|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From INSERM U479 and Service d'Immunologie et
d'Hématologie, CHU X Bichat; and Centre de la
Drépanocytose and INSERM U458, Hôpital Robert-Debré;
both of Paris, France.
Impaired polymorphonuclear neutrophil (PMN) functions during sickle
cell anemia (SCA) may have a pathogenic role in the onset of
vasoocclusive events. We used flow cytometry to study, in whole blood,
the adhesion molecule expression and respiratory burst of PMNs from
children with SCA. Three different clinical groups were studied: (1)
patients with no history of vasoocclusive events (n = 15); (2)
patients with a history of vasoocclusive events (n = 17); and (3)
patients receiving hydroxyurea therapy for severe vasoocclusive events
(n = 9). Unstimulated PMNs showed decreased L selectin expression and
increased H2O2 production whatever the severity
of the disease, reflecting PMN activation. This could contribute to
endothelial activation reflected by abnormal plasma levels of soluble
adhesion molecules (soluble intercellular adhesion molecule-1, sE
selectin, and sL selectin). After stimulation with bacterial N-formyl
peptides (N-formyl-methionyl-leucyl-phenylalanine [fMLP]), PMNs from
untreated patients with a history of vasoocclusive events showed
dysregulated L selectin shedding and increased
H2O2 production. Furthermore, in these
patients, tumor necrosis factor priming followed by fMLP stimulation
induced an H2O2 production significantly higher
than in the other patient groups and controls. These impairments could
immobilize PMNs on the endothelium, thereby inducing reduced blood flow
and fostering microvascular occlusion and vascular damage. In contrast,
children treated with hydroxyurea showed near-normal basal and
poststimulation H2O2 production as well as
normal L selectin shedding after stimulation but no change in plasma
levels of soluble adhesion molecules. To our knowledge, this is the
first report showing major qualitative changes of PMN abnormalities
upon hydroxyurea treatment in SCA patients. This strongly suggests that
PMNs are a primary target of this drug.
(Blood. 2002;99:2297-2303) Sickle cell anemia (SCA) results from a
single point mutation that substitutes valine for glutamic acid at the
sixth position in the RBCs are not the only circulating blood cells to be involved in SCA.
Thrombospondin produced by activated platelets is involved in RBC
adhesion to the endothelium.8 Monocytes from SCA patients activate endothelial cells in vitro, increasing endothelial expression of adhesion molecules and tissue factor.9 Furthermore,
abnormal adhesion between endothelial cells and polymorphonuclear
neutrophils (PMNs) might participate in the early steps of the
vasoocclusive process by decreasing the flow rate in the
microvasculature. Two major lines of evidence point to the involvement
of PMNs in the pathophysiology of SCA. First, a high PMN count is a
characteristic feature of SCA patients at steady state, and
polymorphonuclear leukocytosis is related to an increased risk of early
death, ACSs, and stroke.10,11 Second, HU decreases the PMN
count,12 and this is the biologic parameter most strongly
linked to the beneficial effect of HU,13 suggesting that
PMNs may be a preferential drug target.
