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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Hematology, Oncology and
Transplantation, Department of Medicine, University of Minnesota,
Minneapolis, MN.
Sickle cell anemia is characterized by painful vaso-occlusive
crises. It is hypothesized that monocytes are activated in sickle cell
disease and can enhance vaso-occlusion by activating endothelium. To
test this hypothesis, human umbilical vein endothelial cells (HUVEC)
and human microvascular endothelial cells (MVEC) with sickle and normal
mononuclear leukocytes were incubated, and endothelial activation was
measured. Endothelial cells incubated with sickle mononuclear
leukocytes were more activated than those incubated with normal
mononuclear leukocytes, as judged by the increased endothelial
expression of adhesion molecules and tissue factor and the adhesion of
polymorphonuclear leukocytes (PMNL). Monocytes, not lymphocytes or
platelets, were the mononuclear cells responsible for activating
endothelial cells. Sickle monocytes triggered endothelial nuclear
factor-kappa B (NF- Mutation of the The vascular endothelium plays a critical role in vaso-occlusion
and ischemic organ damage by several mechanisms.1,2 These
include the regulation of hemostasis and vascular tone, adhesion of red
blood cells (RBCs) and leukocytes, and possibly injury caused by
ischemia-reperfusion. Evidence indicates the vascular endothelium is
damaged in sickle disease.2 This damage includes
histopathologic changes in many vascular beds, such as the spleen,
brain, and retina of patients with sickle disease3-6; formation of thromboses at sites of underlying intimal
hyperplasia4-6; abnormal presence of circulating
endothelial cell adhesion molecules, such as intercellular adhesion
molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and
E-selectin in the plasma7; and increased numbers of
circulating endothelial cells in the blood of patients in acute painful
crisis.8-10
Circulating endothelial cells found in patients in crisis
are activated as judged by the expression of adhesion molecules and
tissue factors on their surfaces.10,11 The adhesion
molecules ICAM-1, VCAM-1, P-selectin, and E-selectin and the
procoagulant molecule tissue factor are expressed on activated
endothelium and play key roles in the recruitment of
leukocytes12,13 and the promotion of
thrombosis14 at sites of vascular inflammation.
Activation of endothelial cells is regulated in part by the
translocation of a transcription factor, NF- Reagents were obtained from Sigma Aldrich (St. Louis, MO) or
Gibco BRL (Grand Island, NY) unless otherwise indicated. Buffer A
contained 0.8% (wt/vol) NaCl, 1 mmol/L EDTA, 10 mmol/L HEPES, and
0.5% bovine serum albumin (BSA), pH 7.4. Buffer B contained Hank's
balanced salt solution, 25 mmol/L HEPES, and 0.5% BSA, pH 7.4. Buffer
C contained phosphate-buffered saline (PBS), 0.5% BSA, and 0.1%
Tween-20, pH 7.4.
Subjects
Endothelial cell culture
Mononuclear leukocyte isolation Mononuclear leukocytes used for incubation with endothelial cells were isolated on Ficoll-Hypaque density gradients.23 Mononuclear leukocytes were washed in buffer A and resuspended in buffer B.Monocyte and lymphocyte separation Monocytes and lymphocytes were isolated by rate zonal density gradient centrifugation using OptiPrep medium.24 Monocytes and lymphocytes were washed in buffer A and resuspended in buffer B. Lymphocyte preparations contained less than 1% monocytes and no polymorphonuclear leukocytes (PMNL), and the monocyte preparations contained less than 10% lymphocytes and no PMNL, as judged by fluorescence-activated cell sorting (FACS) forward and side scatter and CD14 immunostaining.Monocytes also were purified by FACS. Mononuclear leukocytes were isolated on Ficoll-Hypaque density gradients. Five percent of the mononuclear leukocytes were labeled with monoclonal anti-CD14-fluorescein isothiocyanate conjugate and analyzed on a FACStar (Becton Dickinson, San Jose, CA). A gate was placed around monocytes based on forward and side scatter and CD14 immunofluorescence. Monocytes were collected within the gate from the remaining unlabeled mononuclear leukocytes. The monocytes were washed in buffer A and resuspended in buffer B. These preparations contained less than 10% lymphocytes and platelets. Platelet isolation Platelet-rich plasma (PRP) was prepared from citric acid-citrate-dextrose (CACD) blood at 130g for 20 minutes. PRP was diluted 1:1 with 0.1 mol/L CACD, pH 6, and centrifuged at 830g for 20 minutes. The platelets were washed in CACD and resuspended in buffer B.Polymorphonuclear leukocyte isolation Normal PMNL were isolated from blood containing heparin anticoagulant using hydroxyethylstarch and Percoll gradients as described.25Endothelial cell incubations with leukocytes and platelets Given the differences in endothelial cell preparations, all experiments were performed on the same day, with the same preparation of endothelial cells, for any given pair of leukocyte or platelet preparations from patients with sickle cell disease and normal subjects. Leukocytes (1-18 mononuclear leukocytes, monocytes, or lymphocytes per endothelial cell), platelets (100 platelets per endothelial cell), or TNF- (10 ng/mL) were added to the endothelial cell media and incubated for 1 to 6 hours. After incubation, the endothelial monolayer was washed 3 times in buffer B.
