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Normal levels of constitutive and death receptor-mediated apoptosis of peripheral blood neutrophils from patients with chronic idiopathic neutropenia
Clinical Immunology (2007) 122, 349–355
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
Normal levels of constitutive and death receptor-mediated apoptosis of peripheral blood neutrophils from patients with chronic idiopathic neutropenia Daniel Garwicz a,b,c,⁎, Jan Palmblad b , Bengt Fadeel a a
Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden Division of Hematology, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden c Division of Clinical Chemistry, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital Solna, 171 76 Stockholm, Sweden b
Received 24 February 2006; accepted with revision 30 October 2006 Available online 22 December 2006 KEYWORDS Apoptosis; Caspase; Chronic idiopathic neutropenia; Death receptor; Granulocyte colony-stimulating factor; Neutrophil; Phosphatidylserine externalization
Abstract To investigate the role of neutrophil apoptosis in the pathogenesis of chronic neutropenia, we examined constitutive and death receptor-mediated apoptosis ex vivo of peripheral blood neutrophils obtained from six chronic idiopathic neutropenia (CIN) patients and six healthy adult blood donors. Apoptosis was quantified based on phosphatidylserine externalization and caspase-3 activation in freshly isolated neutrophils or after overnight cultivation of neutrophils in the absence or presence of pro- or anti-apoptotic factors, including the pan-caspase inhibitor, zVAD-fmk. Neutrophils from CIN patients receiving treatment with granulocyte colony-stimulating factor appeared to be more prone to constitutive apoptosis than cells from untreated patients; however, further investigations in larger cohorts of patients are needed to validate these pilot studies. Overall, the level of neutrophil apoptosis was similar in patient and control groups, thus supporting the notion that the underlying defect in these neutropenia patients lies elsewhere, such as in the bone marrow microenvironment. © 2006 Elsevier Inc. All rights reserved.
Introduction Chronic idiopathic neutropenia (CIN) is a neutrophil disorder characterized by a persistent reduction of the number of ⁎ Corresponding author. Division of Clinical Chemistry, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital Solna, 171 76 Stockholm, Sweden. Fax: +46 8 31 03 76. E-mail address: [email protected] (D. Garwicz).
circulating neutrophils below the lower limit of the normal distribution in the absence of other signs of underlying disease [1–3]. The cause of neutropenia in affected subjects is unknown. However, pro-inflammatory cytokines, such as transforming growth factor (TGF)-β1, released into the bone marrow microenvironment by stromal cells [4], cytokines and chemokines released into the blood by activated macrophages [5], as well as increased apoptosis of bone marrow myeloid progenitor cells or neutrophil precursors [6], have all been
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suggested to be important in the pathogenesis of this disorder. Nonetheless, the contribution of apoptosis of peripheral blood neutrophils from adult patients with chronic idiopathic neutropenia has not been studied to date. Disturbances in the cell death/cell survival balance have been documented in several hematological conditions. Hence, an increased propensity for apoptosis of bone marrow progenitor cells is seen in Shwachman–Diamond syndrome, and is linked to an increased expression of the Fas death receptor, and to hyperactivation of the Fas signaling pathway [7]. Apoptosis dysregulation has also been reported in Diamond–Blackfan anemia [8] and in patients with myelokathexis [9]. In addition, increased apoptosis of bone marrow cells is seen in myelodysplastic syndrome patients [10], and constitutive mitochondrial activation of erythroid progenitor cells with release of cytochrome c into the cytosol was suggested to contribute to the characteristic anemia [11]. Similarly, our recent studies of the autosomal recessive form of severe congenital neutropenia (Kostmann syndrome) have implicated excessive mitochondria-dependent apoptosis of myeloid progenitor cells in the classical “maturation arrest” in the bone marrow of these patients [12]. In the present study, we investigated whether peripheral blood neutrophils obtained from adult CIN patients display any signs of increased spontaneous apoptosis, or enhanced sensitivity to death receptor ligation, upon ex vivo cultivation, as compared to healthy adult controls.
Materials and methods Patients and control subjects Six patients with mild to severe CIN were included in the study (Table 1). The patients did not suffer from any infections at the time of sampling and C-reactive protein (CRP) values were found to be within the normal range. In addition, the CIN patients did not receive any concurrent medication known to affect absolute neutrophil count (ANC) values or granulocyte function, apart from recombinant human G-CSF treatment in 3 cases, as indicated in Table 1. Six healthy adult blood donors served as control subjects; we did not, however, attempt to gender-match patients and controls in this pilot study. The Table 1
study was performed in accordance with the guidelines of the Declaration of Helsinki, with approval by the ethical committee at Karolinska Institutet (Stockholm), and informed consent from all participating subjects.
Reagents The pan-caspase inhibitor, z-VAD-fmk (10 mM stock solution in DMSO, 10 μM final concentration), was from Sigma (St. Louis, MO, USA). The proteasome inhibitor, lactacystin (5 mM stock in dH2O, 2.5 μM final), was obtained from Santa Cruz Biotechnology (CA, USA), and the calpain inhibitor, PD150606 (10 mM stock in DMSO, 25 μM final) was from Calbiochem (San Diego, CA, USA). Staurosporine (2 mM stock, 1 μM final) was obtained from Sigma. Recombinant human TNF-α (tumor necrosis factor-α) (10 μg/ml stock, 10 ng/ml final) was from Pharmingen (San Diego, CA, USA) and recombinant human TRAIL (TNFrelated apoptosis-inducing ligand) (100 ng/ml stock solution, 0.5 ng/ml final) was from Peprotech (London, UK).
