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Download date: 09 Jan 2020
Chapter 3
Inflammatory and endothelial markers during vaso-occlusive crisis in sickle cell disease
Marein Schimmel1,2, Brenda M. Luken 2, Erfan Nur1, Charlotte F.J. van Tuijn1, Joep W. Sins1, Dees P.M. Brandjes3, Sacha S. Zeerleder1,2 and Bart J. Biemond1
1 Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands2 Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, The Netherlands3 Department of Internal Medicine, Slotervaart Hospital, Amsterdam, The Netherlands
Submitted for publication
48 Chapter 3
ABSTR ACT
Vaso-occlusive crisis (VOC) is the hallmark of sickle cell disease (SCD). The patho-
physiology is complex and characterized by inflammation, neutrophil and endothelial
activation. In this prospective study, we aimed to determine changes in biomarkers
of inflammation, neutrophil and endothelial activation during the course of VOC, to
analyze the dynamics of these processes in the pathogenesis of VOC and subsequent
complications, like acute chest syndrome(ACS). In consecutive SCD patients, we ob-
tained blood samples on day 1, 2, 3 and 5 of admission for VOC and in steady state, and
determined biomarkers of inflammation (C-reactive protein(CRP); pentraxin3(PTX3),
neutrophil activation (elastase-alpha1-antitrypsin-complex(HNE-α1-AT); calpro-
tectin), cell dead (nucleosomes) and endothelial activation (von Willebrand factor
antigen(VWF:Ag); VWF propeptide(VWFpp)).
In 32 VOC’s in 24 adult patients (22 HbSS/Sβ0-thalassemia, 10 HbSC/Sβ+-thalassemia),
PTX3 and CRP levels peaked on day 2-3 in the course of VOC, while highest levels
of nucleosomes and HNE-α1-AT were measured on day 1. Although not significant,
a late rise in calprotectin levels was observed. Levels of VWF:Ag were elevated in
steady state and remained stable during VOC course while VWFpp levels remained
within normal range during steady state and VOC, suggesting chronic endothelial
activation. Significant higher levels of HNE-α1-AT and calprotectin levels were ob-
served in patients who developed an ACS.
In conclusion, our results suggest that VOC is an acute inflammatory process with
prompt neutrophil activation, on top of chronic endothelial activation in patients
with SCD. Neutrophil activation markers HNE-α1-AT and calprotectin may aid the
identification of patients at risk for development of ACS.
Inflammatory and endothelial markers during vaso-occlusive crisis in sickle cell disease 49
3
INTRODUCTION
Sickle cell disease (SCD) is characterized by chronic hemolytic anemia and recurrent
vaso-occlusive crisis (VOC).[1, 2] VOC may lead to life-threatening acute complica-
tions such as acute chest syndrome (ACS), multi-organ failure and sudden death.
[3-5] Pathophysiological factors contributing to VOC include endothelial activation,
adhesion of activated sickle red blood cells (sRBCs), leukocytes and platelets, coagu-
lation activation and increased oxidative stress.[6] Even in sickle cell patients without
symptoms of VOC (steady state), a chronic inflammatory state is present as reflected
by increased levels of circulating C-reactive protein (CRP)[7-9] and inflammatory
cytokines like TGF-β-1,[10] TNF-α and IL-8[11] when compared to healthy individuals.
Furthermore, neutrophils of patients with SCD in steady state displayed an activated
phenotype with increased expression of Mac-1 (CD11b/CD18)[12] and decreased ex-
pression of L-selectin (CD62L)[13] when compared to healthy individuals. In addition,
increased circulating levels of von Willebrand factor (VWF) antigen were observed in
SCD patients in steady state, indicating chronic endothelial activation.[14, 15]
During VOC, close interactions between sickle red blood cells (sRBCs), the endothe-
lium and neutrophils are evident.[16-21] Infusion of the redox active heme in SCD
mice, mimicking chronic hemolysis, induced the appearance of VWF on the vessel
wall.[16] It was suggested that intravascular hemolysis leading to oxidative damage
of endothelial cells, may contribute to the development of VOC as adhesive VWF
molecules are able to bind sRBCs.[17] In addition, in another humanized sickle cell
murine model, it was shown that upon induction of VOC with TNF-α, sRBCs directly
interact with circulating neutrophils, which was accompanied by a reduction in local
microcirculatory blood flow and led to a diminished survival of these mice.[18, 19]
Inhibition of interactions between sRBCs, neutrophils and endothelial cells resulted
in a restored local blood flow in different (murine and human) SCD models of VOC.
