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Viral IL-10 Gene Transfer Decreases
Inflammation and Cell Adhesion Molecule
Expression in a Rat Model of Venous
Thrombosis
Peter K. Henke, Lisa A. DeBrunye, Robert M. Strieter,
Jonathan S. Bromberg, Martin Prince, Amy M. Kadell,
Minakshi Sarkar, Frank Londy and Thomas W. Wakefield
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 164:2131-2141; ;
doi: 10.4049/jimmunol.164.4.2131
http://www.jimmunol.org/content/164/4/2131
Viral IL-10 Gene Transfer Decreases Inflammation and Cell
Adhesion Molecule Expression in a Rat Model of Venous
Thrombosis1,2
Peter K. Henke,3* Lisa A. DeBrunye,§ Robert M. Strieter,‡ Jonathan S. Bromberg,¶
Martin Prince,† Amy M. Kadell,* Minakshi Sarkar,* Frank Londy,† and
Thomas W. Wakefield*
D
eep vein thrombosis (DVT)4 sequelae commonly include pulmonary embolism and chronic venous insufficiency. While pulmonary embolism is often dramatic in
presentation and immediately devastating in outcome, it is less
common than venous insufficiency (1). Chronic venous insufficiency affects up to 30% of patients who suffer DVT and may be
severely debilitating in up to 10% (1, 2). Unfractionated i.v. hep*Jobst Vascular Surgery Laboratory, Section of Vascular Surgery, Department of
Surgery, †Division of Magnetic Resonance Imaging, Department of Radiology,
and ‡Division of Pulmonary and Critical Care Medicine, Department of Internal
Medicine, University of Michigan Medical Center, and §Parke Davis Pharmaceutical Division, Ann Arbor, MI 48109; and ¶Racanti-Miller Transplant Institute,
Department of Surgery and Molecular Medicine, Mt. Sinai School of Medicine,
New York, NY 10029
Received for publication August 2, 1999. Accepted for publication December 8, 1999.
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.
1
This work was supported in part by National Institutes of Health, Heart, Lung, and
Blood Institute Grants HL53355 and HL63148 (to T.W.W.), by University of Michigan Medical Center Grant P60-AR20557, and by a Baxter extramural grant and Ed
Mallinkrodt, Jr., Foundation Grants AI42840/Ag15434 (to J.S.B.).
2
A portion of this study was presented at the 85th Annual Meeting of the American
College of Surgeons-Surgical Forum, October 14, 1999, San Francisco, CA.
3
Address correspondence and reprint requests to Dr. Peter Henke, Section of
Vascular Surgery, Department of Surgery, 2210 Taubman Health Care Center, 1500
East Medical Center Drive, Ann Arbor, MI 48109-0329. E-mail address: henke@
umich.edu
4
Abbreviations used in this paper: DVT, deep vein thrombosis; CAM, cell adhesion
molecule; hIL, human IL; vIL-10, viral IL-10; Ad-vIL-10, adenovirus encoding vIL10; ␤-gal, ␤-galactosidase; RSV, Rous sarcoma virus; IVC, inferior vena cava; MRV,
magnetic resonance venography; Mo, monocytes; TF, tissue factor; vWF, von Willebrand factor; PMN, polymorphonuclear cells; X-Gal, 5-bromo-4-chloro-3-indolyl
␤-D-galactopyranoside; TMB, 3,3⬘,5,5⬘-tetramethylbenzidine.
Copyright © 2000 by The American Association of Immunologists
arin effectively enhances natural clot lysis, but has little effect on
perithrombotic venous inflammation (3). Limiting early perithrombotic inflammation may diminish later consequences, such as venous insufficiency and pain.
The molecular pathophysiology of DVT is beginning to be delineated. Thrombosis, like other physiologic processes, initiates a
complex inflammatory mediator-procoagulant cascade that ultimately leads to vein wall damage (4 –7). The process of thrombus
resolution is similar to that of wound healing, with the nidus of
injury being the clot, followed by leukocyte influx and eventually
vein wall fibrosis. Mediators of this process include proinflammatory cytokines such as TNF-␣ and IL-1␤ (from the injured venous
cells and local leukocytes) that serve to further activate leukocytes,
stimulate chemotactic factor release, promote thrombosis, and increase cell adhesion molecule (CAM) expression (4, 6 –9). Furthermore, thrombin acts as a proinflammatory mediator that not
only amplifies the coagulation cascade, but also stimulates procoagulant changes in endothelial cells with concurrently increased
CAM expression (3, 8, 10 –12). The CAMs P- and E-selectin and
ICAM-1 are critical for leukocyte tethering, firm adhesion, and
emigration, respectively. Leukocyte emigration into the vein wall
allows the release of cytotoxic inflammatory intermediates that
cause vein damage (4, 6, 8, 9, 13–15). Prior work in our laboratory
has clarified that TNF-␣ and P-selectin are central mediators of
post-thrombotic vein wall inflammation and direct the magnitude
of thrombus production (6, 13–15).