Neutrophil isolation and cell culture Venous blood neutrophils from patients and controls were isolated by a method of dextran sedimentation and density gradient centrifugation as previously described [13]. Residual erythrocytes were removed by hypotonic lysis. Purified neutrophils were cultured in RPMI-1640 medium (SigmaAldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO-Invitrogen, Paisley, UK). Cells were seeded at 1.0 × 106 cells/ml in 24-well tissue culture plates and were either analyzed immediately (freshly isolated cells) or maintained in the presence or absence of pro- or anti-apoptotic agents, as specified above, overnight in culture (14–17 h in all cases, except for one of the 6 controls, for whom cells were maintained in culture for 20 h), prior to assessment of apoptosis.
Phosphatidylserine exposure Plasma membrane exposure of phosphatidylserine (PS), a common sign of apoptosis [13], was quantified as detailed in
Clinical characteristics of the CIN patients in the present study
Patient a
Gender b
Age c
Severity d
ANC e
G-CSF f
Infections g
1. 2. 3. 4. 5. 6.
F F F F M M
33 35 81 27 62 62
Severe Severe Severe Severe Moderate Mild h
3 (0.2–0.7) 3.2 2.5 0.42 1.0 (0.5–1.1) 1.1 (0.5–2.5)
2 × 0.3 mg 3 × 0.3 mg 1 × 0.3 mg No No No
High High Moderate No Low Low
a
Patient number. Gender: F = female, M = male. c Age (years) at sampling. d Severity of neutropenia based on typical values at diagnosis according to internationally accepted criteria [3]. e Absolute neutrophil counts (ANC) (×109/L) at sampling or shortly before (values in parentheses designate previous or pre-treatment range). f G-CSF = number of weekly subcutaneous injections of recombinant human G-CSF, in most cases filgrastim. Blood sampling was typically performed z48 h after the last G-CSF injection. g Infections = propensity for bacterial infections. h Patient 6 also has a complete lack of complement factor 9 (C9). b
Neutrophil apoptosis in CIN patients the annexin V-FITC apoptosis detection kit (Oncogene Research Products, Cambridge, MA, USA). Cells were costained with propidium iodide (PI) (50 μg/ml stock solution, 125 ng/ml final) prior to analysis on a FACScan (Becton Dickinson, San Jose, CA, USA) equipped with a 488 nm argon laser. Ten thousand events were collected for each sample and analyzed using CellQuest software (Becton Dickinson).
351 turer’s instructions. To validate the method, we used the human Jurkat T cell line (European Collection of Cell Cultures, Salisbury, UK), cultured in RPMI-1640 medium supplemented as above. Since this method was found to correlate well with the DEVD-AMC cleavage assay and our preliminary studies showed that the latter assay yielded more reproducible results for human neutrophils (unpublished findings), only DEVD-AMC results are shown for these cells.
Flow cytometric detection of active caspase-3 Caspase-3-like enzyme activity Expression of the active subunit of caspase-3 was detected by flow cytometry following labeling of cells with FITC-conjugated rabbit anti-caspase-3 monoclonal antibodies (BD Pharmingen, San Diego, CA, USA) according to the manufac-
Cleavage of the fluorogenic peptide substrate DEVD-AMC, indicative of caspase-3-like enzyme activity, was determined as previously described [13]. Briefly, cell lysates and DEVD-
Figure 1 PS externalization in peripheral blood neutrophils from a healthy blood donor (A–F) and a patient with chronic idiopathic neutropenia (CIN) (G–L). Control neutrophils were cultured in vitro in medium alone for 0 h (fresh) (A), overnight (B), or overnight in the presence of zVAD-fmk (C), anti-Fas antibodies (D), TRAIL (E), or TNF-α (F). CIN patient neutrophils (patient 2) were incubated under corresponding conditions (G–L). Viable cells are found in the lower left quadrant of each FACS plot, early apoptotic cells in the lower right quadrant, and late apoptotic cells are located in the upper right quadrant.
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Figure 2 Constitutive and death receptor-mediated apoptosis of neutrophils from adult blood donors compared to CIN patients. Neutrophils from healthy blood donors (HBD) (A) and CIN patients (B) were cultured in vitro for 0 h (fresh), overnight in medium alone, or overnight in the presence of the inhibitors, zVAD-fmk, PD150606, or lactacystin, or the pro-apoptotic agents, anti-Fas antibodies, recombinant TRAIL, or recombinant TNF-α. Apoptosis was determined by flow cytometric detection of annexin V-FITC and PI labeling, and the percentages of early, late, and total apoptosis were calculated. Data shown are mean values ± SD (n = 3–6).
AMC substrate (50 μM) were combined in a standard reaction buffer (100 mM HEPES, 10% sucrose, 5 mM dithiothreitol, and 0.1% CHAPS; pH 7.25) and enzyme-catalyzed release of AMC was measured every 70 s during a 30 min period in a Fluoroscan II plate reader (Labsystems, Stockholm, Sweden) using 355 nm excitation and 460 nm emission wavelengths. Data were analyzed by linear regression and are displayed as pmol of AMC release/min.