[19-21] Therefore, it appears that VOC is an acute aggravation of ongoing inflammation
with neutrophil and endothelial activation, whereby the exact trigger that initiates
VOC, and the sequence of events involved in VOC including the possible progression
to further acute complications such as ACS, are still incompletely understood.
In the current study, we aimed to gain insight in the sequence of events during VOC
and steady state. Therefore, the dynamics in levels of biomarkers of inflammation,
50 Chapter 3
cell death, neutrophil activation and endothelial activation in patients with SCD dur-
ing the course of hospitalization for VOC and in steady state were studied.
Levels of the acute phase proteins CRP and pentraxin3 (PTX3) were determined as
markers of inflammation. Both CRP and PTX3 are members of the pentraxin protein
family,[22] a family of soluble pattern recognition molecules with an important role in
pathogen opsonization and complement activation. Levels of the neutrophil derived
azurophilic protein elastase in complex with its inhibitor α1-antitrypsin (HNE-α1-AT),
and the neutrophil cytosolic protein calprotectin, were determined as markers of neu-
trophil activation. Circulating nucleosomes, the basic units of DNA organization,[23]
were determined as a marker of systemic inflammation and cell death,[24-26] and
have been attributed to the formation of neutrophil extracellular traps (NETs) by ac-
tivated neutrophils.[27-30] Finally, VWF antigen and propeptide (VWF:Ag and VWFpp)
were determined as markers of endothelial activation in this study.
METHODS
Patients
Consecutive adult patients with SCD admitted for VOC to the Academic Medical Center
or the Slotervaart Hospital (Amsterdam, The Netherlands) were approached for par-
ticipation. Inclusion criteria were: age ≥ 18 years with sickle cell anemia (HbSS) or the
compound heterozygous states HbSβ0-thalassemia (HbSβ0-thal), HbSβ+-thalassemia
(HbSβ+-thal) or sickle-hemoglobin C (HbSC). VOC was defined as musculo-skeletal and/
or visceral pain not otherwise explained and recognized as such by the patient. An
acute chest syndrome was defined as experiencing clinical symptoms including fever,
respiratory symptoms, or chest pain, in combination with a new pulmonary infiltrate vis-
ible by X-ray imaging.[31] Exclusion criteria were: pregnancy, active cancer, chronic HIV
infection or blood transfusion in the three months prior to admission. Blood samples
were obtained after overnight fasting on the morning following hospital admission, and
on subsequent mornings (hospitalization days) as indicated. Inclusion was limited to a
maximum of two separate episodes of VOC per patient. Patients included in the study
were seen at the out-patient clinic at least 4 four weeks after discharge for collection
of a steady state blood sample, if at that moment they were not experiencing vaso-
occlusive pain. From all patients a written informed consent was obtained. The study
Inflammatory and endothelial markers during vaso-occlusive crisis in sickle cell disease 51
3
protocol was approved by the Medical Ethical Committee of the participating centers
and conducted in agreement with the Helsinki declaration.
Laboratory analysis
Blood samples were taken by venipuncture. Blood tubes were centrifuged once or
twice (citrated plasma) for 15 minutes at 3000 x g at 4°C to obtain plasma and serum
and stored in aliquots at -80˚C until further analysis. The following markers were
determined only upon admission for VOC: standard blood counts (hemoglobin and
white blood cell count) were determined in EDTA-anticoagulated plasma (Cell-Dyn
4000, Abbott, Illinois, USA) and hemolysis parameters (bilirubin and lactate dehydro-
genase, LDH) were measured with spectrophotometry in heparinized plasma (P800
Modular, Roche, Switzerland). All markers were determined in a longitudinal approach
on the morning upon admission for VOC and on every following day during admission.
