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Prepublished online as a Blood First Edition Paper on January 23, 2003; DOI 10.1182/blood-2002-10-3313.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Medicine, Division of
Hematology, Oncology and Transplantation, and Department of Mechanical
Engineering, University of Minnesota, Minneapolis.
Inflammation may play an essential role in vaso-occlusion in
sickle cell disease. Sickle patients have high white counts and elevated levels of serum C-reactive protein (CRP), cytokines, and
adhesion molecules. In addition, circulating endothelial cells, leukocytes, and platelets are activated. We examined 4 transgenic mouse
models expressing human Sickle cell disease, one of the most common
inherited hematologic diseases, is caused by a single amino acid
substitution in the The development of transgenic mice that express the human
We report here that transgenic sickle mice, like human sickle cell
patients, have an active inflammatory response. We hypothesize that
anti-inflammatory agents may minimize vaso-occlusion and tissue injury
in sickle cell disease. Transgenic sickle mice appear to be good animal
models to test this hypothesis.
Reagents were obtained from Sigma Aldrich (St Louis,
MO) unless otherwise indicated.
Mice
The values in Table 1 for percentage of Normal male and female mice (C57BL/6J) obtained from Jackson Laboratory
(Bar Harbor, ME) were used as controls for sickle mice. Mice were age-
and sex-matched for all studies. The mice used in these studies were
between 10 and 20 weeks of age and were housed in specific
pathogen-free housing to prevent common murine infections that could
cause an inflammatory response. A few normal mice were injected
intraperitoneally with either 10 µg lipopolysaccharide (LPS;
Escherichia coli, serotype 055:B5) or 60 mg thioglycollate
medium to induce an inflammatory response; these animals were used as
positive, proinflammatory controls.
Hematocrits, white blood cell (WBC) counts, and WBC
differentials
Serum amyloid P-component (SAP) Whole blood was collected from the tail vein and allowed to clot for 5 to 10 minutes. The blood cells were spun down at 4000g for 4 minutes, and the serum was collected and frozen at 80°C until
use. Serum SAP levels were measured by enzyme immunoassay (EIA) as
previously described.44 All samples were measured in replicates of 4 or 6, and the resulting values were averaged.
Serum interleukin 6 Serum was collected and stored as described in "Serum amyloid P-component (SAP)." Interleukin 6 (IL-6) was measured by EIA according to the manufacturer's protocol (Endogen, Woburn, MA).Immunohistochemistry of lung tissue Mice were asphyxiated with CO2. The lungs were removed and frozen at80°C in tissue-freezing medium (Baxter
Scientific Products, Chicago, IL). Frozen 5-µm sections were prepared
using a cryostat and mounted onto slides. The sections were air-dried
for 10 minutes, fixed in acetone for 10 minutes at room temperature,
and stored at 80°C until immunostaining. Endogenous peroxides were
removed by immersing slides in 0.3% H2O2 in
phosphate-buffered saline (PBS), pH 7.4, for 10 minutes. Slides were
washed once in Tris (tris(hydroxymethyl)aminomethane)-buffered
saline (TBS), pH 7.5, containing 0.1% Tween 20. Slides were blocked
with 3% bovine serum albumin (BSA) in PBS (blocking buffer)
for 10 minutes at room temperature. Avidin and biotin binding sites
were blocked using specific avidin/biotin blocking reagents according
to the manufacturer's protocol (Vector Laboratories, Burlingame, CA).
Biotinylated primary monoclonal antibodies to mouse VCAM-1,
intercellular adhesion molecule 1 (ICAM-1), and platelet-endothelial
cell adhesion molecule 1 (PECAM-1) (BD Pharmingen, San Diego, CA) were
diluted 1:50 in blocking buffer and incubated with thin sections for 1 hour at 37°C in a humidified chamber. Sections were washed 3 times
with TBS, 0.1% Tween 20. Bound primary antibodies were visualized
using a Vectastain Elite ABC kit containing an avidin/biotin peroxidase complex and a Vector VIP peroxidase substrate kit according to the
manufacturer's protocol (Vector Laboratories). The VIP substrate produces an intense, violet-colored precipitate. Some frozen thin sections from the lungs of NY-S mice were double stained with primary
antibodies to VCAM-1 (BD Pharmingen) and von Willebrand factor
(Cedarlane Laboratories, Hornby, ON, Canada). The primary antibodies
were visualized with appropriate secondary antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA) labeled with
fluorescein isothiocyanate (FITC/green) (VCAM) and tetramethyl rhodamine isothiocyanate (TRITC/red) (von Willebrand factor). The
nuclei were counterstained with 4', 6 diamidino-2-phenylindole (DAPI/blue) (Vector Laboratories).