We have previously characterized an important role for IL-10 as
an anti-inflammatory cytokine in the rat model of DVT. Specifically, neutralization of IL-10 resulted in increased local inflammation, while supplementation with human IL-10 (hIL-10) in a
0022-1767/00/$02.00
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Post-thrombotic inflammation probably contributes to chronic venous insufficiency, and little effective treatment exists. IL-10 is
an anti-inflammatory cytokine that previously has been shown to decrease perithrombotic inflammation and thrombosis. We
investigated in a rat model whether local expression of viral IL-10 (vIL-10) in a segment of vein that undergoes thrombosis would
confer an anti-inflammatory effect and how this effect might be mediated. Rats underwent inferior vena cava isolation, cannulation, and instillation of saline or adenovirus encoding either ␤-galactosidase or vIL-10. Two days after transfection, thrombosis
was induced, 2 days after this the rats underwent gadolinium (Gd)-enhanced magnetic resonance venography exam, and the vein
segments were harvested. Tissue transfection was confirmed by either RT-PCR of vIL-10 or positive 5-bromo-4-chloro-3-indolyl
␤-D-galactopyranoside (X-Gal) staining. vIL-10 significantly decreased both leukocyte vein wall extravasation and area of Gd
enhancement compared with those in controls, suggesting decreased inflammation. Immunohistochemistry demonstrated decreased endothelial border staining of P- and E-selectin, while ELISA of vein tissue homogenates revealed significantly decreased
P- and E-selectin and ICAM-1 levels in the vIL-10 group compared with those in controls. Importantly, native cellular IL-10 was
not significantly different between the groups. However, neither clot weight nor coagulation indexes, including tissue factor
activity, tissue factor Ag, or von Willebrand factor levels, were significantly affected by local vIL-10 expression. These data suggest
that local transfection of vIL-10 decreases venous thrombosis-associated inflammation and cell adhesion molecule expression, but
does not directly affect local procoagulant activity. The Journal of Immunology, 2000, 164: 2131–2141.
2132
vIL10 GENE TRANSFER DECREASES PERITHROMBOTIC INFLAMMATION
dose- and time-dependent fashion decreased not only perithrombotic inflammation but also thrombus size (5). EBV encodes a
homologue to rodent and human IL-10 that is designated viral
IL-10 (vIL-10). vIL-10 has similar anti-inflammatory properties as
hIL-10, but may not have the lymphocyte stimulatory properties
associated with hIL-10 at high concentrations (16, 17). Other
investigators have found that adenovirus encoding vIL-10 (AdvIL-10) transfection is associated with a significant reduction in
alloantigen responsiveness, significantly increased cardiac allograft survival, decreased collagen-induced arthritis inflammation,
and decreased systemic TNF and IL-1␤ release (18 –21). Adenovirus-mediated gene transfer has the advantage of high efficiency,
but is limited by transient gene expression and the host immune
response against the viral infection (22, 23). Theoretically, local
production of IL-10 via transfection has the advantage of minimizing confounding systemic effects, and its constant local expression may further enhance the anti-inflammatory effects compared
with intermittent systemic infusions. In this study we show that
Ad-vIL-10 transfection in a localized venous segment confers an
anti-inflammatory effect during thrombosis and that this effect is
probably mediated by down-regulation of CAM expression, but
not through a reduction in proinflammatory mediator or procoagulant activity.
Materials and Methods
Adenoviral vectors
All vectors are E1 deleted. Ad-CMV encoding ␤-galactosidase (␤-gal) or
vIL-10 gene were under the control of the CMV immediate early promoter.
Ad-RSV-␤-gal was under the control of the RSV immediate early promoter. All vectors were obtained from the vector core of University of
Michigan Medical Center and were produced as previously described (19).
Animal model, transfection, and tissue analysis
This rat model is a modification of a well-characterized method for reproducible stasis induced venous thrombosis (3, 5, 6, 13, 24). In brief, male
Sprague Dawley rats, weighing 250 –300 g, were anesthetized with an inhalation mixture of isoflurane (1–2%) and oxygen (100%) during the procedure. On day 1, aseptic laparotomy was performed, the inferior vena cava
was isolated, and major side branches were ligated (Fig. 1). An ⬃1.2-cm
segment was dissected to allow temporary proximal and distal occlusion
with microvascular clips. After occlusion, the inferior vena cava (IVC) was
cannulated with a 30-gauge needle catheter, the blood was aspirated, and
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FIGURE 1. A, Flow diagram of the rat surgical sequence for analysis of tissue 48 h after thrombus production. B, Schematic for surgery day 1; the rats
underwent laparotomy and temporary occlusion of a segment of the IVC as shown. A 30-gauge catheter was inserted, and the vector or saline solution was
instilled under slight pressure for 30 min. C, Schematic for surgery day 3; the rats underwent repeat laparotomy, the vena cava was gently redissected free,
and a polypropylene ligature was placed to occlude the IVC to cause stasis-induced venous thrombosis.
The Journal of Immunology
the IVC was flushed with a dilute heparinized saline solution. After this,
0.15-ml instillation of saline, Ad-CMV-␤-gal, Ad-RSV-␤-gal or Ad-CMVvIL-10 (all 5 ⫻ 108 PFU/ml final concentration) was performed for 30 min.
The vector solution or saline was aspirated, the clips were removed to
re-establish blood flow, and the midline incision was closed. Two days
after the transfection, the rats were anesthetized in the same fashion, and a
repeat laparotomy was performed. The IVC was then ligated below the
renal veins for establishment of thrombosis. This method produces a clot in
⬎95% of animals, and gradual re-establishment of flow usually occurs by
dilation of posterior collaterals. Two days after thrombosis induction, the
animals underwent gadolinium (Gd)-enhanced magnetic resonance imaging and were sacrificed. The thrombosed IVC segment was harvested below the renal veins and weighed (milligrams), and the weight was corrected
to vein length (centimeters). The vein was then halved, and the proximal
portion was placed in 10% buffered formalin for 24 h, followed by 70%
ethanol for subsequent permanent section processing. The lower segment
of the vein was snap-frozen in liquid N2 and kept at ⫺70°C until
homogenized.
Gd-enhanced magnetic resonance venography (MRV) evaluation
Histopathologic analysis and immunohistochemical staining
Leukocyte infiltration into the vein wall was assessed as previously described (3, 5, 6, 13). In brief, leukocyte morphometric analysis was performed in a blinded fashion on hematoxylin- and eosin-stained sections.
Each rat had three sections reviewed, and the section with the most complete circumference was chosen. Then, five high power fields (⫻1000)
radially around the clot wall interface were assessed using standard criteria
for neutrophils, monocytes (Mo), and lymphocytes. Random sections were
taken from the proximal and middle portions of the vein clot tissue for
analysis.