Results First, we compared apoptosis of peripheral blood neutrophils from controls and CIN patients. To this end, freshly isolated cells and cells cultivated overnight in the presence or absence of anti-apoptotic agents (the broad-spectrum caspase inhibitor, zVAD-fmk, the calpain inhibitor, PD150606, and the proteasome inhibitor, lactacystin) or pro-apoptotic (agonistic anti-Fas antibodies, recombinant TRAIL, recombinant TNF-α) were labeled with annexin V and propidium iodide and analyzed by flow cytometry. Representative data from one control and one patient are shown in Fig. 1, and a summary of the results obtained in 6 healthy blood donors and 6 CIN patients are depicted in Fig. 2. Spontaneous neutrophil apoptosis was increased upon overnight incubation; however, no significant differences were detected between controls and CIN patients (Fig. 2). Of note, the caspase inhibitor was effective in blocking late apoptosis (i.e. cells positive for both propidium iodide and annexin V), yet failed to suppress early apoptosis (cells
Figure 3 Comparison of constitutive apoptosis of neutrophils from untreated CIN patients with neutrophils from patients treated with recombinant G-CSF. Cells were isolated from CIN patients not on G-CSF treatment (n = 3) or on G-CSF treatment (n = 3), and subjected to in vitro cultivation overnight in medium alone. Samples from the G-CSF-treated group were drawn z 48 h after the last G-CSF injection. Apoptosis was determined using the annexin V-FITC assay, and the percentages of early and total apoptosis (as defined in the legend to Fig. 1) were calculated. Data shown are mean values ± SD. An increase of apoptosis of neutrophils was observed in the G-CSF-treated group; however, due to the small number of samples, no statistical analysis was performed.
Neutrophil apoptosis in CIN patients positive only for annexin V) both in controls and CIN patients (Fig. 2), suggesting that cell death was partially caspase-independent. PD150606 or lactacystin, however, had no discernible effect on constitutive apoptosis of control or patient neutrophils, at the time points tested. Fas ligation was found to accelerate apoptosis both of control and CIN patient neutrophils (Fig. 2), whereas TRAIL and TNF-α neither promoted nor blocked PS externalization under these conditions. Overall, no significant differences were observed in death receptormediated apoptosis between controls and CIN patients. However, neutrophils from the 3 patients receiving G-CSF treatment appeared to display an increase in constitutive apoptosis upon overnight incubation, in comparison to cells from patients without ongoing G-CSF treatment (Fig. 3). On the other hand, when the patients were stratified according to severity of disease, irrespective of current
353 treatment status, no such differences were detected (data not shown). We also investigated the degree of caspase activation in neutrophils from neutropenia patients and controls. For this purpose, a comparison was made between a flow cytometric method of detection of caspase-3 (using specific antibodies recognizing the active subunit, but not the pro-form of caspase-3) and the DEVD-AMC method (which is based on the in vitro cleavage of a fluorogenic caspase substrate). There was an excellent correlation between the two methods (Fig. 4), suggesting that the DEVD-AMC cleavage assay faithfully reports caspase-3 activation. For the neutrophil experiments, the latter assay was therefore employed. A significant induction of caspase-3 was observed upon overnight incubation of cells from adult blood donors as well as from CIN patients, and this was effectively blocked by zVAD-fmk (Fig. 5). However, the results obtained in controls and
Figure 4 Comparison of two methods for detection of caspase-3 activity, using the Jurkat T cell line as a model. Cells were cultured in medium alone for 4 h (control), or for 1 to 3 h in the presence of staurosporine (STS), a pro-apoptotic agent. (A) Flow cytometric, antibody-based detection of the active subunit of caspase-3 was performed as described in Materials and methods. The percentages of cells expressing active caspase-3 are shown. (B) Parallel samples from the same experiment were also subjected to quantification of DEVD-AMC cleavage, using a real-time fluorogenic assay. The maximum rate of AMC release (pmol/min) was estimated by linear regression (r2 > 0.99). Data shown are the mean values of duplicate determinations. Linear correlation between the two methods reported in (A) and (B) for detection of caspase-3 activation.
354
Figure 5 Caspase-3 activation detected by the DEVD-AMC cleavage assay in neutrophils isolated from control subjects and CIN patients. Neutrophils from healthy blood donors (HBD) and CIN patients were cultured in medium alone for 0 h (fresh) or overnight in the absence (spontaneous) or presence of inhibitors or pro-apoptotic agents, as indicated. Caspase-3 activation was quantified as outlined in the legend to Fig. 4, and the maximum rate of AMC release (pmol/min) is depicted for each condition. Data shown are mean values ± SD (n = 3).
patients did not differ significantly. Moreover, ligation of Fas using specific antibodies did not appear to enhance caspase3 activation in control or patient cells, at least at the time points tested, whereas treatment of cells from CIN patients with recombinant TNF-α appeared to suppress caspase-3 activation to some extent.
