Neutrophil activation in sickle cell disease L. R. Lard,*†‡ F. P. J. Mul,* M. de Haas,* D. Roos,* and A. J. Duits†‡ *Department of Immunohematology, CLB, Amsterdam, The Netherlands; †Red Cross Bloodbank Curac¸ao; and ‡Clinical Laboratory Department, St. Elisabeth Hospital, Curac¸ao, Netherlands Antilles
Abstract: Vascular occlusion is the main cause of the morbidity and mortality observed in patients with sickle cell disease (SCD). Increasing evidence indicates that (activated) neutrophils could play an important role in the initiation and propagation of vaso-occlusive processes in SCD. In this study, the activation state of neutrophils in sickle cell patients was analyzed by determining the level of expression of neutrophil antigens such as CD62L, CD11b, CD66b, CD63, and Fcg receptors. We also analyzed plasma levels of lactoferrin, elastase, soluble (s)CD16 (sFcgRIII), and serum levels of soluble (s)CD62L (sL-selectin) as neutrophil activation markers in these patients. Significant differences were observed in the activation state of neutrophils in non-symptomatic sickle cell patients compared to healthy HbAA controls as exemplified by significant decrease in L-selectin expression, enhanced expression of CD64, and increased levels of soluble markers like sL-selectin, elastase, and sCD16. During vaso-occlusive crisis the differences were even more pronounced. These results show neutrophils to be activated in sickle cell patients, suggesting a role of importance in the pathophysiology of sickle cell disease. J. Leukoc. Biol. 66: 411–415; 1999. Key Words: sL-selectin · lactoferrin · elastase · sCD16 · CD64
endothelial damage as described in other vascular diseases [9]. Because the neutrophil is larger and more difficult to deform than the red cell, its attachment to the endothelium, particularly in the microcirculation, would impede passage of RBC and WBC, which could increase the risk for vaso-occlusive crises [7, 8]. In this study, the activation state of neutrophils from sickle cell patients was determined by analyzing the expression of several cell surface antigens and levels of circulating proteins released from neutrophils. A significant difference in activation state of neutrophils from sickle cell patients was observed compared to controls, especially in sickle cell patients during a vaso-occlusive crisis.
MATERIALS AND METHODS Study population The study population consisted of non-symptomatic sickle cell patients (n 5 42, HbSS n 5 25, HbSC n 5 15, and HbSb-thal n 5 2), sickle cell patients having a vaso-occlusive crisis (n 5 15, HbSS n 5 11, HbSC n 5 4) and age, race, and sex-matched healthy volunteers (n 5 30, HbAA). Vaso-occlusive crisis was defined as an episode of acute pain in the abdomen and/or the extremities with signs of increased hemolysis. None of the patients were on hydroxyurea or any other kind of treatment. The healthy volunteers were from the Red Cross Bloodbank, Curac¸ao, the Netherlands Antilles. Informed consent was obtained from each patient and volunteer according to protocols established by the Department of Internal Medicine of this hospital.
Blood samples INTRODUCTION Sickle cell disease (SCD), an inherited disorder of the hemoglobin (Hb) structure and synthesis, causes chronic hemolysis, frequent infections, and a variety of other acute and chronic complications that can lead to organ damage, severe pain, disability, and premature death [1, 2]. The main cause of sickle cell-associated morbidity and mortality is (micro)vascular occlusion. Direct adherence of sickle red blood cells (RBC) to the endothelial cell surface plays a major role in this process. This results in delayed passage with concomitant enhancement of RBC sickling, ultimately leading to tissue hypoxia and infarction [3–5]. Increasing evidence suggests that white blood cells (WBC), especially neutrophils, may be involved in the initiation and propagation of vaso-occlusive crisis in SCD [1, 6–8]. Elevated total WBC counts are common in SCD patients and WBC counts of more than 15,000 cells/µL are associated with an increased risk of early death in SCD [8]. Adhesion of (activated) neutrophils to endothelium in patients with SCD may lead to
Venous blood samples were obtained from healthy volunteers and sickle cell patients. Blood samples of non-symptomatic sickle cell patients were taken when they visited the outpatient clinic for routine control; blood samples of the sickle cell patients having a vaso-occlusive crisis were taken on the day of admittance (set as day 0) in the Saint Elisabeth Hospital. The blood samples (EDTA or serum) were immediately put on ice and processed within 24 h for flow cytometric analysis. Plasma or serum was obtained by centrifugation of blood samples for 10 min at 1700 g.