Immunohistochemical analysis was performed on the paraffin-processed
tissue sections (10 ␮m) as previously described (6). In brief, the slides were
deparaffinized with xylene followed by rehydration with graded dilutions
of ethanol. The sections underwent Ag retrieval using 0.1% trypsin/
0.1%CaCl2 for 25 min, followed by quenching of endogenous peroxidase
activity with methanol/3% H2O2 (50/50) for 15 min. On each slide after
blocking nonspecific sites, the sections were incubated with either control
isotype specific Ab (goat IgG) or anti-E-selectin, anti-P-selectin (both 1
␮g/ml), or anti-ICAM-1 (2 ␮g/ml; all from Santa Cruz Biotechnology,
Santa Cruz, CA) for 30 min. An anti-goat biotinylated Ab (1/1000) followed by avidin-peroxidase conjugate was added in sequence, and color
development was performed with addition of diaminobenzidene substrate
(ABC kit, Vector, Burlingame, CA). After color development, the slides
were washed and counterstained with Mayer’s hematoxylin. Qualitative
assessment of staining intensity was compared between the experimental
and control groups.
5-Bromo-4-chloro-3-indolyl ␤-D-galactopyranoside (X-Gal) staining
was performed on the control vector sections as described to confirm transfection (18). In brief, the rat vein sections were embedded with O.C.T.
(Miles Scientific, Naperville, IL), snap-frozen in liquid N2, sectioned at 10
␮M, and fixed in 0.25% glutaraldehyde in PBS for 30 min. After rinsing in
PBS for 30 min, the slides were incubated overnight at 37°C with the
X-Gal solution (0.5 mg/ml X-Gal in 5 mM potassium ferrocyanide, 5 mM
potassium ferricyanide, 2 mM MgCl2, 1 mM spermidine, 0.02 Nonidet
P-40, and 0.01% sodium deoxycholate in PBS.). The sections were then
fixed with 4% formaldehyde and counterstained with eosin.
Cytokine/coagulation factor/CAM analysis
The vein tissue (clot and wall) was thawed, placed in complete lysis buffer
(Boehringer Mannhein, Indianapolis, IN), homogenized with a hand-held
homogenizer, and sonicated for 10 s. The homogenate was centrifuged at
10,000 ⫻ g for 5 min, and the supernatant and sediment fraction were
separated.
Quantification of inflammatory mediators and CAMs were all normalized to total protein in the sample. Total protein quantitation was performed
using a modified Bradford assay according to the manufacturer’s instructions (Pierce, Rockford, IL) with serial dilutions of BSA (Sigma, St. Louis,
MO) as standards. The analysis of TNF-␣ and cellular IL-10 involved
pooling three rat vein tissue segments. All other mediator analysis was
performed on single rat vein tissue samples. Tissue homogenate ELISAs
for the aforementioned mediators were quantified using a double-ligand
technique as previously described (5, 6, 13). In brief, flat-bottom 96-well
microtiter plates (Immuno-Plate I 96-F, Nunc, Naperville, IL) were coated
with 50 ␮l/well (1 ng/ml) of the specific rabbit anti-cytokine Ab in coating
buffer (0.6 mol/L NaCl, 0.26 mol/L H3PO4, and 0.08 N NaOH, pH 9.6) for
16 –24 h at 0°C. Nonspecific sites were then blocked with 2% BSA in PBS
for 60 min at 37°C, followed by sample addition of a 50-␮l aliquot in
duplicate and incubated for 60 min at 37°C. After washing, 50 ␮l of a
biotinylated rabbit polyclonal Ab (3.5 ␮g/ml in PBS pH 7.5, 0.05% Tween20, and 2% FCS) was added and incubated for 45 min at 37°C. No Ab
cross-reactivity between the cytokines was found. Plates were washed,
streptavidin-peroxidase conjugate (1/5000) was added, and the plates were
incubated for 30 min at 37°C. The plates were again washed, the substrate
TMB (3,3⬘,5,5⬘-tetramethylbenzidine, Kierkegaard & Perry, Gaithersburg,
MD) was added for color development, and the reaction was quenched
using 100 ␮l of 1 M H2PO4. Plates were then read at 450 nm with an
automated microplate reader (Bio-Tek Instruments, Winooski, VT). Standards were 0.5 log dilutions of the cytokines from 1 pg/ml to 100 ng/ml
with a sensitivity ⱖ50 pg/ml.
Analysis of the rat cytokine IL-1␤ and prostacyclin (estimated by 6-keto-PGF2␣) levels were performed using a commercial ELISA and enzyme
immunoassay, respectively, according to the manufacturer’s instructions
with a sensitivity of ⱖ10 pg/ml (R&D Systems, Minneapolis, MN). The
protein levels of tissue factor (TF) and von Willebrand factor (vWF) were
analyzed using a human commercial ELISA with a sensitivity of ⱖ50
pg/ml or 10 milliunits/ml, respectively. (American Diagnostics,
Greenwich, CT).
P- and E-selectin and ICAM-1 levels were quantified in the cell homogenate using a direct ELISA method. In brief, 25 ␮l of the vein tissue
homogenate or Ag-specific blocking peptide (for rat P- and E-selectin and
ICAM-1) as standards (serial dilutions from 0.001–10 ng/ml; Santa Cruz
Biotechnology) was mixed with coating buffer, added in duplicate, and
incubated overnight at 0°C. The plates were washed three times with PBS
and blocked with 2% BSA in PBS for 60 min at 37°C. After further washing, 50 ␮l of Ab to P-selectin, E-selectin, or ICAM-1 (2 ␮g/ml; Santa Cruz
Biotechnology) was added and incubated for 60 min at 37°C. After washing three times with PBS, a goat anti-rabbit peroxidase conjugate secondary Ab (1/200 dilution; Santa Cruz Biotechnology) was added and incubated for 45 min at 37°C. The plates were again washed, TMB was added
for color development, and the reaction was quenched using 100 ␮l of 1 M
H2PO4. The sensitivity was ⱖ10 pg/ml, and negligible background was
found when testing the secondary Ab against the adsorbed Ags.
