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Prepublished online as a Blood First Edition Paper on January 2, 2003; DOI 10.1182/blood-2001-12-0289.
RED CELLS
From the Departments of Pharmacology and Medicine,
Center for Thrombosis and Hemostasis, University of North Carolina at
Chapel Hill (UNC-CH); Department of Hematology, Duke University,
Durham, NC
The vasoocclusive crisis is the major clinical feature of sickle
cell anemia, which is believed to be initiated or sustained by sickle
(SS) red blood cell (RBC) adhesion to the vascular wall. SS RBCs, but
not unaffected (AA) RBCs, adhere avidly to multiple components of the
vascular wall, including laminin. Here we report a novel role for
epinephrine and cyclic adenosine monophosphate (cAMP) in the
regulation of human SS RBC adhesiveness via the laminin receptor, basal
cell adhesion molecule/Lutheran (BCAM/Lu). Our data demonstrate that
peripheral SS RBCs contain greater than 4-fold more cAMP than AA RBCs
under basal conditions. Forskolin or the stress mediator epinephrine
further elevates cAMP in SS RBCs and increases adhesion of SS RBCs to
laminin in a protein kinase A (PKA)-dependent manner, with the
low-density population being the most responsive.
Epinephrine-stimulated adhesion to laminin, mediated primarily via the
Sickle (SS) red blood cells (RBCs), unlike
unaffected (AA) RBCs, adhere avidly to components of the vascular wall,
and this abnormal adhesion is believed to contribute to the painful
vasoocclusive crises that occur in patients with sickle cell
anemia. Laminin is an extracellular matrix (ECM) protein distributed
throughout most vascular beds,1 with isoforms 10 and 11 specifically supporting more SS RBC adhesion than other ECM proteins
tested under physiologic shear and flow conditions.2
Patients with sickle cell anemia have extensive endothelial
damage,3 likely bringing the underlying matrix laminin
into direct contact with flowing blood. In addition, plasma levels of
laminin are elevated in sickle cell disease,4 suggesting
that laminin may be deposited on the surface of the endothelium where
it could also serve as an adhesive substrate. Udani et al5
have demonstrated that the basal cell adhesion molecule and its isoform
Lutheran (BCAM/Lu) are the major RBC receptors for laminin under basal
conditions. Furthermore, recent studies demonstrating that the adhesive
state of SS RBCs can be modulated by signal transduction6
set a precedent to explore the regulation of SS RBC adhesion to laminin.
RBCs have traditionally been viewed as simple conduits for oxygen
transport; however, these cells contain a broad range of signaling
molecules.7 Some of the most well-studied signaling pathways in RBCs are mediated by the second messenger cyclic adenosine monophosphate (cAMP),8,9 generated by the conversion of
adenosine triphosphate (ATP) to cAMP via membrane-associated
adenylyl cyclase. The downstream effects of cAMP are primarily
attributed to cAMP-dependent protein kinase A (PKA), the most
well-studied downstream effector of cAMP.10 This pathway
is important in down-regulating the adhesion of other hematopoietic
cells, such as the rapid cAMP- and PKA-dependent reduction in platelet
adhesiveness,11-16 and a similar, albeit more gradual,
reduction in leukocyte adhesiveness mediated by this
pathway.17,18 In contrast, more limited reports describe a
gradual increase in the adhesion of leukocytes to various substrates,19 as well as the adhesion of T lymphocytes to
laminin,20 in response to cAMP- and PKA-mediated signaling
pathways. However, the adhesive effects of this pathway on RBCs,
whether normal or diseased, have never been explored.