PMN extravasation into tissues requires the expression of adhesion
molecules at the PMN surface and their counterparts on endothelial
cells.14,15 In addition, PMNs release large quantities of
reactive oxygen species (ROS) in response to various stimuli, in the
so-called respiratory burst.16 This process is critical for bacterial killing and also potentiates inflammatory reactions, sometimes inducing severe host tissue injury when activation is excessive or inappropriate.17,18 Inaccurate regulation of
adhesion molecule expression could therefore increase PMN binding to
the endothelial surface, leading to inappropriate endothelial
activation. Incubation of sickle erythrocytes (SS-RBCs) with
endothelial cells in various in vitro models increases the expression
of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion
molecule (ICAM)-1, and E selectin,19,20 which are the
endothelial counterreceptors of the blood cell adhesion molecules very
late antigen-4, In this study PMNs from untreated SCA children with and without a
history of vasoocclusive events were analyzed in fresh whole blood to
minimize procedure-related changes in surface receptor expression. We
also studied children treated with HU for major vasoocclusive events to
examine the effects of HU on PMN functions. Flow cytometry, which can
be used to study events at the single-cell level, was chosen to analyze
adhesion molecule expression at the PMN surface (especially L selectin
and CD11b/CD18) and the PMN respiratory burst
(H2O2 production). These parameters were
measured at baseline and after ex vivo stimulation. In parallel, we
determined circulating levels of soluble adhesion molecules (sICAM-1,
sE selectin, and sL selectin), which have been reported to reflect blood cell-endothelium interactions and endothelial
damage.28-30
Patients
All patients were at steady state, ie, were free of any acute
event 4 weeks prior to and after blood sampling, and transfusion-free for 3 months prior to and 1 month after blood sampling. They were all
treated with preventive penicillin V and folate supplementation. No
patients have had stroke or priapism in each category. There was no
crossing over between non-VOC and VOC categories in the 2 years
following the study. The complete medical history was recorded, and no
systemic disease potentially altering PMN functions was identified.
None of the patients had taken curative antibiotics or corticosteroids
during the 6 months prior to blood sampling. Blood samples were
obtained during a regular clinical consultation. Thirty heterozygous AS
(sickle cell trait) subjects (median age, 39 years; range, 26-53 years)
and 11 healthy African AA (normal hemoglobin) subjects (median age, 18 years; range, 5 to 39 years) served as controls. Complete blood
counts including RBC indices and reticulocytes were performed, and
fetal hemoglobin (HbF) was assayed.
After informed consent had been obtained from the patients, the
parents, and the controls, whole blood was sampled, kept on ice, and
transported immediately to the laboratory.
Reagents
Stock solutions of DCFH-DA (50 mM) and fMLP (10 Determination of adhesion molecule expression at the PMN surface Whole-blood samples were either kept on ice or incubated with fMLP (10 6 M) or PBS at 37°C for 5 minutes. To study
CD11b and CD18 expression on CD45high cells, samples (100 µL) from each patient were incubated at 4°C for 30 minutes with the
following reagent combination: PE-anti-CD11b/PerCP-anti-CD45 and
FITC-anti-CD18/PerCP-anti-CD45. To study L selectin expression on
CD45high cells, samples were incubated with nonconjugated
anti-CD62L antibody at 4°C for 30 minutes, washed with ice-cold PBS,
and then incubated at 4°C for 30 minutes with FITC-goat antimouse
antibody; after one wash in ice-cold PBS, samples were incubated with
PerCP-anti-CD45 at 4°C for 30 minutes. Red cells were lysed with
fluorescence-activated cell sorter (FACS) lysing solution (Becton
Dickinson), and white cells were resuspended in 1%
paraformaldehyde-PBS and kept on ice until flow cytometry. Nonspecific
antibody binding was determined on cells incubated with the same
concentration of an irrelevant antibody of the same isotype.
H2O2 production H2O2 production was measured by using a flow cytometric assay derived from the technique described by Bass et al31 and others.32 We checked that this assay could be used to compare PMN oxidative burst in subjects with different PMN counts (data not shown). One milliliter of fresh blood collected onto preservative-free lithium heparinate (10 U/mL) was preincubated for 15 minutes with DCFH-DA (100 µM) in a water bath at 37°C with gentle horizontal agitation. DCFH-DA diffuses into the cells and is hydrolyzed into DCFH; during the PMN respiratory burst, nonfluorescent intracellular DCFH is oxidized to highly fluorescent dichlorofluorescein (DCF) by H2O2 in the presence of peroxidase. The samples were then incubated with TNF-
(100 U/mL) or LPS (5 µg/mL) diluted in PBS, or with PBS alone, at
37°C for 30 minutes. fMLP diluted in PBS (106 M final
concentration) or PBS was added for 5 minutes at 37°C. The reaction
was stopped, and samples were incubated with PerCP-anti-CD45 antibody
at 4°C for 30 minutes. Red cells were lysed with FACS lysing solution
(Becton Dickinson). After one wash (400g, 5 minutes), white
cells were suspended in 1% paraformaldehyde-PBS. The fixed samples
were kept on ice until flow cytometric analysis on the same day. As
previously reported, the FACS lysing solution modified neither the
amount of DCF generated nor the expression of activation markers such
as CR3, as shown by flow cytometry.32 Moreover, PMN
viability was not altered in our conditions, as assessed by means of
flow cytometry in terms of propidium iodide exclusion. Finally, we
checked that DCF did not diffuse out of the cells (data not shown).