Western blots of lung VCAM, ICAM, and PECAM protein expression Mice were asphyxiated with CO2, and the left lobes of the lungs were removed and frozen in liquid N2. Lung tissue homogenate was prepared as previously described.45 The lung tissue, frozen in liquid N2, was broken into small pieces with a hammer between layers of aluminum foil, then transferred to a mortar and reduced to a fine powder in liquid N2. The thawed powder was homogenized on ice in 5 mL 0.6% Nonidet P-40 (Calbiochem, La Jolla, CA), 150 mM NaCl, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) pH 7.9, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride in a 15-ml Dounce tissue homogenizer (Wheaton, Millville, NJ) with 10 strokes of the tight-fitting pestle B. Cell debris was removed by centrifuging the crude homogenate at 500g for 30 seconds at 4°C. The lung supernatant was frozen at80°C. The lung homogenate protein
concentration was determined by the bicinchoninic acid (BCA)
protein assay (Pierce, Rockford, IL) after protein precipitation with
trichloroacetic acid.46 Lung homogenate DNA concentrations were determined by a fluorometric DNA dye-binding assay (Bio Rad, Hercules, CA). The bis-benzimide dye (Hoechst 33258) binds specifically to double-stranded (ds) DNA. RNA does not interfere significantly with
the assay. For Western blotting, lung homogenates, containing 1 µg
lung DNA per well, were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5%).
After SDS-PAGE, the homogenates were transferred electrophoretically to
poly(vinylidene diflouride) (PVDF) membranes, and
immunoblotting was performed with goat anti-VCAM-1, -ICAM-1, and
-PECAM-1 immunoglobulin G (IgG; Santa Cruz Biotechnology, Santa Cruz,
CA). Sites of primary antibody binding were visualized with
alkaline phosphatase-conjugated donkey antigoat IgG (Jackson ImmunoResearch Laboratories). The final detection of
immunoreactive bands was performed using a chemofluorescent
detection substrate (Amersham Biosciences, Piscataway, NJ).
RNA extraction and ribonuclease protection assay (RPA) of VCAM mRNA Total RNA was extracted from the right lobe of the lungs of sickle and normal mice using an RNAqueous kit (Ambion, Austin, TX). RPAs were performed with 10 µg extracted mouse lung RNA using an RPAIII kit (Ambion) and VCAM-1 and glyceraldehyde phosphate dehydrogenase (GAPDH) antisense RNA probe templates (BD Biosciences Pharmingen, San Diego, CA) labeled with [32P]UTP using a T7 Maxiscript kit (Ambion). The 32P-labeled antisense VCAM and GAPDH RNA probes were hybridized to lung mRNA overnight at 56°C and then digested with RNAase at 30°C for 45 minutes. Protected RNA fragments were separated by electrophoresis on a 5% acrylamide/8 M urea/TBE (Tris-borate-EDTA) gel.Electrophoretic mobility shift assay (EMSA) for NF- 80°C until use. Lung extracts were incubated with end-labeled 32P-dsDNA containing a consensus murine NF- B
DNA binding sequence (underlined base pairs):
5'-AGTTGAGGGGACTTTCCCAGGC-3' (Santa Cruz Biotechnology).
DNA-protein binding reactions contained lung extracts from 260 ng lung
homogenate DNA and 70 fmol radiolabeled NF-B consensus dsDNA.
Reactions were carried out in 20 mM HEPES, pH 7.9, 5 mM KCl, 0.5 mM
EDTA, 5% glycerol, 1 mM DTT, 0.5 mM phenylmethyl sulfonyl
fluoride (PMSF), 1 mg/mL BSA, 0.1% NP-40, and 250 ng poly dI/dC
(polydeoxyinosinic-deoxycytidylic acid). Binding reactions were incubated 30 minutes at room temperature. Reaction mixtures were
separated on a 6% nondenaturing polyacrylamide gel using 0.5 × TBE
running buffer. To confirm the identity of NF-B bands, some
reactions were run with an excess of unlabeled NF-B dsDNA for
competition experiments or with antibodies to the p50 or p65 subunit of
NF-B for supershift experiments (Santa Cruz Biotechnology). The
mouse NF-B EMSA bands contained both the p50 and p65 subunits (data
not shown).
Quantitation of Western blots, RPA, and NF- Statistics Results from control and transgenic sickle mice were compared using a Student t test on SigmaStat 2.0 for Windows (SPSS, Chicago, IL).