C-reactive protein (CRP) levels were measured in serum using an automated immu-
noturbidimetric end point assay with a linearity of 0.3-350 mg/l, on a Roche cobas
c702 analyzer. The following markers were measured in EDTA-anticoagulated plasma
using previously described ELISA methods: PTX3,[32] extracellular nucleosomes[24, 33]
and calprotectin (the heterodimer S100A8/S100A9).[34] Human neutrophil elastase
was measured in complex with its inhibitor α1-antitrypsin (HNE-α1-AT), using ELISA
as described previously.[35, 36] VWF:Ag levels in citrated plasma were measured by
ELISA using commercial antibodies (DAKO, Denmark). Normal human pooled plasma
was used as standard and calibrated to the WHO 07/316 6th International Standard
for VWF. VWFpp was measured using an in-house commercially available ELISA as pre-
viously described.[37] Throat swab samples, taken upon admission, were screened for
presence of nucleic acids of Influenza A, Influenza B, adenovirus, Respiratory syncytial
virus (RSV) and enterovirus using multiplex real-time polymerase-chain (RT-PCR) on a
LightCycler 480 (Roche Life Science) using methods described elsewhere.[38]
Statistical analysis
For data analysis a commercial statistical package (IBM SPSS Statistics 21.0, US) was
used. Statistical data analysis was done with data of all included patients. An additional
analysis was performed dividing patients in two groups: patients with the relatively
severe genotypes HbSS and HbSβ0-thalassemia were grouped together forming the
HbSS/HbSβ0-thal group, and patients with the relatively milder HbSC and HbSβ+-
thalassemia genotypes were gathered together to form group HbSC/HbSβ+-thal. [39, 40]
Non-normally distributed data are expressed as median with interquartile range (IQR).
52 Chapter 3
Mann-Whitney rank sum test was used to assess differences between groups, or the
Kruskal-Wallis one-way of analysis of variance when more than two groups were com-
pared. For related sample analysis the related- samples Wilcoxon Signed rank test was
used. Correlation analysis was done using Spearman’s rank correlation (Sr). A P-value
below 0.05 was considered statistically significant. For multiple comparison testing the
Bonferroni correction was used and when applied, the adapted P-value is indicated.
RESULTS
Patients
Twenty-four sickle cell patients admitted for VOC were included in the study, ac-
counting for 32 admissions (22 HbSS/Sβ0-thal and 10 HbSC/Sβ+-thal). Baseline char-
acteristics are presented in table 1. There were no significant differences in baseline
characteristics between patients in VOC or steady state. Median hospital stay was 5.5
(IQR 3-8) days for the whole group, 4 (IQR 3-7) days for the HbSS/Sβ0-thal group and 7
(IQR 6-9) for the HbSC/Sβ+-thal group. Five patients (4 HbSS and 1 HbSβ+-thal patient)
developed an ACS during admission. All patients with ACS received antibiotics and
Table 1.
Sickle cell diseaseVaso-occlusive crisis
First sample after admission
Sickle cell diseaseSteady state
Inclusions n = 32 n = 24
Demographics
HbSS-HbSβ0 // HbSC-HbSβ+ 67% // 33%
Age (y) 26 (21-30) 26 (22-30)
Female/male ratio 16/16 10/11
On hydroxycarbamide (%) 25% 29%
Laboratory measurements
White blood cells (10e9/l) 9.9 (7.2-12.7) 8.6 (6.1-9.6)
Hemoglobin (mmol/ll) 6.1 (4.7-6.9) 6.0 (5.2-7.0)
Bilirubin Total (μmol/l) 30 (23-55) 26 (19-40)
LDH (U/l) 405 (260-492) 362 (218-435)
CRP (mg/l) 3.7 (1.5-10.6) 2.1 (0.8-6.2)
Table 1. Baseline characteristics. Results are shown as median with interquartile range.
*Significant difference between healthy controls and steady state patients with sickle cell disease, P <0.05.