Flow cytometry We used a Becton Dickinson FACSCalibur with a 15-mW, 488-nm argon laser. PMN functions were analyzed using CellQuest software. A first gate was drawn on a forward- and side-scatter dot plot around the granulocyte population. The fluorescence of anti-CD45 antibody was used to identify CD45+ cells and to gate out other cells and debris, such as SS reticulocytes (which are resistant to red cell lysis). A second gate was then drawn on CD45+ cells. The purity of the gated cells was assessed by using FITC- or PE-conjugated CD3, CD14, and CD15 antibodies (Becton Dickinson). Acquisition was performed on the intersection of these 2 gates. A total of 5000 events were counted per sample, and the fluorescence pulses were amplified by 4-decade logarithmic amplifiers. The green fluorescence of DCF and FITC antibodies was recorded from 515 to 545 nm; the red fluorescence of PE-anti-CD11b was recorded from 563 to 607 nm. In all cases, unstained cells were used and the photomultiplier settings were adjusted so that the unstained cell population appeared in the lower left-hand corner of the fluorescence display. For dual-color analysis, single-cell controls were used to optimize signal compensation. All the results were obtained with a constant photomultiplier gain. The mean fluorescence intensity (MFI) was used to quantify cell responses.Cytokine and soluble adhesion molecule assays Blood was collected into sterile ethylenediaminetetraacetic acid-treated vacuum tubes, transported on ice to the laboratory, and immediately centrifuged at 1500g for 15 minutes at 4°C to avoid cytokine synthesis or breakdown in vitro. Plasma samples were stored at 70°C for no longer than 15 days before assay. Cytokines in plasma were assayed in duplicate by
using immunoenzymatic assays for interleukin (IL)-8 and TNF-, with a
detection limit of 10 pg/mL (R&D, Abingdon, United Kingdom, for IL-8;
and Immunotech, Marseille, France, for TNF-). The assays were
standardized by comparing the results to those of standards from the
National Institute for Biological Standards and Control (England).
Cytokine recovery was 102% ± 4% from normal plasma spiked with
recombinant human cytokines. Soluble L selectin, sE selectin, and
sICAM-1 were assayed in duplicate by using immunoenzymatic assays (R&D for sE selectin and sICAM-1; Immunotech for sL selectin) with respective detection limits of 15 ng/mL, 0.1 ng/mL, and 0.35 ng/mL for
sL selectin, sE selectin, and sICAM-1.
Statistical analysis Results are expressed as means ± SEM. The group means were compared using analysis of variance followed by a multiple comparison of means with the Mann-Whitney least-significant-difference procedure, and P values below .05 were considered significant. Correlations were identified by means of the Spearman rank correlation coefficient ( ).
