction of VCAM-l directly by viral
double-stranded RNA and indirectly by increased cytokine
production in response to infection could be beneficial to
the host by mounting an inflammatory response to control
the infection. However, in the case of sickle cell anemia,
VCAM-1 induction is potentially dangerous due to the
higher percentage of circulating reticulocytes expressing the
counter receptor a4pIthat could adhere, leading to the onset
of vascular occlusion.
ACKNOWLEDGMENT
The authors thank the staff of the Grady Memorial Hospital Sickle
Center, especially Joyce Howard Bailey and Patricia Troutman, for
their assistance in obtaining blood specimens.
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From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
1995 85: 2945-2950
Double-stranded RNA induces sickle erythrocyte adherence to
endothelium: a potential role for viral infection in vaso-occlusive pain
episodes in sickle cell anemia
PA Smolinski, MK Offermann, JR Eckman and TM Wick
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