Here, we describe an unusually rapid cAMP- and PKA-dependent activation
of SS RBC adhesion to laminin. The physiologic stress mediator
epinephrine, acting largely through the Reagents
Red blood cell preparation
Arabinogalactan density gradient RBC fractionation Washed RBCs were diluted 1:1 in an isotonic universal diluent (Larex, St Paul, MN) for 5 minutes. Next, 2 mL undiluted arabinogalactan cell separation medium (Larex) was layered at the bottom of an ultracentrifuge tube, followed by 3 mL of 1 part universal diluent to 2 parts cell separation medium solution. Finally, 3 mL of 1 part packed RBCs to 1 part diluent solution was layered on top of the gradient. The RBCs were centrifuged at 74 000g for 45 minutes at 20°C. The top 15% of RBCs (low-density fraction) was carefully aspirated, followed by the bottom 15% of RBCs (high-density fraction). These fractions were packed in PBS at 400g for 10 minutes and resuspended in HBSS.Cyclic AMP enzyme-immunoassay Total cAMP was measured in 1 × 108 RBCs as described in the Amersham Pharmacia Biotechnology (Piscataway, NJ) cAMP enzyme-immunoassay system protocol 4 (total cellular cAMP measurement). Briefly, 160 µL of 1 × 108 RBCs was added per well in a 96-well tissue culture plate. Next, RBCs were incubated at 37°C, and 20 µL desired cAMP-modulating agent was added. RBCs were lysed in 20 µL lysis reagent (2.5% solution of dodecyltrimethylammonium bromide). The 96-well plate was agitated for 30 minutes to ensure complete lysis, and total cAMP levels were measured according to the manufacturer's instructions.Construction, expression, and purification of soluble BCAM/Lu recombinant protein A cDNA construct of the BCAM/Lu extracellular domain was generated from the full-length Lutheran cDNA by polymerase chain reaction (PCR), using the forward oligonucleotide primer 5'-AACATGGAGCCCCCGGACGCA-3' and the reverse primer 5'-CTGGGGGCTCACGGCGC-3'. The resulting cDNA encoding the extracellular domain fragment Met1 to Gln543 was subcloned into the pcDNA3.1/V5-His-TOPO expression vector (Invitrogen, Carlsbad, CA), which provides both a V5 epitope and a polyhistidine C-terminal sequence. This construct was stably transfected into 293 cells with Lipofectin (Invitrogen, Carlsbad, CA) and subsequently adapted to 293 serum-free medium (SFM) II containing 0.5 mg/mL Geneticin (Invitrogen). The secreted recombinant Lutheran protein (BCAM/Lu) produced by transfected 293 cells was purified using the Xpress protein purification system (Invitrogen) under native conditions.Flow adhesion assay RBC adhesion to laminin was measured under physiologic flow conditions using a parallel plate flow chamber as previously described.21,22 Briefly, 0.75 µg purified human laminin in PBS was immobilized on identical wells formed by a silicon gasket seated into a 35-mm polystyrene tissue culture dish (Becton Dickinson, Franklin Lakes, NJ) by incubating for 2 hours at 37°C. A 1% hematocrit of RBCs (1.5 mL) was flowed across the immobilized laminin at a constant flow rate of 1.0 mL/minute and shear stress of 1 dyne/cm2. After a 6-minute wash, adherent RBCs were quantified in 4 representative areas by microscopy (×200). Occasional contaminating WBCs were excluded from all counts.Staining of adherent RBCs following the flow assay Following the flow adhesion assay, the vacuum seal was gently removed followed by separation of the chamber from the underlying tissue culture dish. Each well was gently washed twice with HBSS and twice with PBS. Immediately afterward, adherent RBCs were simultaneously fixed and stained with methylene blue and washed with deionized, distilled (dd) H2O. Adherent RBCs were counted microscopically (40× oil immersion).Data analysis Figures represent averages across patients, shown with standard deviation bars, or selected representative patients. A 2-tailed paired t test was used to assess the statistical significance between cAMP levels or adhesion of treated versus untreated samples where indicated. All other data analyses relied on descriptive and graphical methods.