Characteristics of the study population As shown in Table 1, total leukocyte, PMN, and monocyte and platelet counts were significantly higher in the 3 groups of SCA patients than in healthy African controls. Patients treated with HU had significantly lower leukocyte counts than untreated patients, while the difference in the PMN count was borderline. The Hb level was significantly lower in the patients than in the healthy controls. The percentage of HbF was significantly higher in HU-treated patients than in untreated VOC children (12.4% vs 8.3%), and so was the median of the mean corpuscular volume values (92 vs 82 µm3). The reticulocyte count was significantly lower in HU-treated patients (285 × 109/L) than in untreated patients (389 × 109/L). The data for heterozygous AS parents did not differ from those in the African control group.Expression of adhesion molecules at the PMN surface L selectin expression at the PMN surface is a critical factor in the earlier rolling phase of interaction between PMN surface and endothelial cells leading to transendothelial migration.15 As shown in Table 2, the MFI value for anti-L selectin antibody binding to unstimulated PMNs was significantly lower in all groups of SCA patients than in the healthy AA controls. In addition, L selectin expression correlated negatively with the PMN count ( = 0.5, P = .0004). Incubation
with PBS alone at 37°C did not significantly modify L selectin
expression relative to unstimulated PMNs maintained at 4°C (data not
shown). As expected,33 after stimulation with fMLP
(106 M, 5 minutes, 37°C), L selectin was no longer
detectable on PMNs from control subjects (Table 2). The MFI obtained
with anti-L selectin antibody was lower than the MFI obtained with an
irrelevant antibody of the same isotype (data not shown). L selectin
shedding by PMNs from the non-VOC and HU patient groups was normal. In contrast, L selectin was still detectable after fMLP stimulation of
PMNs from VOC patients, with an MFI significantly higher than that in
control subjects and HU-treated patients.
The
No significant modification of adhesion molecule expression was observed in heterozygous AS subjects compared with control subjects (data not shown). H2O2 production by PMNs in whole blood As shown in Table 4, basal H2O2 production was significantly higher in unstimulated PMNs (samples incubated with DCFH-DA and treated with PBS) from untreated SCA patients than in PMNs from healthy controls, the latter expressing low background fluorescence. This increased basal H2O2 production was not observed in patients receiving HU.
TNF (100 U/mL, 30 minutes) added separately to whole blood induced
barely detectable H2O2 production by PMNs from
control subjects. A small but significant increase was observed in
untreated SCA patients compared with the healthy controls. fMLP
(10 As noted above for adhesion molecule expression, no significant modification of H2O2 production by resting or stimulated PMNs was observed in heterozygous AS subjects. Plasma levels of cytokines and soluble adhesion molecules Because TNF and IL-8 are potent PMN activators,35,36 we measured plasma levels of these 2 cytokines. No significant modification of TNF levels (Table 5) or IL-8 levels (data not shown) was found in SCA patients compared with controls. To investigate endothelial cell activation, we measured circulating levels of soluble adhesion molecules. Soluble ICAM-1 levels and sE selectin levels were significantly increased in both groups of untreated patients as compared with control subjects. We also observed a significant decrease in sL selectin levels in non-VOC patients compared with control subjects. No significant difference in soluble adhesion molecule levels was found between the 2 groups of untreated patients or between VOC patients and HU-treated patients (Table 5).
These results show that circulating PMNs from SCA children are activated whatever the clinical expression of the disease. Indeed, unstimulated PMNs from these patients showed decreased L selectin expression and increased H2O2 production. Concomitantly, we found increased plasma levels of sICAM-1 and sE selectin, reflecting endothelial cell activation. These abnormalities did not increase with disease severity. However, PMNs from untreated patients with a history of vasoocclusive events (VOC patients) showed enhanced H2O2 production after stimulation with bacterial N-formyl peptides or TNF alone and after TNF priming followed by fMLP stimulation. In addition, PMNs from these VOC patients showed reduced L selectin shedding in response to fMLP. Note that values in the heterozygous AS parents did not differ from those in African AA controls (data not shown). HU treatment had a strong beneficial effect on the PMN abnormalities observed in VOC patients. Indeed, HU therapy was associated with near-normal H2O2 production, both at basal state and after priming, and normal L selectin shedding after stimulation. In contrast, HU treatment did not affect sICAM plasma levels. To our knowledge, this is the first report showing qualitative PMN changes upon HU treatment, strongly suggesting a targeted effect of the drug on PMNs. A major step in PMN migration from the circulation to inflammatory
