Inflammatory and endothelial markers during vaso‐occlusive crisis and acute chest syndrome in sickle cell disease
Authors: Marein Schimmel, Brenda M. Luken, Erfan Nur, Charlotte F. J. van Tuijn, Joep W. Sins, Dees P. M. Brandjes, Sacha S. Zeerleder, Bart J. Biemond
Journal: American Journal of Hematology (2017)

Sickle cell disease (SCD) is characterized by chronic hemolytic anemia and recurrent vaso-occlusive crisis (VOC) that may lead to life-threatening acute complications such as acute chest syndrome (ACS). Pathophysiological factors contributing to VOC include endothelial activation, cell adhesion, coagulation activation and increased oxidative stress.1 Even in sickle cell patients without symptoms of VOC a chronic inflammatory state is present as reflected by increased levels of circulating C-reactive protein (CRP), inflammatory cytokines, activated neutrophils, and increased levels of von Willebrand factor (VWF) antigen, indicating chronic endothelial activation. During VOC, an acute aggravation of ongoing inflammation with neutrophil and endothelial activation develops, 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 this sequence of events during VOC by assessing the dynamics in biomarkers of inflammation, cell death, neutrophil activation, and endothelial activation in patients with SCD during the course of hospitalization for VOC and the development of ACS. Levels of the acute phase proteins CRP and pentraxin3 (PTX3) were determined as markers of inflammation. Levels of the neutrophil derived azurophilic protein elastase in complex with its inhibitor α1-antitrypsin (HNE-α1-AT), and the neutrophil cytosolic protein calprotectin, a damage-associated-molecular-pattern protein (DAMP) that acts as an endogenous danger signal to support and aggravate an inflammatory response, were determined as neutrophil activation markers. Circulating nucleosomes, the basic units of DNA organization, were determined as a marker of systemic inflammation and cell death2 and have been attributed to the formation of neutrophil extracellular traps (NETs) by activated neutrophils. Finally, VWF antigen and propeptide (VWF:Ag and VWFpp) were determined as markers of endothelial activation. Consecutive adult patients with SCD (HbSS, HbSβ0-thal, HbSβ+-thal or HbSC) admitted for VOC to the Academic Medical Center or the Slotervaart Hospital (Amsterdam, The Netherlands) were approached for participation. Using a power analysis program an actual sample size of 32 patients was calculated. Statistical data analysis was done with data of all included patients. Additional analyses were performed for patients with the relatively severe genotypes HbSS and HbSβ0 (HbSS/HbSβ0 group) and patients with the relatively milder HbSC and HbSβ+ genotypes (HbSC/HbSβ+ group) separately. Additional information on methods is available in the online supporting information. Twenty-four sickle cell patients admitted for VOC were included in the study, accounting for 32 admissions (22 HbSS/Sβ0 and 10 HbSC/Sβ+). Baseline characteristics are presented in Supporting Information Table S1. Median hospital stay was 5.5 (IQR 3–8) days for the whole group. Five patients (4 HbSS and 1 HbSβ+ patient) developed an ACS during admission. All patients with ACS received antibiotics and two patients (both HbSS) required erythrocytapheresis. 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. During hospitalization the different biomarkers showed clearly distinct temporal patterns (Figure 1 and Supporting Information Table S2). 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 inflammatory 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,3 both forms of inflammatory response seem to be active in VOC. Nucleosome levels peaked at day 1 followed by a rapid decline the days thereafter and mimicked the course of HNE-α1-AT (Figure 1C,D). Calprotectin levels did not significant change compared to steady state in the analysis of all patients. (Figure 1E). Interestingly, plasma levels of all inflammatory biomarkers and in particular markers of neutrophil activation were significantly higher in patients developing ACS. (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. Samples were taken on the first, second, third and fifth morning of VOC. Means with SEM. Levels of all markers 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. 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 admission. 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. Plasma levels of all inflammatory biomarkers and markers of neutrophil activation were significantly higher in patients developing ACS Previously, levels of nucleosomes, HNE- α1-AT4 and PTX35 have been demonstrated to relate with clinical severity. Our current data support these findings, given the fact that the highest levels of nucleosomes, HNE-α1-AT and calprotectin were observed in patients that developed an ACS (Figure 1). Exchange transfusion early in the onset of ACS is considered best treatment, but identifying patients at risk for ACS development is challenging. While only 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 prospectively to identify patients at risk for developing ACS in future studies. Plasma levels of VWF:Ag were increased in the absence of elevated VWFpp and remained stable during the course of VOC and were comparable to levels in steady state. (Figure 1F,G). Since VWF:Ag and VWFpp are released in equimolar amounts but VWFpp has a shorter half-life than VWF:Ag, the ratio between these markers indicates chronic endothelial activation, as has been reported previously.6 Additional data on difference between VOC and steady state and analysis of correlations between markers of inflammation, neutrophil activation, endothelial activation and hemolysis is available in the online Supporting Information (Supporting Information Table S3). 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 patients when compared to HbSC/HbSβ+ patients. This could be explained by the fact that less pronounced hemolysis and its downstream effects such as oxidative damage, inflammatory response 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 complaints may have varied between patients. Secondly, our trial included a limited number of VOC what 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. During VOC, an acute inflammatory response accompanied by neutrophil activation was observed as reflected by elevated CRP, PTX3 and nucleosome levels. Although HNE-α1-AT and calprotectin levels in VOC demonstrated a modest nonsignificant rise compared to steady state, extremely high levels were observed in patients developing ACS. Therefore, neutrophil activation, especially in patients with an ACS, may form an interesting therapeutic target and therefore deserves further investigation. The authors would like to thank B. Bottazzi and A. Mantovani (Istituto Clinico Humanitas, Rozzano, Milan, Italy) for the laboratory determination of pentraxin-3 and useful discussions and prof. dr. T. Vogl (Institute of Immunology, University of Muenster, Germany), for the laboratory determination of calprotectin and useful discussions. Furthermore, the authors would like to thank I. Bulder and G. van Mierlo (Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam) for technical support and useful discussions. M. Schimmel, S.S. Zeerleder and B.J. Biemond had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: MS, EN, BB. Acquisition of data: MS, BL, JS, CF. (Statistical) analysis of data: MS, BB and SS. Interpretation of data: MS, BL, SS and BB Drafting of the manuscript: MS, BL, SS and BB. Critical revision of the manuscript for important intellectual content: all named co-authors. None reported. None reported. M. Schimmel http://orcid.org/0000-0002-0698-4915 Additional Supporting Information may be found online in the supporting information tab for this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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