Discussion The mechanism underlying chronic idiopathic neutropenia (CIN) remains unknown. Our current results suggest that peripheral blood neutrophils from CIN patients do not display an increased propensity for constitutive or death receptor-mediated apoptosis upon in vitro cultivation. However, an increased degree of apoptosis was observed in cells derived from CIN patients receiving G-CSF therapy in comparison to CIN patients without ongoing treatment. Notwithstanding the small number of evaluated patients, one may speculate that the latter findings could be due to a G-CSF withdrawal effect [14], or perhaps an increased vulnerability of neutrophils synthesized under the influence of supraphysiological doses of G-CSF. Alternatively, these patients may harbor an intrinsically increased propensity for neutrophil apoptosis. However, when the CIN patients included in the present study were stratified according to disease severity and not to treatment status, no significant differences in neutrophil apoptosis were seen, thus arguing against an intrinsic defect in peripheral blood neutrophils in this condition. Of note, mice with a targeted disruption of the G-CSF gene display reduced neutrophil numbers in peripheral blood [15], and mice lacking the G-CSF receptor were reported to exhibit impaired production and increased apoptosis of neutrophils [16], hence supporting the notion that G-CSF promotes the survival of these cells. To address this issue conclusively in human subjects, larger cohorts of neutropenia patients treated or not treated with G-CSF, or
D. Garwicz et al. healthy controls or stem cell donors subjected to high doses of G-CSF followed by sudden withdrawal of the growth factor, need to be examined. However, such investigations were beyond the scope of the present pilot study. The life span of neutrophils is subject to regulation by death receptors and their corresponding ligands [13,17]. Liu et al. [18] have previously demonstrated high levels of circulating Fas ligand in the serum of large granular lymphocyte (LGL) leukemia patients, and showed that the resolution of neutropenia was associated with disappearance or marked reduction in Fas ligand levels. However, no significant differences were seen in the effect of Fas ligation on neutrophils from CIN patients versus controls in the present study. TRAIL has been suggested to play a role in the pathogenesis of neutropenia in systemic lupus erythematosus (SLE) patients [19], yet TRAIL is not pro-apoptotic for normal neutrophils under non-inflammatory conditions [20], and our data do not support a role for TRAIL in promoting neutrophil apoptosis in CIN patients. Finally, previous reports have demonstrated that TNF-α may either promote or inhibit apoptosis of neutrophils, depending on the dose and on the kinetics of cell death [21,22]. In the present study, TNF-α stimulation resulted in a decreased caspase activation in some patients as well as in some controls. However, this effect was not reflected in a corresponding decrease of PS exposure, thus confirming previous observations that both caspase-dependent and -independent pathways are involved in the cell surface externalization of PS in neutrophils [13]. Our pilot study, performed in a small number of patients, thus suggests that there are no overall differences in the rate of apoptosis of peripheral blood neutrophils between CIN patients and control subjects, indicating that the underlying defect in this disease lies elsewhere, for instance in the bone marrow microenvironment, as proposed recently by others [23,24]. Indeed, we have recently documented a similar degree of constitutive apoptosis of peripheral blood neutrophils from a WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) patient and control subjects [25], thus pointing to the retention and/or impaired survival of neutrophils in the bone marrow [9] as the primary defect in the latter condition. It is conceivable that only subsets of immature neutrophils are affected in congenital or idiopathic neutropenia patients while the remaining mature neutrophils egress from the bone marrow in response to growth factor treatment; this could then help to explain why there are no marked differences in apoptosis of circulating neutrophils between patients and controls. To conclude, further investigations in larger cohorts of neutropenia patients, and of immature and mature neutrophils obtained in parallel from the bone marrow and peripheral blood compartments of these patients, are needed to address this important question.
Acknowledgments We thank all patients and control subjects who participated in this study. We are also grateful to Ms. Birgitta Wester at the flow cytometry core facility, Department of Microbiology, Tumor and Cellbiology, Karolinska Institutet, for expert advice. The study was supported, in part, by the Swedish
Neutrophil apoptosis in CIN patients Research Council, the Swedish Cancer Foundation, and the Swedish Society of Medicine. D.G. was the recipient of a post doctoral fellowship from the Swedish Society for Medical Research.
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355 [13] B. Fadeel, A. Åhlin, J.-I. Henter, S. Orrenius, M.B. Hampton, Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species, Blood 92 (1998) 4808–4818. [14] Y. Ogawa, T. Kobayashi, A. Nishioka, S. Kariya, E. Garcia Del Saz, H. Seguchi, S. Yoshida, Sudden termination of G-CSF injection leads to apoptosis of a large proportion of increased granulocytes, Oncol. Rep. 8 (2001) 1027–1029. [15] G.J. Lieschke, D. Grail, G. Hodgson, D. Metcalf, E. Stanley, C. Cheers, K.J. Fowler, S. Basu, Y.F. Zhan, A.R. Dunn, Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization, Blood 84 (1994) 1737–1746. [16] F. Liu, H.Y. Wu, R. Wesselschmidt, T. Kornaga, D.C. Link, Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice, Immunity 5 (1996) 491–501. [17] W.C. Liles, P.A. Kiener, J.A. Ledbetter, A. Aruffo, S.J. Klebanoff, Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils, J. Exp. Med. 184 (1996) 429–440. [18] J.H. Liu, S. Wei, T. Lamy, P.K. Epling-Burnette, G. Starkebaum, J.Y. Djeu, T.P. Loughran, Chronic neutropenia mediated by Fas ligand, Blood 95 (2000) 3219–3222. [19] W. Matsuyama, M. Yamamoto, I. Higashimoto, K. Oonakahara, M. Watanabe, K. Machida, T. Yoshimura, N. Eiraku, M. Kawabata, M. Osame, K. Arimura, TNF-related apoptosisinducing ligand is involved in neutropenia of systemic lupus erythematosus, Blood 104 (2004) 184–191. [20] S.A. Renshaw, J.S. Parmar, V. Singleton, S.J. Rowe, D.H. Dockrell, S.K. Dower, C.D. Bingle, E.R. Chilvers, M.K. Whyte, Acceleration of human neutrophil apoptosis by TRAIL, J. Immunol. 