Antibodies The following antibodies were used: irrelevant murine control monoclonal antibody (mAb) of the IgG2a subclass, B2.12 and CBMR1/5 (CD11b), FcRgran1 and 3G8 (CD16), IV.3 (CD32), 22 (CD64), 435 (CD63), B 13.9 (CD66b, formerly named CD67), 8G3 (CD14), Leu-8 (L-selectin), C1A2 (HLA-DR), and fluorescein isothiocyanate (FITC)-conjugated goat-antimouse-Ig (CLB G26M17F). 3G8, IV.3, and 22 were obtained from Medarex (West Lebanon, NH). Leu-8 was obtained from Becton-Dickinson (San Jose,
Correspondence: Dr. A. J. Duits, Red Cross Bloodbank Curac¸ao, Breedestraat 193 (O), Curac¸ao, Netherlands Antilles. E-mail:
[email protected] Received December 19, 1998; revised March 29, 1999; accepted March 30, 1999.
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CA). The mAb CBMR1/5 was a kind gift of Dr. T. A. Springer (Boston, MA). All other mAbs were produced in the CLB; their reactivity was established during the Fourth International Workshop on Human Leukocyte Differentiation Antigens (Vienna, Austria, 1989).
Immunophenotypic analysis Erythrocytes were lysed with ice-cold isotonic NH4Cl solution (155 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA, pH 7.4) for 10 min. The remaining leukocytes were washed in ice-cold phosphate-buffered saline (PBS) and were resuspended in ice-cold PBS supplemented with bovine serum albumin (BSA), 0.2% wt/vol to a concentration of 15 3 106 cells/mL. After isolation, the leukocytes were fixed with 1% wt/vol paraformaldehyde (PFA) for 5 min on ice and preincubated with human IgG (1 mg/mL; CLB, Amsterdam, The Netherlands) for 10 min at 4°C. To analyze the expression of the CBMR1/5 antigen (CD11b) the leukocytes were not fixed and resuspended in incubation medium [132 mmol/L NaCl, 6.0 mmol/L KCl, 1.0 mmol/L MgSO4, 1.2 mmol/L potassium phosphate, 20 mmol/L HEPES, 5.5 mmol/L glucose, and 0.5% (w/v) human serum albumin (HSA), pH 7.4] instead of PBS/BSA. This procedure was found to be necessary to determine the CBMR1/5 expression on neutrophils (data not shown). Subsequently, the leukocytes were incubated for 30 min at 4°C with the mAbs and then washed with 3 mL ice-cold PBS/BSA. Binding of the mAbs was visualized with FITC-conjugated goat-anti-mouse Ig. Flow cytometric analysis was performed with a FACScan (Becton-Dickinson). The mean channel numbers (MCN) provided by the FACScan software, which represent cell-associated fluorescence on a logarithmic scale, were converted to mean cellular fluorescence intensities (MFI) with the formula MFI 5 10(MCN/256). Antigen expression was expressed as MFI (expressed in arbitrary units). Neutrophils were discriminated from lymphocytes and monocytes by their characteristic forward/sideward scatter pattern and CD14 expression.
Measurement of elastase, lactoferrin, sCD16, and sCD62L The plasma levels of elastase, lactoferrin, and sCD16 were measured by enzyme-linked immunosorbent assay (ELISA), as previously described [10, 11]. Serum levels of sCD62L (R & D Systems) were also analyzed by ELISA, following the manufacturer’s procedures.
Statistical analysis Data were analyzed by unpaired Student’s t test. When differences in standard deviation were significant, the alternative (Welch) t test was used. A two-sided P value , 0.05 was considered to indicate a significant difference.