White blood cell counts (Table 2)
were elevated in all of the transgenic sickle mouse models compared
with normal controls except the NY-S mice. White counts were elevated
144%, 164%, and 206% of normal in the Berk-SAntilles
(P
SAP is a well-documented acute-phase reactant in mice with extensive
(60%-70%) sequence homology with human CRP.47,48 Serum SAP was elevated 8- to 12-fold in transgenic sickle mice compared with
normal control mice (P
Frozen thin sections of lungs were prepared from 3 mice in each model
and immunostained with specific IgG against mouse VCAM, ICAM, PECAM,
von Willebrand factor, and nonspecific control IgG. VCAM, ICAM, and
PECAM staining was visually increased in normal mouse lungs 18 hours
after an intraperitoneal injection of 10 µg LPS and in all transgenic
sickle mouse lungs relative to untreated normal control mice (Figure
1A). VCAM staining (green) in NY-S sickle
mouse lungs was often colocalized with an endothelial cell marker, von
Willebrand factor (red) (Figure 1B). The colocalized green and red
fluorescence appears yellow (Figure 1B), indicating VCAM was expressed
on the vascular endothelial cells in the lungs. ICAM staining in the
lungs was diffuse throughout the parenchyma and was not concentrated as
heavily around the blood vessels like VCAM and PECAM (Figure 1A).
Different ICAM antibodies gave a similar diffuse staining pattern
(data not shown). Interestingly, red and white blood cells are visible
in the blood vessels of NY-S/SAntilles mice (Figure 1A,
VCAM and PECAM stains). Some of the visible leukocytes appear to
be marginated.
The increased adhesion molecule staining suggested the lungs of
transgenic sickle mice were inflamed. Western blots were run on lung
homogenates to confirm and quantify increased adhesion molecule
expression in transgenic sickle mice. The lungs of sickle mice had a
significantly higher hemoglobin and protein content than normal mice as
judged by their red color and the protein-DNA ratios of the lung
homogenates (data not shown, P < .05). Therefore, equal
amounts (1 µg) of lung homogenate DNA were loaded onto gel wells to
normalize each lung sample for DNA, a better surrogate of cell number
than protein. On Western blots, VCAM, ICAM, and PECAM were up-regulated
3- to 5-fold (P < .05) in normal mice 18 hours after LPS
treatment (10 µg intraperitoneal) and in transgenic NY-S,
Berk-SAntilles, NY-S/SAntilles, and Berk-S mice
(Figure 2). Some of the ICAM and PECAM
Western blots appeared to show a doublet closely related in molecular weight. Both bands were included in the densitometry quantification. Different antibodies to ICAM and PECAM gave the same banding pattern, suggesting the doublets were related in sequence (data not shown). These data confirm and quantify the up-regulation of adhesion molecules
in transgenic sickle mouse lungs seen by immunohistochemistry.
RPAs (Figure 3) were performed to measure
VCAM mRNA in lung tissue. VCAM mRNA was normalized to GAPDH mRNA
expression. Mean VCAM/GAPDH mRNA ratios were elevated in NY-S and
NY-S/SAntilles lungs (P < .01) and in
LPS-treated (10 µg intraperitoneal, 18 hours) normal lungs
(P < .001) relative to normal untreated control lungs.
VCAM/GAPDH ratios were increased modestly by 21% to 44% in the NY-S,
Berk-SAntilles, and NY-S/SAntilles mice
compared with a 3.7-fold induction in LPS-treated normal mice. These
data suggest chronic VCAM induction in transgenic sickle mice may
depend more on posttranscriptional mechanisms, whereas acute VCAM
induction in LPS-treated normal mice may depend more on transcription
of VCAM mRNA.