** P <0.001 , Ɨ Significant difference between steady state patients with sickle cell disease and patients with
sickle cell disease in painful crisis P <0.05. ** ƗƗ P <0.001
Inflammatory and endothelial markers during vaso-occlusive crisis in sickle cell disease 53
3
two patients (both HbSS) required erythropheresis. The presence of a respiratory viral
infection as trigger for VOC was ruled out in all patients that were tested (27 out of 32
admissions) using multiplex RT-PCR.
Longitudinal analysis of the levels of inflammatory markers, nucleosomes,
neutrophil activation markers and endothelial activation markers
During hospitalization for VOC the levels of markers of inflammatory response,
nucleosomes and markers of neutrophil activation and endothelial activation were
followed. The different markers showed clearly distinct temporal patterns (Figure 1).
The dynamics of plasma levels of both CRP and PTX3 were largely comparable to each
other during the course of admission, with high levels at day 1 of admission when
compared to steady state and reaching peak levels at day 2 and 3 for CRP and PTX3,
respectively (Figure 1A and 1B). Levels of nucleosomes were highest upon admis-
sion and showed a rapid decline on day 2 and further decreased during subsequent
days of admission (Figure 1C). Levels of HNE-α1-AT seemed to peak upon admission,
but were not significantly different from levels in steady state and normalized from
the second day of admission (Figure 1D). Levels of calprotectin showed a more
gradual increase during hospitalization reaching peak levels at day 5 of admission,
and returned to the normal range in steady state (Figure 1E). Plasma levels of the
endothelial activation markers VWF:Ag were comparable to levels in steady state and
remained stable during the course of VOC while VWFpp levels remained within the
normal range (Figure 1F, G). Levels of all markers, but especially CRP, nucleosomes,
HNE-α1-AT and calprotectin, were higher in HbSS/HbSβ0-thal patients compared to
levels in HbSC/HbSβ+-thal patients (Figure 1).
Plasma levels of CRP, PTX3 and nucleosomes, but not neutrophil activation markers
HNE-α1-AT and calprotectin, were significantly increased at day 1 of admission for
VOC when compared to levels of these markers in steady state. Levels of VWF:Ag in
steady state were above the normal range but not significantly different from levels
at day 1. Levels of VWFpp in steady state were within the normal range (2.8-8.3 nM)
[41] and were similar to levels in samples taken during VOC at day 1.(See table 2).
54 Chapter 3
Day1 (28
)
Day2 (23
)
Day3 (22
)
Day5 (14
)
Steady Stat
e (28)
0
20
40
60
80
100
CRP
CR
Pm
g/L
Day1 (31
)
Day2 (27
)
Day3 (23
)
Day5 (13
)
Steady Stat
e (28)
0
5
10
15
PTX3
PTX3
ng/m
l
Day1 (31
)
Day2 (27
)
Day3 (23
)
Day5 (13
)
Steady Stat
e (28)
0
1000
2000
3000
Nucleosomes
Nucl
eoso
mes
U/m
l
All patients
HbSS/HbSb0
HbSC/HbSb+
Day1 (31
)
Day2 (27
)
Day3 (23
)
Day5 (13
)
Steady Stat
e (28)
0
100
200
300
400
500
HNE-a1-AT
HNE
-a1-
ATng
/ml
Day1 (31
)
Day2 (28
)
Day3 (23
)
Day5 (14
)
Steady Stat
e (28)
0
20000
40000
60000
80000
Calprotectin
Calp
rote
ctin
ng/m
l
Day1 (29
)
Day2 (28
)
Day3 (23
)
Day5 (13
)
Steady Stat
e (28)
0
100
200
300
VWF:Ag
VWF:
Ag%
Day1 (29
)
Day2 (28
)
Day3 (23
)
Day5 (13
)
Steady Stat
e (28)
0
5
10
15
VWFpp
VWFp
pnM
Inflammatory response
CRPPTX3
Nucleosomes
Neutrophil activation
HNE-a1-AT calprotectin
Endothelial activationVWF:Ag VWFpp
All patientsHbSS/HbSb0
HbSC/HbSb+
Figure 1. Levels of CRP, PTX3, nucleosomes, HNE-α1-AT, calprotectin, VWF:Ag, and VWFpp, during ad-mission for vaso-occlusive crisis (VOC) and in steady state.