To compare the basal levels of cAMP in SS versus AA RBCs, total
cellular cAMP levels were measured under unstimulated conditions. We
found that SS RBCs exhibited a broad range of basal cAMP levels, 4.4-fold higher on average than the consistently low levels measured in
AA RBCs (Figure 1A). We then asked if
cAMP production could be stimulated beyond the elevated basal levels in
SS RBCs via forskolin-mediated activation of adenylyl cyclase. A time
course of forskolin treatment of SS RBCs from 3 separate patients
demonstrated that cAMP levels increased approximately 3-fold on average
above baseline (Figure 1B). Although the absolute cAMP levels induced by forskolin varied from patient to patient, maximum stimulation consistently occurred within 15 minutes. In contrast, minimal cAMP
production occurred in forskolin-treated AA RBCs (Figure 1B). To our
knowledge, these data represent the first evidence of differential
basal and stimulated cAMP levels between intact peripheral SS and AA
RBCs.
To determine how the stimulated cAMP levels affect adhesion of SS
versus AA RBCs, we again treated SS RBCs with forskolin and found that
SS RBC adhesion to laminin was stimulated to various extents (ie,
greater than 1.5 fold) in 3 patients, a modest stimulation in 1 patient, and no stimulation in 3 other patients (Figure
2A). However, preincubation of SS RBCs
with a protein kinase A inhibitor (PKAI) prior to forskolin
stimulation inhibited forskolin-stimulated adhesion to below basal
levels in most patients (P < .05). These data suggest
that cAMP-mediated PKA activation not only stimulates a more adhesive
state in SS RBCs from some patients but also contributes to basal SS
RBC adhesion to laminin.
We also examined the effects of forskolin stimulation and PKA inhibition on the adhesive state of AA RBCs. It should be noted that absolute basal AA RBC adhesion to laminin was approximately 10-fold lower than basal SS RBC adhesion, consistent with previous observations.2,23 Furthermore, AA RBC adhesion to laminin was unaffected by forskolin treatment (Figure 2A). These results are consistent with the inability of forskolin to stimulate cAMP production in peripheral AA RBCs versus SS RBCs (Figure 1B) and may reflect a reduced capacity of circulating AA RBCs to produce cAMP, potentially because of lower levels and/or activity of adenylyl cyclase. Patients with sickle cell anemia have a much higher percentage of circulating reticulocytes than RBCs from unaffected control subjects, and the average age of their "mature" RBCs is markedly less.24 RBCs lose various signaling components as they age7; thus, their capacity to produce and respond to cAMP may be age dependent. To determine if a particular population of SS RBCs was more responsive to cAMP-mediated activation, SS RBCs were separated into low- (reticulocyte-enriched) and high-density (reticulocyte-depleted) fractions by centrifugation over an arabinogalactan density gradient. When forskolin-stimulated cAMP production and adhesion in each fraction were compared, the low-density SS RBC fraction exhibited a substantially greater increase in both cAMP production (Figure 2B) and adhesion (Figure 2C) versus the high-density fraction. Control experiments confirmed that WBC contamination did not account for the increased cAMP production in the low-density fraction (data not shown). These results suggest that the younger, low-density SS RBCs are most responsive to forskolin-induced increases in cAMP production and adhesion. Next, we asked if RBC adhesion to laminin could be stimulated by
directly introducing cAMP, thus bypassing adenylyl cyclase activation.
Both SS and AA RBCs were preincubated with dibutyryl cAMP (db cAMP), a
stable, membrane-soluble cAMP analog that activates PKA. Like
forskolin, db cAMP treatment elevated SS RBC adhesion to laminin almost
3-fold (Figure 3A), further supporting
the notion that elevated intracellular cAMP increases SS RBC
adhesiveness. Interestingly, db cAMP induced a similar fold elevation
in adhesion in AA RBCs (Figure 3B), suggesting that AA RBCs retain the
signaling machinery downstream of adenylyl cyclase required to increase adhesion to laminin; however, the absolute number of adherent AA RBCs
was 10-fold less than that of SS RBCs.
Next, the potential physiologic implications of cAMP-stimulated
adhesion of SS RBCs were explored. Epinephrine, the major mediator of
the physiologic stress response, elevates cAMP levels in
RBCs25; however, the effects of epinephrine on cAMP levels or adhesion of SS RBCs have never been explored. Treatment of SS RBCs
with epinephrine induced a maximal stimulation of total cellular cAMP
in SS RBCs within 1 minute (Figure 4A)
and a corresponding peak in SS RBC adhesion to laminin within 1 to 5 minutes of treatment (Figure 4B), suggesting a correlation between
these 2 events. Consistent with our previous observations with
forskolin (Figure 2B-C), epinephrine-stimulated cAMP levels were more
pronounced in the low-density population of SS RBCs (data not shown).