Analysis of procoagulant activity
An amidolytic assay was used for the determination of TF activity in the
vein tissue homogenate (25). In brief, 50 ␮l of the vein tissue homogenate
was added in duplicate to a microtiter plate followed by addition of the
enzyme-substrate solution (1 U/ml Proplex T (Baxter-Allegiance Healthcare, McGaw Park, IL) and 200 ␮g/ml of S2222 (Helena Laboratories,
Beaumont, TX) in medium 199 without phenol red (Life Technologies,
Gaithersburg, MD) and with 3.24 mM CaCl2). This mixture was incubated
for 30 min at 37°C in a humidified 5% CO2 incubator and then quenched
with 3% HOAc. The plate was read at 410 nm. A standard of 0.01 nmol/ml
of lipidated TF (American Diagnostics) was run under the same conditions,
and color development was arbitrarily set at 1 ␮/ml. The background of the
homogenate without substrate enzyme mixture was subtracted from the
sample. Negligible color development occurred in the absence of tissue
homogenate.
RT-PCR
After removal of the vein tissue homogenate supernatant, the sediment
fractions were pooled (n ⫽ 2–3) and placed in Trizol (Life Technologies,
Gaithersburg, MD), which uses guanidinium-thiocyanate-phenol to isolate
RNA. The RNA was extracted with chloroform/isoamyl alcohol and precipitated with 70% ethanol. The resulting RNA was quantified spectophometrically, and then 0.5–1 ␮g/ml of RNA was used to synthesize cDNA by
random primer reverse transcriptase (Invitrogen, Carlsbad, CA). The
cDNA was amplified using two specific primers for the vIL-10 gene as
previously described (18, 26): 5⬘-ATGGAGGAGCGAAGGTTAGTG
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On the day of sacrifice, the rats were sedated with Telazol (20 – 40 mg/kg
i.m.) and imaged with a magnetic resonance imaging system (Signa Horizon LX, Milwaukee, WI) using 8.1 software as previously described (24).
Baseline time-of-flight imaging and T1 weighted imaging were performed
followed by tail vein injection of 2 ml of Gd dimeglumine (0.2– 0.4 mg/kg;
Magnevist, Berlix, Wayne, NJ), and T1-weighted images were repeated.
Gd is a heavy metal chelate that shortens the T1 relaxation time, prolonging
signal enhancement. It extravasates into areas of tissue inflammation (capillary leak) with high sensitivity. In a blinded fashion, analysis of four or
five coronal sections of the whole IVC was reviewed, the most intense
Gd-enhanced segment was chosen, and the area of maximum enhancement
was measured in square millimeters in treated and controls rats.
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vIL10 GENE TRANSFER DECREASES PERITHROMBOTIC INFLAMMATION
GTC-3⬘ (upstream) and 5⬘-ACTCTTGTTCTCACACGGCAG-3⬘ (downstream). Forty PCR cycles yielded a 387-bp fragment using an automated
thermocycler (RJ Research, Swedesboro, NJ) with cycles of 96°C for 15 s,
60°C for 30 s, and 72°C for 2 min. The cDNA from a vIL-10 plasmidtransfected COS cell line using the same RT-PCR reaction was used as a
positive control (26). ␤-Actin was amplified from the same cDNA in a
separate reaction using published sequences and the same PCR cycle settings. The product was separated on a 2% agarose gel and visualized with
1% ethidium bromide staining with a UV source.
Statistical analysis and animal care
Statistical evaluation included data presentation as the mean ⫾ SEM. Oneway ANOVA with Tukey’s highest significant difference test for pairwise
comparison or unpaired Student’s t test was used as appropriate. Statistical
significance was assigned for p ⱕ 0.05. All experiments were repeated at
least twice. All animals in this study were housed and cared for at the
University of Michigan Unit for Laboratory Animal Medicine under the
direction of a veterinarian in accordance with the Guide for the Care and
Use of Laboratory Animals (National Institutes of Health Publication 8623, revised 1985).
Results
Gene transfer and expression in the IVC
The rat IVC was isolated, and transfection with the vector encoding ␤-gal or vIL-10 before thrombus induction was performed.
X-Gal staining on Ad-LacZ control IVCs (n ⫽ 4) representing two
separate transfection experiments demonstrated that about
20 –30% of the inner vein wall circumference stained positive for
␤-gal expression (Fig. 2, A and B). RT-PCR analysis (pooled
groups of three separate rat vein prepared segments; n ⫽ 3 separate
transfection experiments) revealed that only the vIL-10-transfected
group produced a 387-bp fragment consistent with vIL-10 (Fig.
2C). Neither the saline nor ␤-gal controls had any bands when
RT-PCR was performed using the specific vIL-10 primers. However, all groups had similar intensity bands of the housekeeping
gene ␤-actin, suggesting adequate starting cDNA.
Perithrombotic inflammation is reduced by vIL-10 expression
Assessment of inflammation was quantified by leukocyte vein wall
emigration and Gd-enhanced MRV. Total subendothelial and medial leukocytes were all decreased with vIL-10 transfection compared with those in control saline and ␤-gal rats (n ⫽ 6 –12; Fig.
3). Specifically, total leukocytes in the vein wall were significantly
reduced by 36% (vIL-10, 33 ⫾ 3; ␤-gal, 47 ⫾ 4; saline, 56 ⫾ 8),
while neutrophils were reduced by 45% (vIL-10, 16 ⫾ 3; ␤-gal,
27 ⫾ 4; saline, 31 ⫾ 6) compared with controls ( p ⱕ 0.05, by
ANOVA, vIL-10 vs saline and ␤-gal; p ⬍ 0.05, by t test, vIL-10
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FIGURE 2. X-Gal staining of Ad-LacZ-transfected vein sections showing blue stain where the endothelial cells have expressed the ␤-gal gene (black
arrows). W, vein wall. A, ⫻400; B, ⫻1000. C, RT-PCR analysis of saline control, ␤-gal-transfected, and vIL-10-transfected vein homogenate sediment
fractions. A typical agarose gel of PCR products is shown. The cDNA-amplified product from a vIL-10 COS-transfected cell line served as a control (⫹,
left column) and generated a 387-bp fragment. No bands were seen at this size in the saline or ␤-gal controls. In contrast, a distinct band at 387 bp was
present in the vIL-10 group, confirming successful transfection. Similar levels of ␤-actin were seen in all groups.