For experiments in which endothelial cell expression of E-selectin, ICAM-1, VCAM-1, and tissue factor were measured, incubations of 4 or 5 hours with mononuclear leukocytes were sufficient for all proteins to be expressed. For E-selectin experiments, 3 hours were sufficient. In time-course experiments, HUVEC were incubated with monocytes or
lymphocytes for 5 minutes to 5 hours. After the indicated incubation
time, HUVEC were washed and replenished with fresh media. All HUVEC
incubations lasted a total of 5 hours, including the initial
incubations with monocytes, lymphocytes, or TNF- In experiments designed to block leukocyte-HUVEC contact, mononuclear leukocytes were incubated above HUVEC monolayers inside cell culture inserts containing polyethylene terephthalate membranes with 0.4-µm pores and pore densities of 1 × 108 pores/cm2 (Becton Dickinson, Franklin Lakes, NJ). These membranes allow high rates of basolateral diffusion. The role of cytokines in leukocyte activation of HUVEC was evaluated
using monoclonal antibodies (10 µg/mL) to block TNF- PMNL adhesion to HUVEC Normal PMNL were isolated as described above and labeled with 50 to 100 µCi 51Cr for 60 minutes. HUVEC were incubated with sickle or normal mononuclear leukocytes, TNF-, or media only for 4 hours. After these initial preincubations, HUVEC were washed and
incubated with 51Cr-labeled PMNL at a concentration of 1 to
5 PMNL per HUVEC for 15 minutes. After the second incubation, HUVEC
were washed 3 times, and the supernatant and washes from each well were
combined. Bound PMNL and HUVEC were removed from wells after overnight
incubation in 1 N NaOH. Bound and unbound
51Cr-labeled-PMNL were counted in a gamma counter. The
percentage of PMNL bound to HUVEC was calculated as CPM bound/(CPM
bound + unbound). Adhesion was measured in either triplicate or
quadruplicate wells for each of the 5 experiments.
Enzyme immunoassays for HUVEC E-selectin, ICAM-1, VCAM-1, and tissue factor Washes were in buffer B for adhesion molecules and in buffer C for tissue factor. After incubation of HUVEC with mononuclear leukocytes, monocytes, lymphocytes, platelets or TNF- in 24-well tissue culture plates, HUVEC were washed 3 times, fixed for 15 minutes
in 4% paraformaldehyde, washed, and permeabilized with cold methanol
( 20°C) for 15 minutes. Tissue factor assays were not permeabilized.
Thus, tissue factor represents cell surface protein, and E-selectin,
ICAM-1, and VCAM-1 represent intracellular and cell surface protein.
Next, HUVEC were washed and incubated 2 hours with primary mouse
monoclonal antibodies (2 µg/mL) against E-selectin, VCAM-1
(Pharmingen, San Diego, CA), ICAM-1 (R& D Systems, Minneapolis, MN), or
tissue factor (a gift from Dr Ron Bach, Veterans Administration Medical
Center, Minneapolis, MN). HUVEC were washed 3 times and incubated for 1 hour with the secondary antibody, donkey antimouse IgG alkaline
phosphatase conjugate (Jackson ImmunoResearch Laboratories, West Grove,
PA). HUVEC were washed 3 times and incubated for 60 minutes with
alkaline phosphatase substrate P-nitrophenyl phosphate in 1 mol/L diethanolamine buffer, 0.5 mmol/L MgCl2, pH 9.8. The
reaction was stopped with 3 mol/L NaOH, and 100-µL aliquots were
transferred in triplicate to 96-well plates and read at 405 nm in a
microtiter plate reader (Molecular Devices, Palo Alto, CA). Background
optical density (OD) values were measured in wells containing
substrate only and subtracted from all other values. Results are
expressed as a percentage of control (media only) HUVEC. Background OD
values from nonspecific binding of secondary antibody in the absence of
primary antibody were small, so these data were not used in the calculations.