RESULTS
To analyze whether in SCD the neutrophils have an activated phenotype, we determined the expression of several activation markers. A very sensitive marker of neutrophil activation is decreased surface expression of CD62L (L-selectin). CD62L is a membrane-bound molecule that is shed on neutrophil activation. Figure 1 shows a significant decrease in the expression of CD62L in non-symptomatic sickle cell patients compared with controls. The expression of CD62L on neutrophils decreased even further in sickle cell patients having a vaso-occlusive crisis. L-selectin shedding was also analyzed by determination of serum sL-selectin levels. Significantly elevated levels of sL-selectin were measured in non-symptomatic- (2024 6 530 ng/mL; P 5 0.04; n 5 7) and vaso-occlusive crisis sickle cell patients (2144 6 725 ng/mL; P 5 0.03; n 5 22) compared with controls (1493 6 290 ng/mL; n 5 7). Journal of Leukocyte Biology
Neutrophil degranulation in sickle cell patients Neutrophil degranulation was analyzed by measurement of plasma membrane expression of markers of specific granules (CD11b and CD66b) and azurophilic granules (CD63). No significant differences were observed in the expression of the degranulation markers CD11b, its activation-specific neoepitope recognized by CBMR1/5 [12] and CD63 in nonsymptomatic sickle cell patients and in sickle cell patients during a vaso-occlusive crisis compared to healthy controls (Table 1). However, there was a significant increment in the expression of the degranulation marker CD66b in sickle cell patients having a vaso-occlusive crisis compared with controls TABLE 1.
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Fig. 1. Expression of CD62L (MFI) on neutrophils and serum levels of sCD62L (ng/mL) of controls (open bars), non-symptomatic sickle cell patients (hatched bars), and sickle cell patients during a vaso-occlusive crisis (filled bars). MFI was determined as described in Materials and Methods with MFI of an irrelevant mAb IgG2a subclass being substracted. *P , 0.005; **P , 0.0001.
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Neutrophil Antigen Expression in Sickle Cell Patients and Controls Control n 5 30
Non-symptomatic n 5 42
Crisis day 0 n 5 15
CD11b (B2.12) 195 6 42 200 6 56b 212 6 68b CD11b (CBMR1/5)a 19 6 14 26 6 16b 24 6 15b b CD66b 32 6 17 34 6 13 50 6 13c CD63 13 6 11 12 6 10b 20 6 19b c CD64 (FcgRI) 3.4 6 3.3 7.4 6 6.4 9.8 6 9.2c CD32 (FcgRII) 226 6 40 208 6 63b 226 6 42b CD16 (FcgRIII/FcRgranI) 1741 6 362 1946 6 510b 1828 6 515b CD16 (FcgRIII/3G8) 735 6 205 637 6 284b 698 6 219b HLA-DR 3.2 6 5.2 3.8 6 6.2b 5.5 6 8.2b CD14 21 6 11 24 6 15b 26 6 14b Expression of the antigens CD11b, CD66b, CD63, Fcg receptors, HLA-DR, and CD14 on neutrophils from controls, non-symptomatic sickle cell patients, and sickle cell patients having a vaso-occlusive crisis. Antigen expression was expressed as MFI. Background MFI, which was determined with the mAb IgG2a subclass, was subtracted.—a n 5 18, n 5 25, and n 5 11, respectively.— b P . 0.05 and c P , 0.05 compared with controls.
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population, the CD32 expression was similar in all three groups (Table 1). Finally, we analyzed the expression of CD16 (FcgRIII). No significant differences were observed in the CD16 expression on neutrophils, either in the group of non-symptomatic sickle cell patients or in the group of sickle cell patients having a vaso-occlusive crisis compared with controls (Table 1). Although no difference in CD16 membrane expression was found, a significant increase in plasma soluble (s)CD16 was observed in both non-symptomatic sickle cell patients and sickle cell patients during a vaso-occlusive crisis compared with healthy controls (Fig. 3). Characteristically for each sickle cell patient, sickle cell vaso-occlusive crisis resolution was accompanied by a marked decrease in leukocyte count (Fig. 4).