Samples were taken on the first, second, third and fifth morning of VOC. Means with SEM. Levels of all mark-
ers were higher in HbSS/HbSβ0-thal patients when compared to levels in HbSC/HbSβ+-thal patients. A and
B. Levels of CRP (A) and PTX3 (B) are increased in the first sample taken on admission when compared to
steady state levels and show a further increment during respectively the first three (PTX3) and two (CRP)
days of admission, thereafter levels tend to return to steady state levels.
Inflammatory and endothelial markers during vaso-occlusive crisis in sickle cell disease 55
3
Relation of inflammatory markers, nucleosomes, neutrophil and endothelial
activation markers to hemolysis
To investigate the contribution of hemolysis and cell death to VOC in this cohort,
plasma levels of bilirubin and lactate dehydrogenase (LDH) were measured upon ad-
mission for VOC. There was a significant relation between the LDH level and the level
of both nucleosomes and HNE-α1-AT, (P=0.028 and P=0.048, Kruskal-Wallis test). CRP
(P=0.087), calprotectin (P=0.070), VWF:Ag (=0.079) and VWFpp (P=0.054), but not
PTX3 (P=0.289), showed a trend to correlation with this marker. Bilirubin levels were
also related to levels of nucleosomes (P = 0.026) and to VWF:Ag (P = 0.002) levels but
not to HNE-α1-AT (P = 0.243), calprotectin (P = 0.575), CRP (P = 0.335), PTX3 (P = 0.398),
and VWFpp (P = 0.218).
Relation between inflammatory markers, nucleosomes and markers for
neutrophil- and endothelial activation
In the steady state, applying Bonferroni correction for multiple correlation testing (P
< 0.007 was considered statistically significant), a correlation was observed between
levels of CRP and VWF:Ag, Sr = 0.518, P = 0.006. No other correlations were observed
between the markers at steady state.
In samples taken the first morning after admission for VOC, strong (Sr ≥0.500) and
highly significant correlations became manifest between almost all selected markers
for chronic endothelial activation, inflammation, nucleosomes and neutrophil activa-
tion. See table 3. Notable exceptions were that VWFpp levels did not correlate with
levels of CRP, PTX3, HNE-α1-AT and calprotection. And PTX3 levels did not show any
correlation with levels of VWF:Ag and nucleosomes.
← Figure 1. Levels of CRP, PTX3, nucleosomes, HNE-α1-AT, calprotectin, VWF:Ag, and VWFpp, during admission for vaso-occlusive crisis (VOC) and in steady state. (continued)
C. Levels of nucleosomes were high upon admission and show already upward of day 1 a rapid decline
followed by a gradual decrease during subsequent days of admission. D. Levels of HNE-α1-AT appear high
upon admission, but levels are not significantly increased, and are already low the second day of admis-
sion. E. Levels of calprotectin show a small gradual increment during subsequent days of admission. F and
G. Levels of VWF:Ag (F) and VWFpp (G) remain stable during admission for VOC and in steady state.
56 Chapter 3
Table 2.