170 (2003) 1027–1033. [21] K. Yamashita, A. Takahashi, S. Kobayashi, H. Hirata, P.W. Mesner, S.H. Kaufmann, S. Yonehara, K. Yamamoto, T. Uchiyama, M. Sasada, Caspases mediate tumor necrosis factor-α-induced neutrophil apoptosis and downregulation of reactive oxygen production, Blood 93 (1999) 674–685. [22] C.Y. Liu, A. Takemasa, W.C. Liles, R.B. Goodman, M. Jonas, H. Rosen, E. Chi, R.K. Winn, J.M. Harlan, P.I. Chuang, Broadspectrum caspase inhibition paradoxically augments cell death in TNF-α-stimulated neutrophils, Blood 101 (2003) 295–304. [23] H.A. Papadaki, K. Stamatopoulos, A. Damianaki, C. Gemetzi, A. Anagnostopoulos, T. Papadaki, A.G. Eliopoulos, G.D. Eliopoulos, Activated T-lymphocytes with myelosuppressive properties in patients with chronic idiopathic neutropenia, Br. J. Haematol. 128 (2005) 863–876. [24] H.A. Papadaki, A.G. Eliopoulos, T. Kosteas, C. Gemetzi, A. Damianaki, H. Koutala, J. Bux, G.D. Eliopoulos, Impaired granulocytopoiesis in patients with chronic idiopathic neutropenia is associated with increased apoptosis of bone marrow myeloid progenitor cells, Blood 101 (2003) 2591–2600. [25] D. Sanmun, D. Garwicz, C.I.E. Smith, J. Palmblad, B. Fadeel, Stromal-derived factor-1 abolishes constitutive apoptosis of WHIM syndrome neutrophils harbouring a truncating CXCR4 mutation, Br. J. Haematol. 134 (2006) 640–644.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
Normal levels of constitutive and death receptor-mediated apoptosis of peripheral blood neutrophils from patients with chronic idiopathic neutropenia Daniel Garwicz a,b,c,⁎, Jan Palmblad b , Bengt Fadeel a a
Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden Division of Hematology, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden c Division of Clinical Chemistry, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital Solna, 171 76 Stockholm, Sweden b
Received 24 February 2006; accepted with revision 30 October 2006 Available online 22 December 2006 KEYWORDS Apoptosis; Caspase; Chronic idiopathic neutropenia; Death receptor; Granulocyte colony-stimulating factor; Neutrophil; Phosphatidylserine externalization
Abstract To investigate the role of neutrophil apoptosis in the pathogenesis of chronic neutropenia, we examined constitutive and death receptor-mediated apoptosis ex vivo of peripheral blood neutrophils obtained from six chronic idiopathic neutropenia (CIN) patients and six healthy adult blood donors. Apoptosis was quantified based on phosphatidylserine externalization and caspase-3 activation in freshly isolated neutrophils or after overnight cultivation of neutrophils in the absence or presence of pro- or anti-apoptotic factors, including the pan-caspase inhibitor, zVAD-fmk. Neutrophils from CIN patients receiving treatment with granulocyte colony-stimulating factor appeared to be more prone to constitutive apoptosis than cells from untreated patients; however, further investigations in larger cohorts of patients are needed to validate these pilot studies. Overall, the level of neutrophil apoptosis was similar in patient and control groups, thus supporting the notion that the underlying defect in these neutropenia patients lies elsewhere, such as in the bone marrow microenvironment. © 2006 Elsevier Inc. All rights reserved.
Introduction Chronic idiopathic neutropenia (CIN) is a neutrophil disorder characterized by a persistent reduction of the number of ⁎ Corresponding author. Division of Clinical Chemistry, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital Solna, 171 76 Stockholm, Sweden. Fax: +46 8 31 03 76. E-mail address: [email protected] (D. Garwicz).
circulating neutrophils below the lower limit of the normal distribution in the absence of other signs of underlying disease [1–3]. The cause of neutropenia in affected subjects is unknown. However, pro-inflammatory cytokines, such as transforming growth factor (TGF)-β1, released into the bone marrow microenvironment by stromal cells [4], cytokines and chemokines released into the blood by activated macrophages [5], as well as increased apoptosis of bone marrow myeloid progenitor cells or neutrophil precursors [6], have all been
1521-6616/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2006.10.015
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suggested to be important in the pathogenesis of this disorder. Nonetheless, the contribution of apoptosis of peripheral blood neutrophils from adult patients with chronic idiopathic neutropenia has not been studied to date. Disturbances in the cell death/cell survival balance have been documented in several hematological conditions. Hence, an increased propensity for apoptosis of bone marrow progenitor cells is seen in Shwachman–Diamond syndrome, and is linked to an increased expression of the Fas death receptor, and to hyperactivation of the Fas signaling pathway [7]. Apoptosis dysregulation has also been reported in Diamond–Blackfan anemia [8] and in patients with myelokathexis [9]. In addition, increased apoptosis of bone marrow cells is seen in myelodysplastic syndrome patients [10], and constitutive mitochondrial activation of erythroid progenitor cells with release of cytochrome c into the cytosol was suggested to contribute to the characteristic anemia [11]. Similarly, our recent studies of the autosomal recessive form of severe congenital neutropenia (Kostmann syndrome) have implicated excessive mitochondria-dependent apoptosis of myeloid progenitor cells in the classical “maturation arrest” in the bone marrow of these patients [12]. In the present study, we investigated whether peripheral blood neutrophils obtained from adult CIN patients display any signs of increased spontaneous apoptosis, or enhanced sensitivity to death receptor ligation, upon ex vivo cultivation, as compared to healthy adult controls.