The Journal of Immunology
FIGURE 3. Vein wall leukocyte counts and differential summed in five
high power fields. Note the significant decreases in total, neutrophil, Mo,
and lymphocyte counts in the vIL-10 group compared with those in the
saline and ␤-gal controls (ⴱ, p ⬍ 0.05, by ANOVA). Few lymphocytes
were present at this time in all groups.
the rats just before sacrifice (n ⫽ 6 –12; Fig. 4). Coronal venous
section analysis of the ␤-gal and saline control rats revealed 31 ⫾
4- and 31 ⫾ 5-mm2 average areas of perithrombotic Gd enhancement, respectively. In contrast, the rats transfected with vIL-10 had
a decreased average coronal vein section area of perithrombotic
enhancement of 17 ⫾ 2 mm2 ( p ⫽ 0.056, by ANOVA, vIL-10
compared with saline and promoter controls; p ⬍ 0.05 by t test,
vIL-10 compared with promoter control). Whereas the Gd enhancement was fairly diffuse in the control groups, the zone of
enhancement was tighter and seemed to be mainly associated with
the clot in the vIL-10 group. These data reflect a decreased amount
of capillary leak and therefore decreased inflammation in the vIL10-transfected group. No significant enhancement was observed
before Gd injection by review of baseline scans (data not shown),
and from previous work, nonclotted IVC has an average Gd enhancement of 1.8 mm2 (24).
Vein tissue cytokine and PGI2 analysis
ELISA analysis of the vein tissue homogenates showed a trend
toward a decreased TNF-␣ level in the vIL-10 group compared
with those in ␤-gal and saline controls, but this did not reach statistical significance (n ⫽ 2– 4 groups of three pooled vein segments; Fig. 5A). In contrast, IL-1␤ was not significantly different
FIGURE 4. Gd-enhanced magnetic resonance imaging of in vivo rat vein coronal sections (T1-weighted images). Note that saline and ␤-gal controls
show fairly diffuse and large perithrombotic enhancement (white arrows). A smaller and more tightly enhanced area was typical in the vIL-10-transfected
animals (lower left panel). Quantitative representation of all the coronal section analysis between the groups in square millimeters confirmed less perithrombotic enhancement in the vIL-10 group compared with that in controls (ⴱ, p ⫽ 0.056, by ANOVA; subgroup comparison by t test was p ⫽ 0.026,
saline vs vIL-10, ␤-gal vs vIL-10).
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vs. ␤-gal). Similarly, Mo extravasation was reduced by 27% ( p ⬍
0.05), and lymphocytes in the vein wall were rare in all groups.
To corroborate in vivo the reduction in vein wall leukocytes as
well as physiologic effect, Gd-enhanced MRV was performed on
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vIL10 GENE TRANSFER DECREASES PERITHROMBOTIC INFLAMMATION
Thrombus size and procoagulant parameters are not affected by
vIL-10 transfection
FIGURE 5. A, Vein tissue (clot and wall) TNF-␣ and IL-1␤ levels in
picograms per milligram of protein. Rat saline control, ␤-gal control, and
vIL-10 transfection are represented. A trend is noted toward decreased
TNF-␣ levels in the vIL-10 group, but this did not reach statistical significance. B, Vein tissue 6-keto-PGF-1␣ levels reflect PGI2 levels. Note that
vIL-10 transfection is associated with the highest levels and is significantly
greater than that in the promoter control (ⴱ, p ⬍ 0.05, by t test).
between the groups (n ⫽ 5– 6; p ⬎ 0.05, by ANOVA). To test that
endogenous rat IL-10 was not accounting for the differential effects
observed, an ELISA for murine IL-10 that does not cross-react
with vIL-10 was used. Indeed, no significant differences were
found in local IL-10 levels, although a trend toward lower concentrations was found in the transfected animals (n ⫽ 2– 4 groups
of three pooled animals; saline, 918 ⫾ 43 pg/mg protein; ␤-gal,
569 ⫾ 185 pg/mg protein; vIL-10, 604 ⫾ 90 pg/mg protein).
Prostacyclin was estimated as its stable breakdown product,
6-keto-PGF1␣, and is an antithrombotic mediator and an indirect
marker of inflammation. Interestingly, PGI2 levels were significantly higher in the vIL-10-transfected group compared with those
in the ␤-gal control (47 ⫾ 10 vs 21.9 ⫾ 3 ng/mg protein; p ⱕ 0.05,
by t test; Fig. 5B) and was not significantly ( p ⫽ 0.3) different
from that in the saline control (n ⫽ 5– 6).
Viral IL-10 transfection is associated with decreased CAM
levels
We focused on endothelial-luminal-clot border staining, as leukocytes probably migrate inward from the retracted clot-vein wall
Clot weight normalized to length was not reduced by vIL-10 expression (saline, 79 ⫾ 8 mg/cm; ␤-gal, 65 ⫾ 7 mg/cm; vIL-10,
65 ⫾ 7 mg/cm; n ⫽ 10 –15). No difference in vein-clot gross
morphology was found between the groups. In accordance with the
anatomical findings, functional TF activity, TF protein levels, and
vWF levels were not significantly different in the vIL-10 group
compared with the controls (n ⫽ 3–5; Fig. 8). Nonclotted vein wall
analysis showed 2- to 3-fold less TF activity and Ag levels (data
not shown). Thus, no direct difference in functional or biochemical
measures of tissue coagulant activity was found with vIL-10 expression or adenovirus transfection.