sites is the modulation of adhesion molecule expression on both PMNs
and endothelial cells. In particular, proinflammatory mediator-induced
shedding of L selectin (CD62L) and increased expression of Our results confirm the decrease in L selectin expression at the surface of resting PMNs reported by Lard et al26 in asymptomatic SCA patients. Diminished L selectin expression related to a shedding process has been reported to occur during PMN activation.15 A similar decrease in L selectin expression, associated with normal CD11b/CD18 surface expression, has previously been reported in patients with inflammatory diseases other than SCA.37,38 L selectin shedding depends at least in part on the action at the PMN surface of a protein disulfide isomerase, which regulates the susceptibility of L selectin cleavage by a metalloprotease.39 This protein disulfide isomerase-mediated mechanism did not alter the expression of several other cell surface molecules. Because L selectin interacts with carbohydrates of the endothelial membrane, the decreased expression of this molecule at the PMN surface may lead to a decrease in the marginal pool of PMNs and thereby contribute to the hyperleukocytosis observed in SCA patients. Indeed, we found a negative correlation between L selectin expression and the PMN count. The impairment of L selectin expression by unstimulated cells did not increase with disease severity. In contrast, after stimulation with N-formyl peptides, significant L selectin dysregulation was observed on PMNs from VOC patients: L selectin was still detectable at the surface of stimulated PMNs from VOC patients, while it disappeared from the surface of stimulated PMNs from controls and non-VOC patients. Blockade of L selectin shedding has been reported to increase leukocyte exposure to the inflammed endothelium.40 The dysregulated L selectin shedding observed in VOC patients could therefore immobilize PMNs on the endothelium and play a key role in the initiation and propagation of vasoocclusive events by slowing blood through the microvasculature, delaying the SS-RBC transit time, and increasing the likelihood of HbS polymerization within the microvasculature. We also observed increased basal H2O2 production by whole- blood PMNs from SCA patients relative to PMNs from African healthy control subjects. In addition, H2O2 production by PMNs from SCA patients was barely increased after stimulation with TNF or fMLP alone. The contrast between this latter result and reports of an unaltered respiratory burst in PMNs from SCA patients23,25 could at least in part be due to methodologic factors. Indeed, we studied PMNs in whole blood, while other studies used PMNs isolated from their blood environment by various procedures that may affect surface receptor expression and thereby alter cell responses. Activation due to isolation procedures might thus mask differences between patients and healthy controls. After TNF priming followed by fMLP stimulation, H2O2 production by PMNs from VOC patients was increased by the same degree as in controls and reached a level significantly higher than in the other patient groups, in which the effects of the 2 stimuli were only additive. This difference may be related to a relative response defect in the non-VOC group, although acquired PMN hyperresponsiveness in the VOC group cannot be ruled out at this stage. The increased ROS production observed after priming of PMNs from VOC patients may be related at least in part to potentiation of the oxidative burst by L selectin, which did not disappear after fMLP stimulation and has been reported to act as a signaling molecule.41 Decreased L selectin expression associated with increased
H2O2 production in SCA patients could result
from local production of proinflammatory cytokines such as TNF- Dysregulation of adhesion molecule expression and increased ROS production by resting and stimulated PMNs from SCA patients could increase endothelial activation and vascular damage. As previously reported by Blei et al,48 we found increased levels of sICAM-1 and sE selectin, which have been reported to reflect endothelial damage.28,29 The observed decreased sL selectin levels may reflect sL selectin sequestration by widespread binding to activated endothelium in microvascular beds.49 This is supported by immunohistochemical findings showing that sL selectin specifically binds to the luminal surface of high endothelial venules at sites of inflammation.49,50 Thus, increased levels of sICAM-1 