Materials and methods Patients and control subjects Six patients with mild to severe CIN were included in the study (Table 1). The patients did not suffer from any infections at the time of sampling and C-reactive protein (CRP) values were found to be within the normal range. In addition, the CIN patients did not receive any concurrent medication known to affect absolute neutrophil count (ANC) values or granulocyte function, apart from recombinant human G-CSF treatment in 3 cases, as indicated in Table 1. Six healthy adult blood donors served as control subjects; we did not, however, attempt to gender-match patients and controls in this pilot study. The Table 1
study was performed in accordance with the guidelines of the Declaration of Helsinki, with approval by the ethical committee at Karolinska Institutet (Stockholm), and informed consent from all participating subjects.
Reagents The pan-caspase inhibitor, z-VAD-fmk (10 mM stock solution in DMSO, 10 μM final concentration), was from Sigma (St. Louis, MO, USA). The proteasome inhibitor, lactacystin (5 mM stock in dH2O, 2.5 μM final), was obtained from Santa Cruz Biotechnology (CA, USA), and the calpain inhibitor, PD150606 (10 mM stock in DMSO, 25 μM final) was from Calbiochem (San Diego, CA, USA). Staurosporine (2 mM stock, 1 μM final) was obtained from Sigma. Recombinant human TNF-α (tumor necrosis factor-α) (10 μg/ml stock, 10 ng/ml final) was from Pharmingen (San Diego, CA, USA) and recombinant human TRAIL (TNFrelated apoptosis-inducing ligand) (100 ng/ml stock solution, 0.5 ng/ml final) was from Peprotech (London, UK).
Neutrophil isolation and cell culture Venous blood neutrophils from patients and controls were isolated by a method of dextran sedimentation and density gradient centrifugation as previously described [13]. Residual erythrocytes were removed by hypotonic lysis. Purified neutrophils were cultured in RPMI-1640 medium (SigmaAldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO-Invitrogen, Paisley, UK). Cells were seeded at 1.0 × 106 cells/ml in 24-well tissue culture plates and were either analyzed immediately (freshly isolated cells) or maintained in the presence or absence of pro- or anti-apoptotic agents, as specified above, overnight in culture (14–17 h in all cases, except for one of the 6 controls, for whom cells were maintained in culture for 20 h), prior to assessment of apoptosis.
Phosphatidylserine exposure Plasma membrane exposure of phosphatidylserine (PS), a common sign of apoptosis [13], was quantified as detailed in
Clinical characteristics of the CIN patients in the present study
Patient a
Gender b
Age c
Severity d
ANC e
G-CSF f
Infections g
1. 2. 3. 4. 5. 6.
F F F F M M
33 35 81 27 62 62
Severe Severe Severe Severe Moderate Mild h
3 (0.2–0.7) 3.2 2.5 0.42 1.0 (0.5–1.1) 1.1 (0.5–2.5)
2 × 0.3 mg 3 × 0.3 mg 1 × 0.3 mg No No No
High High Moderate No Low Low
a
Patient number. Gender: F = female, M = male. c Age (years) at sampling. d Severity of neutropenia based on typical values at diagnosis according to internationally accepted criteria [3]. e Absolute neutrophil counts (ANC) (×109/L) at sampling or shortly before (values in parentheses designate previous or pre-treatment range). f G-CSF = number of weekly subcutaneous injections of recombinant human G-CSF, in most cases filgrastim. Blood sampling was typically performed z48 h after the last G-CSF injection. g Infections = propensity for bacterial infections. h Patient 6 also has a complete lack of complement factor 9 (C9). b
Neutrophil apoptosis in CIN patients the annexin V-FITC apoptosis detection kit (Oncogene Research Products, Cambridge, MA, USA). Cells were costained with propidium iodide (PI) (50 μg/ml stock solution, 125 ng/ml final) prior to analysis on a FACScan (Becton Dickinson, San Jose, CA, USA) equipped with a 488 nm argon laser. Ten thousand events were collected for each sample and analyzed using CellQuest software (Becton Dickinson).
351 turer’s instructions. To validate the method, we used the human Jurkat T cell line (European Collection of Cell Cultures, Salisbury, UK), cultured in RPMI-1640 medium supplemented as above. Since this method was found to correlate well with the DEVD-AMC cleavage assay and our preliminary studies showed that the latter assay yielded more reproducible results for human neutrophils (unpublished findings), only DEVD-AMC results are shown for these cells.
Flow cytometric detection of active caspase-3 Caspase-3-like enzyme activity Expression of the active subunit of caspase-3 was detected by flow cytometry following labeling of cells with FITC-conjugated rabbit anti-caspase-3 monoclonal antibodies (BD Pharmingen, San Diego, CA, USA) according to the manufac-
Cleavage of the fluorogenic peptide substrate DEVD-AMC, indicative of caspase-3-like enzyme activity, was determined as previously described [13]. Briefly, cell lysates and DEVD-
Figure 1 PS externalization in peripheral blood neutrophils from a healthy blood donor (A–F) and a patient with chronic idiopathic neutropenia (CIN) (G–L). Control neutrophils were cultured in vitro in medium alone for 0 h (fresh) (A), overnight (B), or overnight in the presence of zVAD-fmk (C), anti-Fas antibodies (D), TRAIL (E), or TNF-α (F). CIN patient neutrophils (patient 2) were incubated under corresponding conditions (G–L). Viable cells are found in the lower left quadrant of each FACS plot, early apoptotic cells in the lower right quadrant, and late apoptotic cells are located in the upper right quadrant.