Discussion
IL-10 decreases perithrombotic inflammation
In this animal model of venous thrombosis, we found that local
expression of vIL-10 decreased inflammation. This finding correlates well with the results of our previous study in which hIL-10
given at time of thrombus induction significantly reduced inflammation (5). The absolute reduction in leukocytes (and the differential) was similar whether IL-10 was intermittently administered
or constantly expressed, as presented here, suggesting a limit for
the maximal anti-inflammatory effect. That no significant difference in rat cellular IL-10 levels was found between the groups
suggests that the observed anti-inflammatory effect was specifically mediated by vIL-10 and not by endogenous IL-10 up-regulation. Indeed, the transfected control and vIL-10 rats had lower
native IL-10 levels than saline controls. Although the local vIL-10
tissue levels were not directly determined, an unquestionable effect
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interface. By 2 days postligation, about one-third of the clot is
retracted from the vein wall, and some blood flow around and in
the clot is present (from dilated collaterals and channels). Immunohistochemistry was performed on permanent tissue sections to
qualitatively assess CAM intensity between the groups (n ⫽ 4).
These data suggested qualitatively less intense luminal and medial
staining of P- and E-selectin (Fig. 6). Low intensity staining of
ICAM-1 was noted in all groups, with no obvious difference seen
between the groups (data not shown). Importantly, background
staining was negligible and carefully controlled with each section
(data not shown). Some positive medial staining of P- and E-selectin in leukocytes was noted in all groups, suggesting possible
internalization of Ag by the emigrating cells. Vein tissue section
hematoxylin and eosin staining not only yielded the already discussed quantitative leukocyte counts, but also spatially revealed
leukocytes in various stages of emigration, again with greater numbers in the saline and ␤-gal control groups compared with the
vIL-10-transfected rats.
To better quantify CAM expression in the vIL-10 group compared with the control groups, an ELISA for the solubilized CAMs
was performed. In accordance with the immunohistochemical
analysis, all CAMs were reduced with vIL-10 expression (n ⫽
5– 6; Fig. 7). Specifically, E-selectin was most markedly reduced
(nearly 14-fold), while P-selectin was reduced 4-fold compared
with levels in the control groups ( p ⱕ 0.05). ICAM-1 was also
significantly reduced (3-fold) compared with the controls ( p ⱕ
0.05). However, this analysis does not take into account which cell
types specifically have reduced CAM expression. Nonclotted vein
tissue homogenates had negligible levels of CAMs (data not
shown), reflecting low basal expression as well as the fact that no
clot (or inflammatory nidus) was present.
The Journal of Immunology
2137
was seen by two parameters of inflammation, vein wall leukocyte
emigration and Gd-MRV. It is important to note that the aforementioned measures of inflammation correlate well with other
measures of inflammation, including Evan’s blue dye extravasation (capillary leak) and myeloperoxidase activity (marker of polymorphonuclear cells (PMN)) (4 – 6, 13). Also congruent with our
prior study was the finding that vIL-10 reduces PMN extravasation
to the greatest extent of all the leukocytes. Neutrophils are the most
prevalent early leukocyte after thrombosis and are probably a primary mediator of vein wall damage (5–7).
Other investigators have found similar anti-inflammatory efficacy with vIL-10 transfection in various experimental settings.
For example, the collagen-induced arthritis rat model is similar
to our model, in that a chronic inflammatory stimulus is present.
With Ad-vIL-10 transfection, significantly decreased arthritic
inflammation compared with that in controls was observed (20).
Other animal models in which an inflammatory nidus is not
present also support the thesis that local vIL-10 expression is
anti-inflammatory. Specifically, in a murine cardiac allograft
transplant model, retrovirally mediated vIL-10 transfection improved allograft survival by 3-fold (18). In a similar model
using adenovirus-mediated vIL-10 transfer, vIL-10 expression
accounted for a 2.5-fold increase in the duration of allograft
survival (19). The authors also suggest that incorporating the
vIL-10 gene may decrease the host immune response to the
adenovirus itself, a significant problem with this method of
gene transfer (22, 23, 27–29). The anti-inflammatory effect of
vIL-10 in these studies was diminished T cell responsiveness
and cytotoxicity. We did not specifically look at these T cell
parameters, but it is likely that this effect plays only a small role
in the diminished inflammation that was observed because lymphocytic infiltration is quantitatively a small component of the total vein wall leukocytes. We also did not find any evidence of
increased inflammation in the Ad-␤-gal control group, perhaps because a significant inflammatory stimulus was already present and
“hid” any additional inflammatory effect of the viral transfection.
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FIGURE 6. Composite photomicrographs of immunohistochemistry and hematoxylin- and eosin-stained stains of rat vein segments. W, vein wall. A,
Vein wall section stained for E-selectin in a saline control rat (⫻1000). Note endothelial lining staining of modest intensity (black arrows) with a few stained
medial leukocytes. B, Vein wall section stained for E-selectin in a ␤-gal control rat (⫻1000). Most staining is on the endothelial border, as shown by black
arrows. C, Vein wall section stained for E-selectin in the vIL-10 group (⫻1000). Minimal luminal staining is seen compared with that in A and B (as
demarcated by black arrow). D, A vein wall section stained for P-selectin in a saline control rat is shown (⫻1000). Note the extensive fluffy enhancement
luminally (black arrows; E). A vein wall section stained for P-selectin in a ␤-gal control is shown (⫻1000). The staining intensity was similar to that in
D. F, Vein wall section stained for P-selectin in a vIL-10-transfected rat (⫻1000). Minimal staining was found (black arrow, G). Hematoxylin and eosin
staining of a saline control rat vein section is shown (⫻400), illustrating a heavy infiltration of leukocytes in the subendothelial (white arrowhead) and
medial areas (white arrows). The endothelial border is marked by a black arrow. H1, Hematoxylin and eosin staining (⫻1000) of a ␤-gal control, illustrating
PMN infiltration of subendothelial region (white arrow) and media (white arrowhead). H2, Hematoxylin and eosin staining (⫻1000) of a ␤-gal control,
illustrating subendothelial Mo extravasation. I, Hematoxylin and eosin staining of a vIL-10-transfected rat vein section (⫻400), showing a few scattered
leukocytes (white arrows) in the media and a relatively intact endothelial layer (black arrows).