RNA extraction and reverse transcription-polymerase chain reaction RNA was extracted from washed endothelial cells using a chloroform phenol extraction kit, RNAzol B (Tel-Test, Friendswood, TX). RNA quantity and quality were measured spectrophotometrically at 260/280 nm (DU-70; Beckman, Fullerton, CA). An extinction coefficient of 12 mg × µL 1 × OD1 × cm1
at 260 nm was used to calculate RNA concentrations.
All reagents for cDNA synthesis and amplification were obtained from Life Technologies, (Gaithersburg, MD). One microgram total RNA was used for cDNA synthesis using SuperScript II RT and a poly dTTP primer. Polymerase chain reactions (PCR) were conducted in an Attotech (Minneapolis, MN) thermocycler. A 1000-bp region of E-selectin cDNA was amplified using the following primer sequences: 5'-GCGGTACCCCTGTACATTTGACTGTGAAG-3' and 5'-GCTCTAGAAAGGCTTTGGCAGCTGCTGGCA-3'. A 462-bp region of tissue factor cDNA was amplified using the following sequences: 5'-ACTCCCCAGAGTTCACACCTTACC-3' and 5'-GGAGCTGTGGCATTTGTGGTCA-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control. A 600-bp region of GAPDH cDNA was amplified using primers 5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-GGTGGACCTGACCTGCCGTCAAGA-3'. cDNA products were separated on 2% agarose gels in TAE buffer containing 40 mmol/L Tris-acetate, 2 mmol/L Na2 EDTA, pH 8.5, in the presence of 1µg/mL ethidium bromide. Extraction of endothelial cell nuclear proteins Nuclear proteins were extracted from washed (3×) endothelial cells (5.5 × 106) using a modification of previously described methods.26 Cells were harvested with trypsin-EDTA and washed once in ice-cold media. Cells were washed twice more in PBS and resuspended 5 minutes in 100 µL cell lysis buffer containing 10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.3 mol/L sucrose, 1.5 mmol/L MgCl2, 0.5 mmol/L dithiothreitol, 0.5% NP-40 (Calbiochem, La Jolla, CA), and protease inhibitors. Nuclei were pelleted at 16 000g and washed in 100 µL cell lysis buffer. Nuclear proteins were extracted into 100 µL nuclear extraction buffer containing 20 mmol/L HEPES, pH 7.9, 0.1 mol/L KCl, 0.1 mol/L NaCl, 0.5 mmol/L dithiothreitol, 20% glycerol, and protease inhibitors. Protein concentrations were measured by bicinchoninic acid protein assay after trichloroacetic acid precipitation.27Electrophoretic mobility shift assay One hundred nanograms of ssDNA 5'-TCTCAACAGAGGGGACTTTCCGAGAGGCCATCTGG-3' containing the consensus sequence of the NF- B DNA binding site (underlined) was
made into radioactive dsDNA by fill-in labeling with
-[32P]dGTP, -[32P]dCTP, dATP, and
dTTP according to the manufacturer's protocol (Boehringer Mannheim,
Indianapolis, IN). DNA-protein binding reactions contained 10 µg
nuclear protein extract and 2 ng labeled dsDNA. Reactions were carried
out in 20 mmol/L HEPES, pH 7.9, 5 mmol/L KCl, 0.5 mmol/L EDTA, 5%
glycerol, 1 mmol/L dithiothreitol, 0.5 mmol/L PMSF, 1 mg/mL BSA, 0.1%
NP-40, and 250 ng poly dI/dC. Some reactions contained a 25-fold excess
of unlabeled NF-B-specific dsDNA to serve as a cold competitor with
32P-labeled dsDNA or 2 µg of goat anti-NF-B p65 or
p50 IgG (Santa Cruz Biotechnology, Santa Cruz, CA) to supershift the
NF-B band. Binding reactions were incubated for 30 minutes at room
temperature and separated on a 6% nondenaturing polyacrylamide gel
using 0.5× TBE running buffer containing 44.5 mmol/L Tris, 44.5 mmol/L
boric acid, and 1 mmol/L EDTA, pH 8.