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Figure 2 Constitutive and death receptor-mediated apoptosis of neutrophils from adult blood donors compared to CIN patients. Neutrophils from healthy blood donors (HBD) (A) and CIN patients (B) were cultured in vitro for 0 h (fresh), overnight in medium alone, or overnight in the presence of the inhibitors, zVAD-fmk, PD150606, or lactacystin, or the pro-apoptotic agents, anti-Fas antibodies, recombinant TRAIL, or recombinant TNF-α. Apoptosis was determined by flow cytometric detection of annexin V-FITC and PI labeling, and the percentages of early, late, and total apoptosis were calculated. Data shown are mean values ± SD (n = 3–6).
AMC substrate (50 μM) were combined in a standard reaction buffer (100 mM HEPES, 10% sucrose, 5 mM dithiothreitol, and 0.1% CHAPS; pH 7.25) and enzyme-catalyzed release of AMC was measured every 70 s during a 30 min period in a Fluoroscan II plate reader (Labsystems, Stockholm, Sweden) using 355 nm excitation and 460 nm emission wavelengths. Data were analyzed by linear regression and are displayed as pmol of AMC release/min.
Results First, we compared apoptosis of peripheral blood neutrophils from controls and CIN patients. To this end, freshly isolated cells and cells cultivated overnight in the presence or absence of anti-apoptotic agents (the broad-spectrum caspase inhibitor, zVAD-fmk, the calpain inhibitor, PD150606, and the proteasome inhibitor, lactacystin) or pro-apoptotic (agonistic anti-Fas antibodies, recombinant TRAIL, recombinant TNF-α) were labeled with annexin V and propidium iodide and analyzed by flow cytometry. Representative data from one control and one patient are shown in Fig. 1, and a summary of the results obtained in 6 healthy blood donors and 6 CIN patients are depicted in Fig. 2. Spontaneous neutrophil apoptosis was increased upon overnight incubation; however, no significant differences were detected between controls and CIN patients (Fig. 2). Of note, the caspase inhibitor was effective in blocking late apoptosis (i.e. cells positive for both propidium iodide and annexin V), yet failed to suppress early apoptosis (cells
Figure 3 Comparison of constitutive apoptosis of neutrophils from untreated CIN patients with neutrophils from patients treated with recombinant G-CSF. Cells were isolated from CIN patients not on G-CSF treatment (n = 3) or on G-CSF treatment (n = 3), and subjected to in vitro cultivation overnight in medium alone. Samples from the G-CSF-treated group were drawn z 48 h after the last G-CSF injection. Apoptosis was determined using the annexin V-FITC assay, and the percentages of early and total apoptosis (as defined in the legend to Fig. 1) were calculated. Data shown are mean values ± SD. An increase of apoptosis of neutrophils was observed in the G-CSF-treated group; however, due to the small number of samples, no statistical analysis was performed.
Neutrophil apoptosis in CIN patients positive only for annexin V) both in controls and CIN patients (Fig. 2), suggesting that cell death was partially caspase-independent. PD150606 or lactacystin, however, had no discernible effect on constitutive apoptosis of control or patient neutrophils, at the time points tested. Fas ligation was found to accelerate apoptosis both of control and CIN patient neutrophils (Fig. 2), whereas TRAIL and TNF-α neither promoted nor blocked PS externalization under these conditions. Overall, no significant differences were observed in death receptormediated apoptosis between controls and CIN patients. However, neutrophils from the 3 patients receiving G-CSF treatment appeared to display an increase in constitutive apoptosis upon overnight incubation, in comparison to cells from patients without ongoing G-CSF treatment (Fig. 3). On the other hand, when the patients were stratified according to severity of disease, irrespective of current
353 treatment status, no such differences were detected (data not shown). We also investigated the degree of caspase activation in neutrophils from neutropenia patients and controls. For this purpose, a comparison was made between a flow cytometric method of detection of caspase-3 (using specific antibodies recognizing the active subunit, but not the pro-form of caspase-3) and the DEVD-AMC method (which is based on the in vitro cleavage of a fluorogenic caspase substrate). There was an excellent correlation between the two methods (Fig. 4), suggesting that the DEVD-AMC cleavage assay faithfully reports caspase-3 activation. For the neutrophil experiments, the latter assay was therefore employed. A significant induction of caspase-3 was observed upon overnight incubation of cells from adult blood donors as well as from CIN patients, and this was effectively blocked by zVAD-fmk (Fig. 5). However, the results obtained in controls and
Figure 4 Comparison of two methods for detection of caspase-3 activity, using the Jurkat T cell line as a model. Cells were cultured in medium alone for 4 h (control), or for 1 to 3 h in the presence of staurosporine (STS), a pro-apoptotic agent. (A) Flow cytometric, antibody-based detection of the active subunit of caspase-3 was performed as described in Materials and methods. The percentages of cells expressing active caspase-3 are shown. (B) Parallel samples from the same experiment were also subjected to quantification of DEVD-AMC cleavage, using a real-time fluorogenic assay. The maximum rate of AMC release (pmol/min) was estimated by linear regression (r2 > 0.99). Data shown are the mean values of duplicate determinations. Linear correlation between the two methods reported in (A) and (B) for detection of caspase-3 activation.