VoC
First sample
Steady state
At least 4 weeks past VOC
Inflammatory response
CRPmg/L
7.0 (1.5-33.6) * 3.9 (2.0-7.1)
HbSS/beta0 22.4 (7.8-58.0) * 5.3 (2.8-9.6)
HbSC/beta+ 1.7 (1.2-4.4) 1.8 (0.6-4.5)
PTX3ng/ml
6.6 (3.4-8.7) * 3.5 (3.0-6.7)
HbSS/beta0 8.6 (3.2-10.6) * 3.5 (2.9-6.8)
HbSC/beta+ 6.8 (3.7-8.4) 6.1 (3.4-8.2)
Nucleosomes
Neutrophil activation markers
Nucleosomes U/ml
26 (8-114) * 18 (5-23)
HbSS/beta0 48 (24-771) * 23 (5-25)
HbSC/beta+ 16 (5-31) * 5 (5-5)
HNE-α1-ATng/ml
65.1 (47.1-97.6) 59.9 (37.1-94.5)
HbSS/beta0 87.3 (52.0-310.8) 60.7 (43.3-111.0)
HbSC/beta+ 51.2 (32.4-69.8) 48.7 (28.3-60.6)
Calprotectin ng/ml
1876 (792-4813) 1587 (915-2459)
HbSS/beta0 2551 (922-6909) 1486 (902-2459)
HbSC/beta+ 1466 (95-2209) 1688 (1308-2524)
Endothelial activation VWF:Ag%, (50-150)
169 (131-250) 181 (144-268)
HbSS/beta0 216 (155-270) 227 (155-285)
HbSC/beta+ 123 (85-155) * 147 (109-173)
VWFpp nM, 2.8-8.3
6.8 (4.7-8.7) 7.2 (5.8-7.8)
HbSS/beta0 6.8 (4.8-9.3) 7.3 (6.2-8.3)
HbSC/beta+ 5.3 (3.5-7.4) 6.1 (5.1-7.5)
Table 2. Levels of CRP, PTX3, nucleosomes, HNE-α1-AT, calprotectin and VWF:Ag and VWFpp in samples of patients with SCD as markers for inflammation, cell death, neutrophil activation endothelial activa-
tion.
Values are depicted as median with interquartile range.
* Significant difference between levels in steady state and VOC.
Inflammatory and endothelial markers during vaso-occlusive crisis in sickle cell disease 57
3
Table 3.
CRP PTX3 Nucleosomes HNE-α1-AT Calprotectin VWFAg VWFpp
CRP - 0.558P=0.002
0.596P=0.001
0.731P<0.001
0.852P<0.001
0.590P=0.002
NS
PTX3 - NS 0.521P=0.002
0.624P<0.001
NS NS
Nucleosomes - 0.622P<0.001
0.602P<0.001
0.699P<0.001
0.498P=0.006
HNE-α1-AT - 0.793P<0.001
0.571P=0.001
NS
Calprotectin - 0.523P=0.004
NS
VWFAg - 0.604P=0.001
VWFpp -
Table 3. Correlations between the markers for inflammation, nucleosomes and markers for neutrophil and endothelial activation on first day after admission for VOC. Only significant correlations are de-picted here (according Bonferroni correction for multiple comparison a significant value here is when P ≤0.007).
Relation with acute chest syndrome (ACS)
In the five patients that developed an ACS during admission for VOC, levels of nucleo-
somes, HNE-α1-AT, calprotectin and VWF:Ag in the samples the first morning after
admission for VOC were significantly higher compared to levels of these markers in
patients without ACS (nucleosomes: 2220.0 (IQR 568.0-19875.0) vs 26.4 (IQR 5.0-50.6)
U/ml, P=0.01; HNE-α1-AT: 534.0 (IQR 222.4-2805.0) vs 58.7 (IQR 42.4-89.5) ng/ml ,
P=0.004; calprotectin: 9440.0 (IQR 3996.5-191500.0) vs 1875.5 (IQR 691.5-3453.8) ng/
ml, P = 0.02 and VWF:Ag: 315 (IQR 220-365) vs 167 (IQR 129-231) % , P=0.006). See
Figure 2. Levels of CRP, PTX3 and VWFpp were not different in patients with and those
without ACS. (CRP: 88.4 (IQR 20.2-291.1) vs 8.1 (IQR 1.6-44.5) mg/L, P = 0.07; PTX3: 8.8
(IQR 6.8-18.5) vs 6.8 (IQR 3.3-8.9) ng/mL, P = 0.13 and VWFpp: 10.6 (IQR 5.7-24.6) vs 6.5
(IQR 4.6-8.2) nM , P=0.05).