2138
vIL10 GENE TRANSFER DECREASES PERITHROMBOTIC INFLAMMATION
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FIGURE 7. Quantitative representation of CAM levels in vein tissue
homogenates. A, The E-selectin level (picograms per milligrams of protein)
is significantly less than those in the ␤-gal and saline controls. B, The
P-selectin level (nanograms per milligram of protein) is significantly less
than those in saline and ␤-gal controls. C, The ICAM-1 level (picograms
per mg protein) is significantly less than those in saline and ␤-gal controls.
For all graphs: ⴱ, p ⬍ 0.05, by ANOVA. Note the relative absolute differences between the CAM concentrations; P-selectin is greatest, followed
by E-selectin, and ICAM-1 is the least.
FIGURE 8. Graphical representation of vein tissue coagulant markers.
No difference was found between the groups in any of these parameters
(p ⬎ 0.05, by ANOVA). A, Vein tissue homogenate procoagulant activity
as reflective of functional TF activity in milliunits per milligrams of protein. B, Tissue factor Ag levels in picograms per milligrams of protein. C,
vWF levels in vein tissue homogenates in milliunits per milligrams of
protein.
The Journal of Immunology
Also, at 4 days after transfection an adoptive immune response is
not expected.
Viral IL-10 decreases CAM up-regulation
lymphocyte adhesion (37). In contrast, IL-10 administration in a
human microvascular endothelial cell line actually stimulated Eselectin expression (38). These data suggest possibly a direct effect
of IL-10 on endothelial CAM expression. However, experiments
with human umbilical vascular endothelial cells in our laboratory
have failed to show any direct modulating effect of IL-10 (over a
wide concentration range) on E-selectin, ICAM-1, or procoagulant
activity (unpublished observations). These results underscore the
important functional heterogeneity of endothelial cells as well as
the local environmental influences (4, 7, 8). As such, we believe
that vIL-10 probably works indirectly on CAM expression.
The observation that P-selectin localizes rapidly and specifically
to the endothelial border after thrombin stimulation underlies the
importance of its role in the initial clot formation and subsequent
inflammation (12, 39). Our immunohistochemical data suggest decreased luminal expression of the selectins in the vIL-10-transfected animals, and we hypothesize that luminal expression of the
selectins is most important for leukocyte emigration. IL-10 may
decrease the CAM expression on other cell types besides endothelial cells, and our results with the homogenate ELISA were not
able to differentiate specific cell type levels. For example, IL-10
exerts an inhibitory effect on Mo ICAM-1 expression (40) that,
again, may confer an anti-inflammatory effect by counterligand
inhibition.
Viral IL-10 and its relationship to other inflammatory mediators
IL-10 is a pleiotropic cytokine derived primarily from Mo and T
lymphocytes that has many other anti-inflammatory actions besides CAM modulation and may also account for the decreased
inflammation observed in these studies. Importantly, IL-10 inhibits
Mo Ag-presenting capacity and IL-1␤ and TNF-␣ production, as
well as increases IL-1R antagonist levels and lymphocyte IL-4
production (16, 17, 41). Furthermore, the neutrophils in the vein
wall may have a diminished release of reactive oxygen intermediates, while Mo nitric oxide and chemotactic chemokines are reduced by IL-10 (42, 43). TNF-␣ is an important proximal stimulus
for CAM up-regulation (4, 8, 9), and although we only found a
trend in decreased TNF-␣ in the vIL-10-transfected animals, this
may be physiologically important. Two hypotheses may account
for the nonsignificant differences found in TNF-␣. First, the clot
environment is more static than that of other inflammation models;
thus, the TNF-␣ Ag may not be cleared as readily (falsely elevated
local levels). Secondly, the measured TNF-␣ levels may not correlate with functional activity. Alternatively, other inflammatory
mediators, such as C5a, IL-6, and IFN-␥, may also be down-regulated by IL-10 and indirectly account for the decreased CAM
levels (16, 17).
Interestingly, PGI2 was significantly less in ␤-gal compared
with vIL-10 animals. PGI2 is a vasodilatory and antithrombotic PG
that is adaptively up-regulated in chronic thrombosis (44). IL-1␤
levels correlated with PGI2 levels (although not significantly different between the groups), and it is well documented that IL-1␤
is a proximal stimulus for PGI2 production (45, 46). Thus, it seems
that vIL-10 expression preserves and may stimulate the antithrombotic PGI2 response, and that adenovirus transfection inhibits this
compensatory response to chronic thrombosis compared with that
in saline controls. Furthermore, our in vivo data are supported by
in vitro experiments showing that IL-1␤-stimulated PGI2 production is dissociated from vWF production (47). Additionally, PGI2
has anti-leukocyte adhesive properties (48) that may have contributed to the observed decrease in vein wall leukocytes in addition to
the decreased CAM expression.
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Cell adhesion molecule up-regulation occurs in the setting of multiple biologic events, such as wound healing, atherosclerosis, and
cancer. Indeed, the regulation of CAMs is very complex and controlled in part by the balance of pro- and anti-inflammatory cytokines (4, 8, 9). However, while the specific stimuli for increased
CAM expression are fairly well defined, inhibitors of expression
are less well known. In the setting of thrombosis, increased CAM
expression is seen at the onset of thrombus induction and facilitates stabilization of the fibrin-platelet leukocyte complex as well
as allows the proper recruitment of leukocytes for clearance and
resolution of the inflammatory process (8, 9, 11, 12). Most experimental data of CAM kinetics have been made in flow situations,
whereas it is less clear what occurs in a blood stasis model. Furthermore, the kinetics and response of the CAMs are different
when comparing in vitro to in vivo situations. For example, Pselectin is rapidly mobilized after a stimulus such as thrombin,
whereas E-selectin expression increases several hours later (4, 8).