354
Figure 5 Caspase-3 activation detected by the DEVD-AMC cleavage assay in neutrophils isolated from control subjects and CIN patients. Neutrophils from healthy blood donors (HBD) and CIN patients were cultured in medium alone for 0 h (fresh) or overnight in the absence (spontaneous) or presence of inhibitors or pro-apoptotic agents, as indicated. Caspase-3 activation was quantified as outlined in the legend to Fig. 4, and the maximum rate of AMC release (pmol/min) is depicted for each condition. Data shown are mean values ± SD (n = 3).
patients did not differ significantly. Moreover, ligation of Fas using specific antibodies did not appear to enhance caspase3 activation in control or patient cells, at least at the time points tested, whereas treatment of cells from CIN patients with recombinant TNF-α appeared to suppress caspase-3 activation to some extent.
Discussion The mechanism underlying chronic idiopathic neutropenia (CIN) remains unknown. Our current results suggest that peripheral blood neutrophils from CIN patients do not display an increased propensity for constitutive or death receptor-mediated apoptosis upon in vitro cultivation. However, an increased degree of apoptosis was observed in cells derived from CIN patients receiving G-CSF therapy in comparison to CIN patients without ongoing treatment. Notwithstanding the small number of evaluated patients, one may speculate that the latter findings could be due to a G-CSF withdrawal effect [14], or perhaps an increased vulnerability of neutrophils synthesized under the influence of supraphysiological doses of G-CSF. Alternatively, these patients may harbor an intrinsically increased propensity for neutrophil apoptosis. However, when the CIN patients included in the present study were stratified according to disease severity and not to treatment status, no significant differences in neutrophil apoptosis were seen, thus arguing against an intrinsic defect in peripheral blood neutrophils in this condition. Of note, mice with a targeted disruption of the G-CSF gene display reduced neutrophil numbers in peripheral blood [15], and mice lacking the G-CSF receptor were reported to exhibit impaired production and increased apoptosis of neutrophils [16], hence supporting the notion that G-CSF promotes the survival of these cells. To address this issue conclusively in human subjects, larger cohorts of neutropenia patients treated or not treated with G-CSF, or
D. Garwicz et al. healthy controls or stem cell donors subjected to high doses of G-CSF followed by sudden withdrawal of the growth factor, need to be examined. However, such investigations were beyond the scope of the present pilot study. The life span of neutrophils is subject to regulation by death receptors and their corresponding ligands [13,17]. Liu et al. [18] have previously demonstrated high levels of circulating Fas ligand in the serum of large granular lymphocyte (LGL) leukemia patients, and showed that the resolution of neutropenia was associated with disappearance or marked reduction in Fas ligand levels. However, no significant differences were seen in the effect of Fas ligation on neutrophils from CIN patients versus controls in the present study. TRAIL has been suggested to play a role in the pathogenesis of neutropenia in systemic lupus erythematosus (SLE) patients [19], yet TRAIL is not pro-apoptotic for normal neutrophils under non-inflammatory conditions [20], and our data do not support a role for TRAIL in promoting neutrophil apoptosis in CIN patients. Finally, previous reports have demonstrated that TNF-α may either promote or inhibit apoptosis of neutrophils, depending on the dose and on the kinetics of cell death [21,22]. In the present study, TNF-α stimulation resulted in a decreased caspase activation in some patients as well as in some controls. However, this effect was not reflected in a corresponding decrease of PS exposure, thus confirming previous observations that both caspase-dependent and -independent pathways are involved in the cell surface externalization of PS in neutrophils [13]. Our pilot study, performed in a small number of patients, thus suggests that there are no overall differences in the rate of apoptosis of peripheral blood neutrophils between CIN patients and control subjects, indicating that the underlying defect in this disease lies elsewhere, for instance in the bone marrow microenvironment, as proposed recently by others [23,24]. Indeed, we have recently documented a similar degree of constitutive apoptosis of peripheral blood neutrophils from a WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) patient and control subjects [25], thus pointing to the retention and/or impaired survival of neutrophils in the bone marrow [9] as the primary defect in the latter condition. It is conceivable that only subsets of immature neutrophils are affected in congenital or idiopathic neutropenia patients while the remaining mature neutrophils egress from the bone marrow in response to growth factor treatment; this could then help to explain why there are no marked differences in apoptosis of circulating neutrophils between patients and controls. To conclude, further investigations in larger cohorts of neutropenia patients, and of immature and mature neutrophils obtained in parallel from the bone marrow and peripheral blood compartments of these patients, are needed to address this important question.
Acknowledgments We thank all patients and control subjects who participated in this study. We are also grateful to Ms. Birgitta Wester at the flow cytometry core facility, Department of Microbiology, Tumor and Cellbiology, Karolinska Institutet, for expert advice. The study was supported, in part, by the Swedish
Neutrophil apoptosis in CIN patients Research Council, the Swedish Cancer Foundation, and the Swedish Society of Medicine. D.G. was the recipient of a post doctoral fellowship from the Swedish Society for Medical Research.
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