Measurement of monocyte activation by FACS Leukocytes were obtained from blood after lysis of RBCs in isotonic ammonium chloride buffer.28 They were labeled with monoclonal anti-CD14-FITC (Becton Dickinson, San Jose, CA) and either anti-CD11b-phycoerythrin (PE; Becton Dickinson), anti-TNF--PE (Pharmingen, San Diego, CA), or anti-IL-1 -PE (R&D
Systems) conjugate antibodies. Leukocytes were fixed and permeabilized
(Pharmingen) before immunostaining for CD14/TNF- or CD14/IL-1.
Labeled cells were washed and resuspended in 0.5% formaldehyde
in PBS-EDTA.
FACS was performed on a FACSCalibur (Becton Dickinson). Mononuclear
leukocytes were identified and gated by forward and side scatter.
Monocytes were identified within the gate using CD14-specific immunofluorescence. Monocyte activation was measured as the mean fluorescent intensity of monocyte TNF- C-reactive protein measurement C-reactive protein (CRP) was measured by rate nephelometry on a Beckman Coulter Array 360 system (Beckman Coulter, Brea, CA) in the clinical laboratory of Fairview University Medical Center.Statistics Normal and sickle groups were compared using a Student t test or a Mann-Whitney rank sum test run on SigmaStat 2.0 for Windows (SPSS, Chicago, IL). Data presented without statistics are from individual experiments, which are representative of 2 or more experiments.
Pretreatment of HUVEC for 4 hours with mononuclear leukocytes from
patients with sickle disease increased the adhesion of normal PMNL to
HUVEC 3-fold compared with HUVEC pretreated with mononuclear leukocytes
from normal subjects (P < .05) and 8-fold compared with
control HUVEC (Figure 1). TNF-
Sickle and normal mononuclear leukocytes were compared for their
ability to activate adhesion molecule and tissue factor expression on
endothelial cells. HUVEC were incubated 5 hours with sickle or normal
mononuclear leukocytes. Adhesion molecule and tissue factor protein
expression on HUVEC were measured by enzyme immunoassay (Figure
2). Sickle mononuclear leukocytes
increased E-selectin expression 1.8-fold compared with normal
mononuclear leukocytes (594% vs 329%, P < .05; control
HUVEC = 100%). ICAM-1 expression was increased 1.3-fold (338% vs
254%, P < .05) VCAM-1 expression was increased 1.4-fold
(354% vs 248%, P < .05), and tissue factor expression
was increased 1.9-fold (495% vs 263%, n = 2). TNF-
The data presented for E-selectin, ICAM-1, and VCAM-1 represent total protein expression (intracellular + cell surface expression) by HUVEC because the cells were permeabilized with methanol during the immunoassay procedure. However, the tissue factor data represent cell surface expression by HUVEC because the cells were not permeabilized for the tissue factor immunoassays. In one study, HUVEC were stimulated with sickle mononuclear leukocytes, and subsequent immunoassays for E-selectin and VCAM-1 were run with and without cold methanol permeabilization to differentiate between intracellular and cell surface protein expression. Intracellular and cell surface E-selectin expression peaked at 4 and 5 hours, respectively (data not shown). Virtually all the E-selectin was found on the cell surface after 5 hours. Intracellular and cell surface VCAM-1 expression peaked at 5 and 6 hours, respectively (data not shown); however, VCAM-1 expression was not measured beyond 6 hours. Virtually all the VCAM-1 protein was found on the cell surface at 6 hours. HUVEC E-selectin mRNA levels were examined by reverse transcription
(RT)-PCR after incubation for 5 hours with sickle and normal
mononuclear leukocytes. E-selectin mRNA levels were up-regulated in
HUVEC incubated with sickle and normal mononuclear leukocytes compared
with control HUVEC (Figure 3). E-selectin
up-regulation was greater in HUVEC incubated with sickle mononuclear
leukocytes than in normal mononuclear leukocytes. HUVEC pretreated with
TNF-
Because both monocytes and endothelial cells can express tissue factor
in response to an inflammatory stimulus,29-31 it
was possible that the observed tissue factor expression was associated with monocytes bound to the endothelial monolayer. However, this possibility was unlikely because virtually all the mononuclear cells
were removed from endothelial cells by washing 3 times after incubation. We used immunofluorescence staining and laser confocal microscopy to confirm that tissue factor was expressed on endothelial cells. Tissue factor immunofluorescence was strongly up-regulated on
endothelial cells after incubation with TNF- Genes for E-selectin, ICAM-1, VCAM-1, and tissue factor all have
NF-
The data presented in Figures 1 to 4 were generated using mononuclear
leukocytes prepared by Ficoll-Hypaque density gradient centrifugation.