As further confirmation of lung inflammation in transgenic sickle mice,
we measured lung NF-
Like human sickle cell patients, transgenic sickle mice that
express the human There were few statistically significant differences in inflammation
markers between the 4 sickle mouse models despite differences in human
Over the past 2 decades the role of the endothelium and its
interactions with sickle red and white blood cells has led to a revised
paradigm for the understanding of vasoocclusive phenomena in sickle
cell disease.3-5,51-55 Interactions of sickle red and white blood cells with vascular endothelium depend on a variety of
factors,56 including agents that promote the expression of adhesion molecules on the vessel wall. These agents such as
inflammatory cells, cytokines, oxidants, hypoxic stress, and infection
result in an adhesive, inflammatory phenotype that augments sickle red and white blood cell adherence to the endothelium. Clinical conditions, including infections, surgery, and pregnancy (all states that are
"stressors" and in some aspects proinflammatory), are associated with more vasoocclusive crises.8 The acute chest syndrome
frequently is associated with an atypical pneumonia, fever, chest pain,
and a rise in the white count and soluble VCAM.8,9,34 In
these situations, proinflammatory factors such as cytokines could
further activate endothelium and promote changes in vascular tone and permeability, anticoagulant-procoagulant balance, changes in leukocyte trafficking, induction of acute-phase reactants, and promotion of red
and white cell adhesion to an activated endothelium. High-dose intravenous dexamethasone ameliorates acute chest syndrome in children
and adolescents with sickle cell disease.57 This
anti-inflammatory agent can act on inflammation in a variety of ways,
including decreasing the production of cytokines, decreasing the
activation of leukocytes, and inhibiting NF- Even in the steady state, absolute monocytosis is seen in nearly all
sickle patients.58 Moreover, sickle leukocytes have abnormal adhesion and activation.13,19,20,59-61 White
counts are significantly elevated and are highly correlated with stroke in children with sickle cell anemia.62 High white count
may even cause sickle crisis.63 Several case reports have
linked granulocyte colony-stimulating factor (G-CSF) with induction of severe or fatal vasoocclusive crisis.63-65 In the large
multicenter trial of hydroxyurea to ameliorate sickle cell crisis,
it was noted that a rise in hemoglobin F levels inversely correlated with the frequency of crises, acute chest syndrome, leg ulcers, and
early mortality.62,66-69 However, before hemoglobin F
increases, there is a marked drop in the white blood count, and the
reduction of total white blood count is a predictor of clinical
response to hydroxyurea.68 Thus, the white cell appears to
be playing an important role in vasoocclusive crisis. Recently,
intravital microscopy of blood flow in the cremasteric venules of mice
expressing human sickle It is unclear from these studies whether the vascular inflammation is a
primary response to the polymerization of sickle hemoglobin or a
secondary response to tissue injury or infection. Infection was not a
likely contributor to inflammation in these studies, as the mice were
housed in specific pathogen-free housing with careful monitoring and
precautions taken against common murine pathogens. The inflammatory
response could be a response to both tissue injury and sickle
hemoglobin polymerization. The cellular and molecular events that
translate the Another proinflammatory family of molecules is the endothelins. Endothelin-1 is a potent vasoconstrictor and proinflammatory agonist that is significantly elevated in sickle cell disease.73-75 Endothelial cells increase their production of endothelin-1 in response to hypoxia76 and after incubation with sickled, but not unsickled, RBCs.77 Ischemia-reperfusion injury is another potential mechanism that could promote vascular inflammation and tissue injury. Sickle cell disease involves repeated, lifelong, and perhaps near-constant development of transient ischemic episodes.3 Reperfusion of tissues after interruption of their blood supply can cause free radical generation and lead to vigorous inflammatory responses and subsequent tissue damage.78 Transgenic sickle mice exhibit biochemical footprints consistent with excessive free radical generation even at ambient air and following transient induction of enhanced sickling.4,41 An ongoing reperfusion injury would be consistent with the chronic inflammatory response seen in sickle cell patients and transgenic sickle mice. Novel anti-inflammatory and antioxidant treatments may provide fruitful therapies for sickle cell disease. Transgenic sickle mice appear to be good models to study the potential benefit of anti-inflammatory therapies to prevent vaso-occlusion in sickle cell disease.
We thank Stephana Choong for breeding and characterizing the transgenic sickle mice used for these studies.
Submitted November 5, 2002; accepted January 2, 2003.
Prepublished online as Blood First Edition Paper, January 23, 2003; DOI 10.1182/blood-2002-10-3313.
Supported by National Heart, Lung, and Blood Institute grant HL67367.
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, University of Minnesota, Department of Medicine, Division of Hematology, Oncology and Transplantation, 420 Delaware St SE, MMC 480, Minneapolis, MN 55455; e-mail: belcher{at}umn.edu.
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Top
Abstract
Introduction
- and sickle 
-globin genes to determine
if they mimic the inflammatory response seen in patients. These mouse
models are designated NY-S, Berk-SAntilles,
NY-S/SAntilles (NY-S × Berk-SAntilles),
and Berk-S. The mean white counts were elevated 1.4- to 2.1-fold (P 
.01) in the Berk-SAntilles,
NY-S/SAntilles, and Berk-S mice, but not in the NY-S mice
compared with controls. Serum amyloid P-component (SAP), an acute-phase
response protein with 60% to 70% sequence homology to CRP, was
elevated 8.5- to 12.1-fold (P 
3)
more than twice that of
normal mice (Table