Because previous data indicated that the degree of adhesive response to
cAMP stimulation varied from patient to patient, the effects of
epinephrine on SS RBC adhesion to laminin was measured in samples
obtained from a larger group of patients (n = 28). A statistically
significant stimulation of SS RBC adhesion above basal levels was
observed (P < .01). SS RBCs from a subset of patients
(46%) exhibited more than 1.5-fold (50%) elevation in adhesion to
laminin and were classified as responders (Figure 5A), whereas adhesion was slightly
decreased in a smaller subset of patients (11%). These data suggest
that almost half of patients with sickle cell anemia may be susceptible
to epinephrine-stimulated adhesion of SS RBCs.
Next, the mechanism of this epinephrine-stimulated adhesion was further
explored in the responsive patient population. Previous studies
suggested that the Because the most well-described downstream effector of cAMP is PKA, we explored the role of PKA in epinephrine-mediated SS RBC adhesion. As shown in Figure 5C, epinephrine-stimulated adhesion was completely blocked by PKAI in 3 responders (patients 1, 2, and 4) and partially blocked in one responder (patient 3). These data suggest that PKA activation is required for epinephrine-stimulated adhesion to laminin in the responsive population of patients with sickle cell anemia. Studies by Udani et al5 and Zen et al29 have shown that the primary laminin receptor on unstimulated SS RBCs is basal cell adhesion molecule and its isoform Lutheran (BCAM/Lu); we, therefore, asked if BCAM/Lu also mediated the epinephrine-stimulated adhesion. Immobilized laminin was incubated with recombinant, soluble BCAM/Lu, which significantly inhibited the epinephrine-stimulated portion of SS RBC adhesion by an average of 96% (n = 4, P < .05), whereas a control protein, soluble vascular cell adhesion molecule (VCAM), had no statistically significant effect on stimulated adhesion (n = 2) (Figure 5D). These data indicate a role for BCAM/Lu in epinephrine-stimulated SS RBC adhesion to laminin. Because BCAM/Lu contains several serine residues that represent potential PKA phosphorylation sites, we asked if PKA directly affects BCAM/Lu function via serine phosphorylation. However, we were unable to detect serine phosphorylation of BCAM/Lu when immunoprecipitated from epinephrine- or forskolin-stimulated SS RBCs and Western blotted with an antiphosphoserine antibody (data not shown), suggesting that PKA activation is an intermediate step in a pathway that modulates BCAM/Lu function. Reticulocytes, which are RBC precursors, retain more of their signaling
machinery than other more mature circulating RBCs and exist at higher
levels in the blood of patients with sickle cell anemia. To determine
if the observed variability in epinephrine-stimulated SS RBC adhesion
(Figure 5A) was directly related to patient reticulocyte counts per se,
the relationship between fold change in adhesion versus percentage of
reticulocyte count was examined. The percentage of reticulocyte values
did not independently predict SS RBC responsiveness to epinephrine
between patients (Figure 6A). Moreover,
when individual patients were followed over time, there was no
consistent correlation between reticulocyte counts within a given
patient and stimulated adhesion (Figure 6B). Further, when adherent SS
RBCs were fixed and stained with methylene blue following the flow
adhesion assay, the percentage of adherent reticulocytes actually
decreased following epinephrine stimulation (Figure 6C). Thus, the
major epinephrine-responsive population of SS RBCs is not the
RNA-containing reticulocytes detected with our staining procedure, but
likely a subpopulation between the methylene blue-detectable
reticulocyte-senescent SS RBC continuum (Figure 6D).
Next, we explored the possibility that epinephrine-stimulated adhesion
may be a generalized feature of clinical conditions displaying
reticulocytosis (eg, acute blood loss, nonsickle cell hemolytic
anemias). To test this possibility, we obtained RBCs from 4 patients
with elevated reticulocyte counts but without sickle cell anemia and
observed their adhesive response following epinephrine treatment.