and sE selectin, associated with low levels of sL selectin, probably reflect diffuse activation of the endothelium. These results are in keeping with previous data demonstrating the presence of increased numbers of activated circulating endothelial cells51 and increased plasma levels of sVCAM-1.52,53 Moreover, ROS generated by activated PMNs have been reported to stimulate VCAM-1 up-regulation on the vascular endothelium, a known attachment site for sickle reticulocytes.54 Increased ROS levels produced by activated neutrophils could lead to endothelial damage, increased production of proinflammatory mediators, and increased adherence between endothelial cells and SS-RBCs through VCAM-1, leading to amplification of the process. HU is the only drug known to decrease the frequency of painful crisis in patients with SCA.6 It was given initially with the aim of increasing HbF production.55 However, there is no correlation between the increase in HbF levels, which is late and highly variable from patient to patient, and the clinical benefit, which is early and at least almost constant.56 Moreover, Bridges et al57 demonstrated that HU decreased SS reticulocyte adhesion to endothelial cells and that this change preceded the increase in HbF. Following on from reports that HU reduces the PMN count,12,13 we show for the first time that this drug also qualitatively affects PMN functions in SCA. Indeed, PMNs from children treated with HU for past major vasoocclusive events showed a correction of the decreased L selectin shedding found in untreated VOC children. This could limit PMN adhesion to the endothelium and explain the reduced severity and frequency of vasoocclusive events on HU. In addition, basal and stimulated H2O2 production by PMNs from patients treated with HU showed a return to control values, and this could also reduce endothelial injury. This result is in accordance with previous data demonstrating that, in vitro, HU decreases the oxidative burst of activated PMNs from healthy subjects.58 The persistently high sICAM level in HU-treated patients could be due to interactions other than PMN endothelium. In conclusion, we present evidence that whole-blood PMNs from homozygous SCA patients are activated, even in the absence of clinical complications. Reduced L selectin shedding and increased ROS production could immobilize PMNs on the endothelium, thereby inducing reduced blood flow, microvascular occlusion, and vascular damage. HU has a strong beneficial effect on these PMN abnormalities. Our data strongly suggest that PMNs are a primary target of this drug and that this beneficial effect on PMNs may be a key component of its clinical efficiency. Further studies of the mechanism of action of HU on PMNs may lead to the identification of new therapeutic targets.
The authors are grateful to M. Ameri, O. Fenneteau, and the Flow Cytometry Laboratory technicians for their assistance. They thank the children and their families for the very kind and often enthusiastic participation.
Submitted July 25, 2001; accepted November 13, 2001.
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: Carole Elbim, Laboratoire d'Immunologie et d'Hématologie, CHU Xavier Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France; e-mail: pocidalo{at}bichat.inserm.fr.
1. Serjeant GR. Sickle Cell Disease. Oxford, England: Oxford University Press; 1992. 2. Embury SH, Hebbel RP, Mohandas N, Steinberg MH. Sickle Cell Disease: Basic Principles and Clinical Practice. New York, NY: Raven Press; 1994.
3.
Platt OS, Thorington BD, Brambilla DJ, et al.
Pain in sickle cell disease 4. Hebbel RP. Adhesive interactions of sickle erythrocytes with endothelium. J Clin Invest. 1997;99:2561-2564[Medline] [Order article via Infotrieve]. 5. Kaul DK, Hebbel RP. Hypoxia/reoxygenation causes inflammatory response in transgenic sickle mice but not in normal mice. J Clin Invest. 2000;106:411-420[Medline] [Order article via Infotrieve].
6.
Charache S, Torrin ML, Moore RD, et al.
Effect of hydroxyurea on frequency of painful crises in sickle cell anemia.
N Engl J Med.
1995;332:1317-1322 7. Maier-Redelsperger M, Labie D, Elion J. Long-term hydroxyurea treatment in young sickle cell patients. Curr Opin Hematol. 1999;6:115-120[CrossRef][Medline] [Order article via Infotrieve].
8.
Sugihara K, Sugihara T, Mohandas N, Hebbel RP.
Thrombospondin mediates adherence of CD36+ sickle reticulocytes to endothelial cells.
Blood.
1992;80:2634-2642
9.
Belcher JD, Marker PH, Weber JP, Hebbel RP, Vercellotti GM.
Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion.
Blood.
2000;96:2451-2459
10.
Platt OS, Thorington BD, Rosse WF, et al.
Mortality in sickle cell disease: life expectancy and risk factors for early death.
N Engl J Med.
1994;330:1639-1644
11.
Miller ST, Sleeper LA, Pegelow CH, et al.
Prediction of adverse outcomes in children with sickle cell disease.
