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Sickle cell diseasePathogenesis and biomarkersSchimmel, M.

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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


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


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.


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


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.


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


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.


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


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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