These preparations contained monocytes, lymphocytes, and platelets. To
determine which cell type(s) was responsible for endothelial cell
activation and to exclude the possibility that the observed differences
were caused by an artifact or contamination of the mononuclear
leukocyte preparations, 2 additional isolation techniques were
used To exclude the possibility that lymphocytes contributed to endothelial
cell activation, lymphocytes and monocytes from patients with sickle
disease were separated by rate zonal density gradient centrifugation
using OptiPrep medium. The lymphocyte preparations contained less than
1% monocytes and no PMNL, and the monocyte preparations contained less
than 10% lymphocytes and no PMNL, as judged by flow cytometry analysis
of forward and side scatter and immunostaining of CD14. However, some
CD61+ platelets remained within the monocyte preparation.
HUVEC were incubated with these purified sickle monocytes, lymphocytes,
or TNF-
Mononuclear leukocyte preparations obtained by Ficoll-Hypaque density
gradient centrifugation and the monocyte preparations obtained by
OptiPrep rate zonal density gradient centrifugation contained
platelets. However, preparations from patients with sickle disease and
normal subjects contained similar numbers of platelets. Mononuclear
leukocytes isolated by Ficoll-Hypaque from normal subjects contained on
average 57 ± 41 (mean ± SD, n = 3) platelets per mononuclear
leukocyte, and preparations from patients with sickle disease contained
40 ± 28 (n = 3). To exclude the possibility that platelets were
contributing to endothelial cell activation, purified platelets from
patients with sickle disease and normal subjects were incubated with
HUVEC for 4 hours in the presence and absence of mononuclear
leukocytes. Incubation of HUVEC with sickle or normal platelets (100 platelets per HUVEC) for 4 hours did not stimulate HUVEC expression of
E-selectin and ICAM-1 above control HUVEC incubated with media alone
(Table 1). Moreover, in a separate
representative experiment, the addition of platelets to mononuclear
leukocytes did not augment, and in most cases slightly decreased, HUVEC
expression of E-selectin and ICAM-1 (Table 1). These data suggest that
platelets did not contribute to endothelial cell activation.
Because sickle monocytes were extremely potent in their ability to
activate endothelium, we wondered whether cell-to-cell contact of
monocytes and endothelial cells was required to achieve activation. To
prevent cell-to-cell contact, cell culture inserts separated monocytes
and endothelial cells with 0.4-µm, high-pore density membranes during
a 4-hour incubation. HUVEC E-selectin, ICAM-1, VCAM-1, and tissue
factor expression were increased by 400% to 600% above control HUVEC
(Figure 6). These results demonstrate that HUVEC can be activated by sickle monocytes without cell-to-cell contact. However, when HUVEC were incubated with sickle monocytes in
the absence of a cell culture insert membrane, E-selectin, ICAM-1,
VCAM-1, and tissue factor expression was increased 800% to 1300%
above control HUVEC (Figure 6), indicating that HUVEC activation is
further enhanced by cell-to-cell contact of sickle monocytes and
endothelial cells.
Because monocytes are known to secrete cytokines TNF-
We measured TNF-
CD11b expression on the monocyte cell surface was measured as an
additional marker of activation. Leukocytes were obtained from blood
after the lysis of RBCs. Leukocytes were immunostained for surface
expression of CD11b and CD14. CD11b, an
Another marker of systemic inflammation is serum CRP, an acute-phase reactant. CRP was measured in 13 patients and normal subjects. Patients had mean ± SD CRP of 3.0 ± 5.1 mg/dL, and normal subjects had mean CRP of 0.3 ± 0.1 mg/dL (P = .003) (data not shown). Taken together, these data indicate that the vasculature of patients with sickle cell disease is in a state of inflammation.