However, in vivo, persistent up-regulation of both P and E selectin
may occur, depending on the inflammatory stimulus (30, 31). In
the setting of venous thrombosis, an inflammatory stimulus (clot)
is present that modulates CAM up-regulation, with P-selectin
present in greatest magnitude.
Although we have only shown an association, our data strongly
argue that vIL-10 mediates its anti-inflammatory action through
down-regulation of CAM expression. In support of this contention,
other investigators have found that IL-10 mediates some of its
anti-inflammatory activity through decreased CAM expression.
Specifically, in an acute lung injury model, IL-10, but not IL-4,
significantly decreased lung injury (hemorrhage and myeloperoxidase activity), and this was associated with decreased ICAM-1
levels, probably due to concurrently decreased TNF-␣ levels (32).
An IL-10 knockout mouse model further enforces the close relationship between IL-10 and selectin regulation. After a low grade
septic insult, the IL-10 knockout animals had markedly increased
leukocyte adhesion in mesenteric venules and increased capillary
leak and myeloperoxidase activity compared with wild-type animals. Importantly, when anti-P- and anti-E-selectin Abs were
given, reduction of the leukocyte adherence was observed, suggesting unbalanced CAM up-regulation that was not seen in the
wild-type mice (33). Even more convincingly, administration of
either Ab to P-selectin or a specific soluble P- and E-selectin antagonist, P-selectin glycoprotein ligand-1, significantly reduced inflammation and thrombus, as measured biochemically, anatomically, and by Gd-MRV in a non-human primate model of venous
thrombosis (14, 15). Both of these models involved experimental
analysis beyond 2 days, suggesting that early inhibition of the
selectin-mediated events may confer later anti-inflammatory effect
and preserve vein wall integrity. Other models support the interrelation between IL-10 and CAMs. IL-10 similarly blunted inflammation secondary to myocardial ischemia-reperfusion through an
ICAM-1 mechanism (34), while in a mouse septic shock model,
IL-10 reduced liver injury and mortality through an ICAM-1 and
VCAM mechanism (35).
In vitro cell culture systems have yielded some conflicting insights into the modulatory action of IL-10 on CAM expression. In
an LPS-stimulated glial cell line, IL-10 significantly reduced the
expression of ICAM-1 but not its message level (36). Similarly, an
IL-1-stimulated human umbilical vascular endothelial cell line had
ICAM-1 and VCAM expression significantly reduced by IL-10 in
a dose-dependent manner and correlated with diminished Mo and
2139
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vIL10 GENE TRANSFER DECREASES PERITHROMBOTIC INFLAMMATION
Viral IL-10 does not affect tissue coagulation mechanisms
Acknowledgments
The interrelationship between CAM up-regulation and thrombosis
is well defined (7–9, 11–15). P-selectin may be particularly important, as it directly links platelets, leukocytes, and endothelial
cells to propagate thrombus. Our prior experiments support the
interrelation between inflammation and thrombus production, as
exogenous IL-10 decreased clot weight compared with control values (5). In the current study, however, expression of vIL-10 did not
inhibit thrombus production as assessed by clot weight, nor did it
affect any of the clotting parameters measured. This might relate to
a difference in receptor binding constants between vIL-10 and endogenous cellular IL-10 despite the high sequence homology between these molecules (41). IL-10 decreases Mo TF activity (49),
and thus, systemic administration may more fully deactivate this
than when vIL-10 is localized at the thrombus site. This hypothesis
is also supported by the fact that only IL-10 given at the time of
thrombus induction was an effective antithrombotic agent (5). It is
more likely that adenovirus transfection itself stimulated the procoagulant state that was not overcome by vIL-10. Adenovirus
transfection (with or without ␤-gal expression) in normal rabbit
arteries is associated with a significant inflammatory response, increased CAM expression, and the development of neointimal hyperplasia compared with that in nontransfected cells (22). The host
inflammatory response to adenovirus infection may occur even in
the absence of gene expression (27) and does not appear to be
specifically related to ␤-gal expression (22). This inflammatory
response may be reduced by adventitial rather than luminal delivery of the vector (29). Certain viral vectors may also promote
procoagulant changes in transfected endothelial cells (28). We did
not find that either procoagulant activity or TF Ag was significantly elevated in the ␤-gal control transfected animals, but this
may be because the maximum thrombus and local inflammatory
milieu stimulus was already present.
We thank J. Ford, M. Varma, and S. Wrobleski for their technical
assistance.
Local expression of vIL-10 effectively reduces early vein wall inflammation, probably by indirect down-regulation of P- and Eselectin and ICAM-1 expression. The important question remains,
however, as to whether this reduces late vein wall damage or affects the development of chronic venous insufficiency. Additional
experiments to better delineate the differences between vIL10 and
rIL10 are planned by systemic administration, which might abrogate differences in gene expression variability. Using specific
CAM knockout mice would also clarify the actions of vIL10 in this
system. Although our methodology of segmental transfection of an
anti-inflammatory mediator was used here as a tool to delineate its
effect and mechanisms, clinical applicability is foreseeable. However, before clinical use of such an anti-inflammatory cytokine,
further physiologic experimental measures, such as distal femoral
venous pressure, ultrasound analysis of clot resolution and vein
wall thickness, and vein wall analysis for fibrosis, should be undertaken. Furthermore, longer duration transfection studies to determine the durability of the effect of anti-inflammatory cytokines
would be important before any clinical use. Given the unremitting
nature of chronic venous insufficiency (CVI) and the increased
likelihood of rethrombosis in an anatomically diseased segment,
local gene-specific anti-inflammatory or anti-coagulant mediators
are appealing. The complications from long term anticoagulation
are significant (1), and selectively preventing vein wall rethrombosis and/or inflammatory damage without systemic anticoagulation is important and worth further investigation.
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