Although one patient (reticulocyte count = 6.5%) exhibited an
elevated adhesion to laminin in response to epinephrine, the other 3 (reticulocyte counts ranging from 2.8% to 9.4%) demonstrated no
significant increase (data not shown). Thus, epinephrine stimulation
did not cause a consistent change from basal adhesion. However, it
would be premature to suggest that epinephrine-stimulated adhesion is a
property limited mainly to SS RBCs but may be a function of the mean
cell age in any given peripheral RBC population. Our results are
summarized in a model that illustrates the role of the
In this study, we provide the first evidence that
cAMP/PKA-mediated signaling pathways can promote RBC adhesion to a
substrate Unlike AA RBCs, SS RBCs exhibit significantly greater basal and forskolin-stimulated levels of cAMP. Because AA RBC adhesion (albeit to much lower levels) could be stimulated with an exogenously added cAMP analog, we speculate that reduced activity or levels of adenylyl cyclase may be the molecular point of divergence between peripheral AA and SS RBCs. SS RBCs have an expanded population of young RBCs, and our data are consistent with reports demonstrating that mature RBC membranes contain only 10% to 20% of the adenylyl cyclase activity of younger RBC membranes.34 The ability of epinephrine to stimulate SS RBC adhesion to laminin is of particular clinical interest. Epinephrine is elevated during periods of stress, and previous studies have shown a direct relationship between stress and sickle cell-associated pain.35,36 These data, therefore, suggest a biologic link between stress and the pathophysiology of sickle cell anemia. Also of interest is that epinephrine mediates this stimulated adhesion in a specific subset of patients (46%), suggesting that there may be an identifiable population of patients predisposed to epinephrine-mediated increases in SS RBC adhesion. Although our study focused primarily on patients with sickle cell anemia, these results illustrate the potential importance of understanding the genetic background on which the sickle hemoglobin mutation is expressed to better predict an individual patient's physiologic response. Comparison of the genetic and protein expression profiles from each subset of patients may yield insight into the factors responsible for the variable responses to epinephrine. One factor that may contribute to the patient-to-patient variability observed in SS RBC adhesive responses to epinephrine is a difference in adenylyl cyclase copy number or enzymatic activity in RBCs from different patients, because we can bypass adenylyl cyclase in AA RBCs and observe a response. Additionally, we demonstrate that epinephrine acts primarily on the
Our data also demonstrate that immature SS RBC populations are more responsive to cAMP-mediated adhesion to laminin, compared with more mature SS RBC populations (Figure 2C), suggesting that patients with a younger mean RBC age (hemolytic anemia, blood loss, etc) may be more susceptible to cAMP-stimulated adhesion. In fact, we also observed epinephrine-stimulated adhesion in a patient with a hemolytic anemia independent of sickle cell disease (data not shown). Interestingly, stimulated SS RBC adhesion is not directly related to reticulocyte counts per se, as increased reticulocyte counts did not correlate with an increase in stimulated adhesion (Figure 6A-B), and reticulocyte adhesion did not increase in response to epinephrine (Figure 6C). Because reticulocytes retain more signaling capacity, this may appear counterintuitive. However, the increased signaling capacity may also be manifested as more active inhibitory pathways that prevent BCAM/Lu activation. As is the case with many other signaling pathways, potential inhibitory pathways may become lost as the reticulocytes mature into the intermediate RBC population illustrated in Figure 6D, explaining how this intermediate population could be the most responsive to stimulated adhesion. Taken together, these data suggest that the responsiveness of young circulating RBCs to agonists resulting in increased adhesiveness might not be specific to sickle cell anemia. However, why patients with sickle cell anemia are predisposed to vasoocclusive crises and other patients are not, is likely due to other factors that are more common features of this disease. For