These studies support our hypothesis that monocytes from patients
with sickle cell anemia are activated and can enhance vaso-occlusion through an endothelial inflammatory response promoted by the
NF- We observed that blocking antibodies to TNF- One may ask, how are sickle monocytes activated? How does the
mutation in hemoglobin translate into monocyte activation? In any given
patient, multiple pathways for monocyte activation may be operational.
Perhaps unique features of the sickle erythrocyte promote activation of
the monocyte. One possibility is that increased erythrophagocytosis of
senescent sickle RBCs by monocytes stimulates monocyte activation.
Another pathway could be RBC microparticles; human RBCs shed plasma
membrane-derived exocytic microvesicles or microparticles in vivo. RBC
microparticles have been found in increased numbers in several vascular
diseases, including sickle cell disease and
An important mechanism whereby vascular inflammation could contribute to vaso-occlusive crisis is through the up-regulation of tissue factor expression. Tissue factor is the coagulation system's triggering mechanism.14 Previous studies have shown that whole blood tissue factor procoagulant activity associated with mononuclear leukocytes is elevated in sickle disease.44 Tissue factor expression is significantly elevated on circulating endothelial cells isolated from patients with sickle cell disease11 compared with normal subjects. Expression is greater when patients have acute vaso-occlusive episodes. Elevated tissue factor expression on activated monocytes and endothelium in sickle cell disease may be a powerful determinant of vaso-occlusive crisis. Moreover, platelets from patients with sickle cell disease are activated45-47 providing ideal conditions for vaso-occlusive crisis. Platelets can adhere to monocytes through thrombospondin cross-linking of glycoprotein IV on the surface of both kinds of cells.48 Mononuclear leukocyte preparations obtained by Ficoll-Hypaque density gradient centrifugation contained substantial numbers of platelets and lymphocytes. However, there were no significant differences in platelet or lymphocyte contamination between sickle and normal mononuclear leukocyte preparations. Moreover, the addition of purified sickle or normal platelets or lymphocytes to endothelial cells in culture in the presence and absence of sickle or normal monocytes did not augment the expression of endothelial cell adhesion proteins. In addition to activated monocytes, platelets, endothelial cells, and circulating cytokines, there are other signs of inflammation in sickle disease. These include elevated serum CRP (this paper and 49-51) and the abnormal presence of adhesion molecules in plasma, such as E-selectin, ICAM-1, and VCAM-1.7 Moreover, many clinical features of sickle cell disease are similar to other inflammatory processes, such as fever, local edema, erythema, leukocytosis, and elevated erythrocyte sedimentation rate. These observations provide a molecular framework for treating sickle
cell disease as an inflammatory disease of the vasculature. An
important question pertains to the old chicken-and-egg dilemma: which
comes first, inflammation or vaso-occlusion? Does vascular inflammation
The authors are deeply indebted to the many patients with sickle cell disease and the control subjects who donated blood for these studies. We thank Ms Jean Harkness for her help in coordinating and procuring blood samples from our patients. We also thank Dr Steve Nelson at the Minneapolis Children's Hospital and Dr Chris Moertel at St. Paul Children's Hospital for coordinating and procuring blood samples from their patients. We thank Mr Chris Bryant for his excellent technical skills and perseverance in the laboratory and Ms Julia Nguyen for her extraordinary help and skills in the isolation and culture of endothelial cells.
Submitted October 18, 1999; accepted May 24, 2000.
Supported by National Institutes of Health grant HL55552.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: John D. Belcher, Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Box 480 UMHC, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: belcher{at}tc.umn.edu.
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Top
Abstract
Introduction
monocytes isolated by rate zonal density gradient centrifugation
using OptiPrep medium and monocytes isolated by FACS. These monocyte
preparations were highly enriched with CD14 monocytes and had
less contamination with CD3 lymphocytes and CD61 platelets. These
monocyte preparations from patients with sickle cell disease continued
to activate HUVEC adhesion molecule expression more than preparations
from normal subjects (data not shown).