N Engl J Med.
2000;342:83-89 12. Rogers ZR. Hydroxyurea therapy for diverse pediatric populations with sickle cell disease. Semin Hematol. 1997;34:42-47[Medline] [Order article via Infotrieve]. 13. Charache S. Mechanism of action of hydroxyurea in the management of sickle cell anemia in adults. Semin Hematol. 1997;34:15-21[Medline] [Order article via Infotrieve]. 14. Anderson DC, Springer TA. Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150-95 glycoproteins. Annu Rev Med. 1987;38:175-194[CrossRef][Medline] [Order article via Infotrieve]. 15. Bevilacqua MP, Nelson RM. Selectins. J Clin Invest. 1993;91:371-387.
16.
Babior BM.
Oxidants from phagocytes: agents of defense and destruction.
Blood.
1984;64:959-966
17.
Harlan JM.
Leukocyte-endothelial interactions.
Blood.
1985;65:513-525
18.
Marx JL.
Oxygen free radicals linked to many diseases.
Science.
1987;235:529-531
19.
Swerlick RA, Eckman JR, Kumar A, Jeitler M, Wick TM.
20.
Shiu YT, Udden MM, McIntire LV.
Perfusion with sickle erythrocytes up-regulates ICAM-1 and VCAM-1 gene expression in cultured human endothelial cells.
Blood.
2000;95:3232-3241
21.
Hofstra TC, Kalra VK, Meiselman HJ, Coates TD.
Sickle erythrocytes adhere to polymorphonuclear neutrophils and activate the neutrophil respiratory burst.
Blood.
1996;87:4440-4447 22. Boghossian SH, Nash G, Dormandy J, Bevan DH. Abnormal neutrophil adhesion in sickle cell anaemia and crisis: relationship to blood rheology. Br J Haematol. 1991;78:437-441[Medline] [Order article via Infotrieve]. 23. Dias-Da-Motta PM, Arruda VR, Muscara MN, et al. The release of nitric oxide and superoxide anion by neutrophils and mononuclear cells from patients with sickle cell anaemia. Br J Haematol. 1996;93:333-340[CrossRef][Medline] [Order article via Infotrieve].
24.
Fadlon E, Vordermeier S, Pearson TC, et al.
Blood polymorphonuclear leukocytes from the majority of sickle cell patients in the crisis phase of the disease show enhanced adhesion to vascular endothelium and increased expression of CD64.
Blood.
1998;91:266-274
25.
Mollapour E, Porter JB, Kaczmarski R, Linch DC, Roberts PJ.
Raised neutrophil phospholipase A2 activity and defective priming of NADPH oxidase and phospholipase A2 in sickle cell disease.
Blood.
1998;91:3423-3429 26. Lard LR, Mul FPJ, de Haas M, Roos D, Duits AJ. Neutrophil activation in sickle cell disease. J Leukoc Biol. 1999;66:411-415[Abstract]. 27. Macey MG, McCarty DA, Vordermeier S, Newland AC, Brown KA. Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leukocytes. J Immunol. 1995;181:211-219. 28. Leeuwenberg JF, Smeets EF, Neefjes JJ, et al. E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro. Immunology. 1992;77:543-549[Medline] [Order article via Infotrieve]. 29. Boehme MW, Raeth U, Scherbaum WA, Galle PR, Stremmel W. Interaction of endothelial cells and neutrophils in vitro: kinetics of thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1): implications for the relevance as serological disease activity markers in vasculitides. Clin Exp Immunol. 2000;119:250-254[CrossRef][Medline] [Order article via Infotrieve]. 30. Donnelly SC, Haslett C, Dransfield I, et al. Role of selectins in development of adult respiratory distress syndrome. Lancet. 1994;344:215-219[CrossRef][Medline] [Order article via Infotrieve]. 31. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol. 1983;130:1910-1917[Abstract].
32.
Elbim C, Chollet-Martin S, Bailly S, Hakim J, Gougerot-Pocidalo MA.
Priming of polymorphonuclear neutrophils by tumor necrosis factor
33.