example, there is a large inflammatory component that contributes to an activated and denuded endothelium.3 The highly adhesive RBCs in sickle cell anemia are much more likely to mediate vasoocclusion in this pro-adhesive environment than reactive RBCs in other disease settings in which these contributing factors are generally absent. In this report, we also implicate BCAM/Lu as the receptor mediating stimulated adhesion (Figure 5D). Although cAMP-mediated PKA activation has been reported to induce integrin-mediated neutrophil adhesion,19 our results with BCAM/Lu suggest that PKA may elevate cell adhesion via a broader range of adhesion receptors than previously realized. Further studies are needed to understand the role of the BCAM/Lu-laminin interaction in vivo. Elucidation of the specific pathways between PKA activation and stimulated BCAM/Lu adhesion may uncover additional potential therapeutic targets. In conclusion, cAMP signaling may play a significant role in the
pathophysiology of sickle cell anemia. In support of this, we provide
the first evidence that the adhesive state of RBCs can be regulated by
cAMP and demonstrate a role for BCAM/Lu in mediating this activated
adhesion. Further, these studies show that epinephrine, acting
primarily via the
We thank Sheritha Lee, Julia Brittain, and Chris Anderson for their invaluable scientific and technical assistance; Dell Strayhorn, Sandra Santucci, and the UNC Comprehensive Sickle Cell Center for their assistance in patient recruitment and clinical perspectives; and Paul Stewart (Department of Biostatistics, UNC-CH) for statistical consultation.
Submitted December 19, 2001; accepted November 25, 2002.
Prepublished online as Blood First Edition Paper, January 2, 2003; DOI 10.1182/blood-2001-12-0289.
Supported by the United Negro College Fund (UNCF)-Merck Graduate Science Research Dissertation Fellowship (P.C.H.), a grant from the March of Dimes (M.J.T.), and grants HL58939 (M.J.T. and L.V.P.), HL067440 (L.V.P.), HL63409 (M.J.T.), and RR0046 (E.P.O.) from the National Institutes of Health.
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: Leslie V. Parise, Department of Pharmacology, University of North Carolina at Chapel Hill, CB#7365, Chapel Hill, NC 27599-7365; e-mail: parise{at}med.unc.edu.
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© 2003 by The American Society of Hematology.
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Top
Abstract
Introduction
2-adrenergic receptor, occurred in SS RBC samples from 46% of
patients and was blocked by recombinant, soluble BCAM/Lu, implicating
this receptor as a target of cAMP signaling. Thus, these studies
demonstrate a novel, rapid regulation of SS RBC adhesion by a
cAMP-dependent pathway and suggest that components of this pathway,
particularly PKA, the 
) and high-density (HD,
) SS RBCs to laminin was measured in the flow adhesion assay
("Materials and methods") in response to forskolin (80 µM for 15 minutes) in SS RBCs from 3 separate patients. Basal adhesion is
normalized to 1.
2 µM epinephrine and lysed after 1, 5, 15, or
30 minutes as indicated. Total cAMP levels were measured in
1 × 108 SS RBCs/condition by enzyme-immunoassay
("Materials and methods"). (B) Adhesion of SS RBCs from 2 different
patients to immobilized laminin was measured using the flow adhesion
assay after treatment for 1, 5, 15, and 30 minutes with
1 × 10
s,
which stimulates adenylyl cyclase (AC). This enzyme catalyzes the
conversion of ATP to cAMP, leading to PKA activation, an intermediate
step in the up-regulation of BCAM/Lu-mediated adhesion.
(Phosphodiesterase [PDE] breaks down cAMP into AMP.) Events between
PKA activation and enhancement of BCAM/Lu-mediated adhesion are
currently unknown. Although this pathway may not be limited to RBCs
from patients with sickle cell anemia, the vasculature of patients with
sickle cell anemia probably exists in a much more pro-adhesive state.
These factors acting in concert suggest that epinephrine-stimulated
adhesion may play a clinically significant role in the context of
sickle cell anemia.
in this case, laminin, which may be particularly important
in the context of sickle cell anemia. The stimulation of adhesion by cAMP and PKA was somewhat unexpected, because PKA activation is widely
described as a negative regulator of cell adhesion in various cell
types.