Fig. 2. Plasma levels of lactoferrin and elastase (ng/mL) in controls (open bars), non-symptomatic sickle cell patients (hatched bars), and sickle cell patients during a vaso-occlusive crisis (filled bars). *P , 0.006; **P , 0.01.
and non-symptomatic sickle cell patients (P 5 0.0009 and 0.0002, respectively; Table 1). We next measured the plasma levels of lactoferrin and elastase in these sickle cell patients (Fig. 2). Neutrophil degranulation leads to release of lactoferrin and elastase from the specific and azurophil granules, respectively. There were no significant differences in the lactoferrin levels in nonsymptomatic sickle cell patients compared with controls (P 5 0.0747). However, a significant increase was observed in the lactoferrin level from sickle cell patients having a vasoocclusive crisis compared with controls and non-symptomatic sickle cell patients. Analysis of elastase levels showed a significant increase in non-symptomatic sickle cell patients compared with controls (Fig. 2). In sickle cell patients having a vaso-occlusive crisis the elastase levels were further increased compared with controls and non-symptomatic sickle cell patients (Fig. 2).
DISCUSSION In recent years much research has been carried out to clarify the pathophysiology of SCD. Several studies indicate that neutrophils, in addition to factors such as plasma proteins, endothelial abnormalities, and sickle RBC, could have a pivotal role in the initiation and/or propagation of vaso-occlusion in SCD [1, 6–8, 16]. WBC counts are correlated with the severity of crisis and risk of premature death. Also, treatment with anti-inflammatory agents such as methylprednisolone decreases the duration of the vaso-occlusive crisis period [8]. Furthermore, Hofstra et al. showed that sickle RBC specifically bind to neutrophils via an IgG (and RGDS)-mediated process, which activates the neutrophils in sickle cell patients [6]. Our aim was to investigate whether neutrophils show an activated phenotype in sickle cell patients during their non-symptomatic period and during a vaso-occlusive crisis. We observed a severely decreased surface expression of
Expression of maturation-dependent and cytokine-induced antigens on neutrophils from sickle cell patients CD64 (FcgRI) is an Fcg receptor expressed on immature neutrophils. During maturation of neutrophils, the CD64 expression normally disappears, although it can persist under the influence of granulocyte colony-stimulating factor or interferon-g, or even be induced by cytokine stimulation [13–15]. A significant increase in CD64 expression on neutrophils from non-symptomatic sickle cell patients and from sickle cell patients during a vaso-occlusive crisis was observed compared to controls (P 5 0.0025 and 0.0294, respectively; Table 1). HLA-DR and CD14 are also antigens whose expression can be induced by cytokines [13]. No significant differences were observed in the expression of the antigens HLA-DR and CD14 in either non-symptomatic sickle cell patients or sickle cell patients during a vaso-occlusive crisis compared with controls (Table 1). We next determined the expression of CD32 (FcgRII), which is constitutively expressed on the cell surface. In our study
Fig. 3. Plasma levels sCD16 (sFcgRIII) (ng/mL) in controls (open bars), non-symptomatic sickle cell patients (hatched bars), and sickle cell patients during a vaso-occlusive crisis (filled bars). *P , 0.02; **P , 0.0001.
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Fig. 4. Total leukocyte count of sickle cell patients during a vaso-occlusive crisis and after crisis resolution.
CD62L on neutrophils from patients, especially when measured during a vaso-occlusive crisis. A concomitant increase in the soluble (s)CD62L serum levels was measured in nonsymptomatic sickle cell patients and sickle cell patients during a vaso-occlusive crisis. High levels of lactoferrin and elastase were measured in the plasma of these sickle cell patients, indicating fusion of the specific and azurophilic granules, respectively, with the plasma membrane [17]. The specific granule activity was phenotypically only partially reflected on neutrophils by enhanced CD66b expression in sickle cell patients having a vaso-occlusive crisis with CD11b expression remaining unaltered. Azurophilic granule activity was not observed phenotypically because no significant increase in the CD63 expression was observed. Also, the CD11b activationepitope CBMR1/5 was not present on the circulating neutrophils in these sickle cell patients. An explanation for this phenomenon might be that in vivo, strongly activated neutrophils may have left the circulation and are therefore underrepresented in the blood samples used for flow cytometric analysis [13, 18]. We did not find a change in the CD16 expression on the neutrophils in these sickle cell patients, whereas the soluble (s)CD16 levels in plasma were significantly increased. sCD16 levels correlate with the turnover of neutrophils and therefore form a reflection