58 Chapter 3
No ACSACS
1
10
100
1000
CRP
CR
Pm
g/L
P = 0.07
No ACSACS
1
10
100
PTX3
PTX3
ng/m
l
P = 0.13
No ACSACS
1
10
100
1000
10000
Nucl
eoso
mes
U/m
l
30000P = 0.01
Nucleosomes
No ACSACS
10
100
1000
10000
HNE-a1-AT
HNE
-a1-
ATng
/ml
P = 0.004
No ACSACS
100
1000
10000
100000
1000000
CalprotectinCa
lpro
tect
inng
/ml
P = 0.02
No ACSACS
0
100
200
300
400
500
VWFAg
VWF:
Ag%
p= 0.006
No ACSACS
-10
0
10
20
30
40
VWFpp
VWFp
pnM
p= 0.051
Inflammatory response
CRPPTX3
Nucleosomes
Neutrophil activation
HNE-a1-AT calprotectin
Endothelial activationVWF:Ag VWFpp
Figure 2. Levels of CRP, PTX3, nucleosomes, HNE-α1-AT, calprotectin, VWF:Ag and VWFpp in samples of
patients during VOC who developed an acute chest syndrome (ACS) during admission for VOC and pa-
tients without ACS. Levels of nucleosomes (C), HNE-α1-AT(D), calprotectin(E) and VWF:Ag(F) were signifi-
cantly elevated in patients who developed an acute chest syndrome during admission compared to levels
in patients without acute chest syndrome.
Inflammatory and endothelial markers during vaso-occlusive crisis in sickle cell disease 59
3
DISCUSSION
Sickle cell disease (SCD) is characterized by recurrent painful vaso-occlusive crises
(VOC). The pathophysiology of these VOC is complex and multi-factorial involving
hemolysis, an inflammatory response, endothelial activation, neutrophil activation
and enhanced cellular adhesion leading to increased cell-cell interactions between
sickle erythrocytes, leukocytes, platelets and endothelial cells. In the present study,
we describe the dynamics in biomarkers of inflammation, cell death, neutrophil and
endothelial activation on consecutive days during the course of VOC and in compari-
son to steady state.
Levels of CRP and PTX3 and were significantly elevated and respectively peaked on
day 2 and 3 of the VOC indicating that VOC is accompanied by an acute inflamma-
tory response. Since CRP is considered to be a reflection of systemic inflammation in
response to interleukin-6 and PTX3 levels mainly reflect local inflammation, as it is
released locally by neutrophils and endothelial cells,[42] both forms of inflammatory
response seem to be active in VOC. This observation is different from findings in
patients with more local forms of inflammation such as acute myocardial infarction
wherein PTX3 levels showed a distinct earlier plasma peak followed by an increase in
levels of CRP.[43] Nucleosome levels peaked at day 1 followed by a rapid decline the
days thereafter. As nucleosomes represent free DNA due to cell death, we observe
a peak of cell death already early in the VOC process. Circulating nucleosomes have
also been related to Neutrophil Extracellular Traps (NETs). NETs can be released by
activated neutrophils with the capacity to capture and kill pathogens, but NETs can
also inflict damage to cells of the host.[30] In the present study, during VOC, levels
of neutrophil activation marker HNE-α1-AT mimicked the course of nucleosomes
levels, similar to our previous study.[29] And even though we cannot exclude that
nucleosomes detected by our ELISA originate from cells other than neutrophils, these
findings suggest that neutrophil activation is related to the release of nucleosomes.