Elbim C, Prevot MH, Bouscarat F, et al.
Polymorphonuclear neutrophils from human immunodeficiency virus-infected patients show enhanced activation, diminished fMLP-induced L-selectin shedding, and an impaired oxidative burst after cytokine priming.
Blood.
1994;84:2759-2766 34. Lund-Johanson F, Terstappen LWMM. Differential surface expression of cell adhesion molecules during granulocyte maturation. J Leukoc Biol. 1993;54:47-55[Abstract].
35.
Elbim C, Bailly S, Chollet-Martin S, Hakim J, Gougerot-Pocidalo MA.
Differential priming effects of proinflammatory cytokines on human neutrophil oxidative burst in response to bacterial N-formyl peptides.
Infect Immun.
1994;62:2195-2201
36.
Topham MK, Carveth HJ, McIntyre TM, Prescott SM, Zimmerman GA.
Human endothelial cells regulate polymorphonuclear leukocyte degranulation.
FASEB J.
1998;12:733-746
37.
Gainet J, Dang PMC, Chollet-Martin S, et al.
Neutrophil dysfunctions, IL-8, and soluble L-selectin plasma levels in rapidly progressive versus adult and localized juvenile periodontitis: variations according to disease severity and microbial flora.
J Immunol.
1999;163:5013-5019 38. Taieb J, Mathurin P, Elbim C, et al. Blood neutrophil functions and cytokine release in severe alcoholic hepatitis: effect of corticosteroids. J Hepatol. 2000;32:579-586[CrossRef][Medline] [Order article via Infotrieve].
39.
Bennett TA, Edwards BS, Sklar LA, Rogelj S.
Sulfhydryl regulation of L-selectin shedding: phenylarsine oxide promotes activation-independent L-selectin shedding from leukocytes.
J Immunol.
2000;164:4120-4129
40.
Hafezi-Moghadam A, Thomas KL, Prorock AJ, Huo Y, Ley K.
L-selectin shedding regulates leukocyte recruitment.
J Exp Med.
2001;193:863-872
41.
Waddell TK, Fialkow L, Chan CK, Kishimoto TK, Downey GP.
Potentiation of the oxidative burst of human neutrophils. A signaling role for L-selectin.
J Biol Chem.
1994;269:18485-18491 42. Bourantas KL, Dalekos GN, Makis A, Chaidos A, Tsiara S, Mavridis A. Acute phase proteins and interleukins in steady state sickle cell disease. Eur J Haematol. 1998;61:49-54[Medline] [Order article via Infotrieve].
43.
Graido-Gonzalez E, Doherty JC, Bergreen EW, Organ G, Telfer M, McMillen MA.
Plasma endothelin-1, cytokine, and prostaglandin E2 levels in sickle cell disease and acute vaso-occlusive sickle crisis.
Blood.
1998;92:2551-2555 44. Raghupathy R, Haider MZ, Azizieh F, D'Souza TM, Abdelsalam R, Adekile AD. Tumor necrosis factor-alpha is undetectable in the plasma of SS patients with elevated HbF. Am J Hematol. 2000;64:91-94[CrossRef][Medline] [Order article via Infotrieve]. 45. Oh SO, Ibe BO, Johnson C, Kuranstin-Mills J, Raj JU. Platelet-activating factor in plasma of patients with sickle cell disease in steady state. J Lab Clin Med. 1997;130:191-196[CrossRef][Medline] [Order article via Infotrieve]. 46. Wang RH, Phillips G, Medof ME, Mold C. Activation of the alternative complement pathway by exposure of phosphatidylserine on erythrocytes from sickle cell disease patients. J Clin Invest. 1993;92:1326-1335. 47. Mold C, Tamerius JD, Phillips G. Complement activation during painful crisis in sickle cell anemia. Clin Immunol Immunopathol. 1995;76:314-320 |
Top
Abstract
Introduction
-globin chain and produces hemoglobin S (Hb
S).
rates and risk factors.
N Engl J Med.
1991;325:11-16