of the total body neutrophil mass and the production of neutrophils in the bone marrow [19, 20]. Because WBC count has been shown to be increased in sickle cell patients [6, 8] and even further during sickle cell crisis (Fig. 4 [21]), the increased plasma sCD16 levels observed could be due to an elevated total neutrophil number. However, we cannot exclude the possibility that the elevated sCD16 plasma level may also be due to activation of neutrophils in these patients [22]. The unaltered CD16 expression observed in the sickle cell patients may argue against activation-induced shedding of CD16, but this could be a combination of shedding of CD16 and up-regulated CD16 expression by fusion of secretory vesicles with the plasma membrane [20, 23]. CD64 expression was observed on neutrophils of the majority of the sickle cell patients. Because no left shifting was observed in these sickle cell patients (data not shown), and neutrophil CD16 expression remained unaltered, this would probably indicate CD64 expression resulting from cytokine induction 414
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instead of neutrophil immaturity [13, 24]. Therefore, we measured the HLA-DR antigen expression, which also can be up-regulated by cytokines such as interferon-g [13]. No significant increases were observed in the HLA-DR expression in these sickle cell patients. On the other hand, correlation studies showed that most patients with HLA-DR-positive neutrophils also expressed CD64 (r 5 0.3431, two-tailed P 5 0.0281, data not shown), suggesting that the CD64 and HLA-DR expression may still be cytokine-induced. Our results demonstrate that neutrophils are activated in sickle cell patients, especially during a vaso-occlusive crisis. Several sickle cell disease-specific events could result in the neutrophil activation observed. We recently reported increased serum levels of the potent neutrophil activator interleukin-8 in sickle cell patients during a vaso-occlusive crisis [25]. Several studies described complement activation in (non-symptomatic) sickle cell patients [26]. Wang et al. demonstrated that the membrane phospholipid changes that occur during sickling of RBC lead to alternative pathway complement activation [27]. Complement activation products, in particular C3a and C5a, are potent activators of neutrophils. Furthermore, autologous IgG binding to sickled RBC due to membrane alterations caused by the sickling process [28] could result in neutrophil activation [6]. Sickling of RBC is a continuous process in sickle cell patients with 2–30% of the circulating RBC irreversibly sickled [29]. Thus, it might be that sickle RBC continuously activate neutrophils in sickle cell patients, either directly via an IgG-mediated process and/or by complement activation. Next to RBC, activated endothelium could be (indirectly) involved in neutrophil activation. Chronically enhanced sVCAM-1 levels indicate a persistent activated endothelium state [7, 30–33]. Several reports have suggested that the adhesion of sickle RBC to endothelial cells leads to direct endothelium damage in sickle cell patients [34]. Activated endothelial cells can produce various cytokines that can stimulate neutrophils. Indeed, several studies have shown increased levels of interleukin-1, interleukin-6, and tumor necrosis factor a in non-symptomatic sickle cell patients and in sickle cell patients during a vaso-occlusive crisis [8, 33, 35]. In addition, sickle cell patients have a diminished or absent splenic function [36], which may lead to a chronic inflammatory state due to insufficient clearance of bacteria and subsequent neutrophil activation. Recently, Fadlon et al. [34] analyzed sickle cell patient polymorphonuclear leukocyte adhesion to vascular endothelium. In contrast to our results the authors observed no activation-dependent change in antigen expression, although an increase in neutrophil adhesion profile was observed. The reason for this discrepancy is unclear, however, next to antigenic profiles the presence of enhanced soluble levels of neutrophil specific enzymes and adhesion molecules also support neutrophil activation. Furthermore, other neutrophil characteristics (like raised phospholipase A2 activity) indicative of ongoing activation in sickle cell patients have recently been reported [37]. The observed neutrophil activation in sickle cell patients may lead to increased adherence to the endothelium in the microcirculation of sickle cell patients as shown in several http://www.jleukbio.org
studies [34, 38, 39]. This will cause an increased risk for vaso-occlusive crisis due to a subsequent delayed passage of RBC and WBC in the microcirculation. We therefore postulate neutrophils to have an important role in the initiation and propagation of vaso-occlusive crises in sickle cell disease.
ACKNOWLEDGMENTS This work was supported by a grant from the Netherlands Antilles Society for Higher Clinical Education (NASKHO).
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