This is further supported by the strong (Sr≥0.500) and highly significant correla-
tions (P<0.007) that were found between the levels of nucleosomes and levels of
neutrophil activation markers HNE-α1-AT and calprotectin during VOC suggesting an
increased presence of NETs during VOC.[27, 28] In Berkeley sickle mice, NETs were
identified as important players in the pathogenesis of VOC including related lung
complications.[51] Increased levels of nucleosomes and HNE- α1-AT, [29] and also
PTX3 [50] during VOC and their relation with clinical severity indicate that NETs may
60 Chapter 3
be involved in the pathogenesis of VOC in sickle cell patients.[29] Our current data
indeed support these findings, since the highest levels of nucleosomes, HNE-α1-AT
and calprotectin were observed in patients with ACS. In our study cohort, 15% of
patients admitted with VOC developed an ACS, of which all recovered. Generally,
ACS has a high mortality rate.[52] Exchange transfusion early in the onset of ACS is
considered the best treatment, but identifying patients at risk for ACS development is
challenging.[53] While only a modest elevation of HNE-α1-AT and calprotectin levels
in our patients with VOC were observed in this study, very high levels were found in
the patients developing ACS. It would be interesting to investigate the value of these
markers to identify patients at risk for developing ACS in future studies. In addition,
the use of anti-inflammatory drugs that reduce neutrophil activation in patients with
VOC in an attempt to prevent the development of ACS might be considered. Espe-
cially drugs that block the integrin Mac-1 (CD11b/CD18) might be of interest. Mac-1
is upregulated on the surface of neutrophils of patients with SCD,[12] and Mac-1 has
been identified as an actor both in neutrophil adhesion to endothelial cells,[54] and
in the formation of NETs.[55]
Calprotectin is a damage-associated-molecular-pattern (DAMP) that acts as an en-
dogenous danger signal to support and aggravate an inflammatory response and is
also considered to be a marker of neutrophil activation as it is abundant in neutrophil
cytosol.[44] Surprisingly and in contrast with the course of nucleosome and HNE-
α1-AT plasma levels, a gradual, non-significant rise in plasma levels of this biomarker
was noticed during admission suggesting that neutrophil activity continues at lower
degree during the further course of VOC, possibly due to ischemia-reperfusion in-
duced oxidative stress. However, the calprotectin plasma levels were modest in
comparison with observations in patients with sepsis or typhoid fever, in which high
plasma levels of calprotectin have demonstrated to correlate with disease severity,
indicating less pronounced, or possibly another way of neutrophil activation in VOC
than in these conditions.[45, 46].
Whilst biomarkers of inflammation (CRP, PTX3) and nucleosomes were significantly el-
evated during VOC when compared to levels in steady state, plasma levels of VWF:Ag
and VWFpp did not change during admission for VOC as compared to steady state
levels. In normal conditions, both VWF:Ag and VWFpp are released upon endothelial
cell activation. Since VWF:Ag and VWFpp are released in equimolar amounts[47] but
VWFpp has a shorter half-life than VWF:Ag,[48] the ratio between these markers can
Inflammatory and endothelial markers during vaso-occlusive crisis in sickle cell disease 61
3
differentiate between acute and chronic endothelial activation.[49] In the present
study, we found increased levels of VWF:Ag in the absence of elevated VWFpp indi-
cating chronic endothelial activation, as has been reported previously.[41, 50]
In our study, levels of all biomarkers during admission for VOC, but especially CRP,
nucleosomes, HNE-α1-AT and calprotectin levels, were higher in HbSS/HbSβ0-thal
patients when compared to HbSC/HbSβ+-thal patients. In fact, hardly any increase in
plasma levels of nucleosomes, HNE-a1-AT and calprotectin was observed in patients
with HbSC/HbSβ+-thal. This could be explained by the fact that less pronounced
hemolysis and its downstream effects such as oxidative damage, inflammatory re-
sponse and neutrophil activation takes place in these generally milder forms of SCD.
Some limitations of our study have to be mentioned. Firstly, day 1 in our study was
defined as the day of admission in hospital while the exact beginning of the com-
plaints may have varied between patients since patients sometimes delay the mo-
ment to seek medical help for a VOC. Secondly, our trial included a limited number of
VOC which may have resulted in the loss of power to detect correlations or significant
elevations of some of the markers.
In conclusion, this study provides increased insight in the sequence of events during
VOC. Biomarkers of endothelial activation did not show an acute-on-chronic eleva-
tion during VOC, indicating chronically activated, or perhaps exhausted, endothelium
in patients with SCD. During VOC, an acute inflammatory response accompanied by
neutrophil activation was observed as reflected by elevated CRP, PTX3 and nucleo-
some levels. Although HNE-α1-AT and calprotectin levels in VOC demonstrated a
modest non-significant rise compared to steady state, extremely high levels were
observed in patients developing ACS. Neutrophil activation, especially in patients
with an ACS, may form an interesting therapeutic target for anti-inflammatory drugs
in further studies.
62 Chapter 3
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