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. 2007 Dec;150(3):539–545. doi: 10.1111/j.1365-2249.2007.03524.x

Mechanisms of neutrophil death in human immunodeficiency virus-infected patients: role of reactive oxygen species, caspases and map kinase pathways

S Salmen *, H Montes †, A Soyano ‡, D Hernández §, L Berrueta *
PMCID: PMC2219366  PMID: 17956581

Abstract

Neutrophils from human immunodeficiency virus-positive (HIV+) patients have an increased susceptibility to undergo programmed cell death (PCD), which could explain neutropenia during advanced disease. In this work, key steps of PCD have been evaluated in neutrophils from HIV+ patients. The role of caspase-3, caspase-8, mitogen activated protein kinase (MAPK) and reactive oxygen species (ROS) was analysed. Spontaneous neutrophil death is dependent upon caspase-3 but independent of caspase-8, suggesting that the intrinsic pathway is involved as a pathogenic mechanism of PCD. Inhibition of ROS decreased spontaneous PCD and caspase-3 hydrolysis, connecting oxidative stress and caspase-3 activation with neutrophil PCD in HIV-infected patients. Additionally, an increased neutrophil death was observed in HIV+ patients, following inhibition of p38 MAPK, suggesting a role for p38 MAPK in cell survival during the disease. We conclude that oxidative stress secondary to HIV infection can accelerate neutrophil death.

Keywords: caspases, cell death, HIV, neutrophils, ROS

Introduction

Programmed cell death (PCD) is an essential process for development, normal growth and tissue homeostasis. Many eukaryotic cells that die and are removed in a programmed manner undergo a stereotypical series of biochemical and morphological changes that mainly involve the activation of caspases, chromatin condensation and the display of phagocytosis markers on the cell surface. This process has been called apoptosis to delineate it clearly from other cell death programmes [1]. Neutrophils are unique in their susceptibility to undergo rapid spontaneous apoptosis once released from the bone marrow, resulting in their clearance from the circulation within a few hours [2].

As a first line of defence against bacterial and fungal infections, neutrophils are recruited rapidly to inflammatory sites, where the expression of their constitutive cell death programme can be delayed or accelerated by a number of agents [3–6]. It has been reported previously that during advanced HIV infection neutrophil PCD is accelerated markedly, which could explain the neutropenia and impaired neutrophil function observed in these patients [7, 8]. Furthermore, we have demonstrated that accelerated neutrophil death in HIV-infected patients may be explained, in part, via a Fas-dependent mechanism, which correlated with viral load [9].

Several molecules are involved in regulated PCD, including cytokines, Fas ligand (FasL), reactive oxygen species (ROS), caspases and mitogen-activated protein kinase (MAPK) pathways [10]. There are three major types of MAPKs in mammalian cells, which have been proved to participate in neutrophils PCD: p42/44 extracellular signal-related protein kinase (ERK), p38 MAPK and c-Jun N-terminal kinase/stress-activated (JNK) MAPK [2]. The functional role of these kinases can be studied by using inhibitors, for instance PD98059 inhibits Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK), thereby preventing the phosphorylation and activation of ERK [11], and SB902190 or SB203580, which are used to inhibit p38MAPK [12].

On the other hand, it has been demonstrated extensively that ROS play an important role in controlling the neutrophil life span [6, 13]. Several studies suggest that ROS affect the intrinsic apoptotic pathway (mitochondrial pathway), with mitochondria (through aerobic metabolism) being the major source [14–17]. ROS generation may change the redox status of cells with a subsequent effect on specific kinases, phosphatases and transcription factors that alter the sensitivity of the cell to death stimuli [2, 18–22].

Caspases are major executors of the apoptotic programme in most cell types, including human neutrophils. Both spontaneous and Fas receptor-mediated neutrophil apoptosis involve the activation of caspases, a family of cysteine proteases, which cut cellular substrates at an obligatory aspartic acid within a preferred sequence [23].

Because the mechanisms of spontaneous and Fas-induced cell death seem to be separated and yet remain unclear, we have explored mechanisms that modulate the cell death programme in neutrophils during HIV infection by looking at MAP kinase pathways, caspase activation and ROS effects in spontaneous neutrophil death. We found that: (i) spontaneous cell death is increased by the amount of generated ROS; (ii) inhibition of caspase-3 decreased spontaneous cell death in neutrophils from HIV-infected patients and caspase-3 hydrolysis is reduced by inhibiting ROS; and (iii) the p38 MAPK is phosphorylated constitutively in neutrophils from HIV-infected patients. Our results may contribute to understanding of the pathogenesis of neutrophil dysfunction during HIV infection.

Materials and methods

Media and reagents

Complete media consisted of RPMI-1640 supplemented with 2 mM l-glutamine, 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin; all reagents were purchased from Gibco life Technologies, Inc.. SB203580 (p38 MAPK inhibitor), SP600125 (JNK MAPK inhibitor), PD98059 (ERK MAPK inhibitor), IETD-CHO (a caspase-8 inhibitor), DEVD-CHO (a caspase-3 inhibitor), catalase and superoxide dismutase (SOD) were purchased from Calbiochem (San Diego, CA, USA). Rabbit anti-p38, rabbit anti-phospho-p38 (Tyr182), mouse anti-ERK, mouse anti-phospho-ERK (Thr202/Tyr204), rabbit anti-JNK (C-17), goat anti-pJNK (Thr183/Tyr185), annexin-V–fluorescein isothiocyanate/propidium iodide (FITC/PI) and donkey–anti-goat horseradish peroxidase (HRP)-conjugate (2020) were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse polyclonal anti-caspase-8 (1C12) and caspase-3 were purchased from Cell Signalling Technology (Beverly, MA, USA). Mouse IgG1 FITC-conjugated, phycoerythrin (PE) and peridinin chlorophyll (PerCp) and TriTest (anti-CD4, anti-CD8, anti-CD3) were purchased from Becton Dickinson Co. (San Jose, CA, USA). The monoclonal antibody IgM anti-Fas ZB4 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY, USA); dihydrorhodamine-123 (DHR-123) was purchased from Molecular Probes (Eugene, OR, USA) and HRP-conjugated anti-mouse and anti-rabbit were purchased from (Pierce, Rockford, IL, USA).

Human blood samples

Peripheral blood samples were obtained from 28 HIV-positive patients and 25 control subjects. The diagnosis of HIV infection was established by enzyme-linked immunosorbent assay (ELISA) and Western blot (Organon Teknika, Durham, NC, USA). The viral load was established by Amplicor Kit (Roche Diagnostics, Indianapolis, IN, USA). Disease classification was established according to Center for Disease Control (CDC) criteria. All seronegative controls were tested by HIV-1 ELISA on the day their blood was drawn. The experimental protocol was approved by the Ethics Committee of the University of Los Andes, and written informed consent was obtained from all the subjects.

Isolation of polymorphonuclear cells (PMN)

Citrated venous blood was mixed with 6% dextran solution (mol. wt 500·000) and incubated at room temperature for 30 min. Leucocyte-enriched supernatant was collected, diluted with RPMI-1640 and layered on Ficoll-Hypaque (1077, Sigma, St Louis, MO, USA). After density gradient centrifugation at 400 g for 30 min, PMN were obtained from the bottom. Red blood cells contained in PMN pellets were eliminated by hypotonic lysis using cold distilled water. This procedure resulted consistently in a highly purified polymorphonuclear cell population (98%), visualized with acridine orange. Purified PMN (98% viable by trypan blue exclusion) were resuspended at 2 × 106 cells/ml in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin.

Superoxide production

Flow cytometric analysis of neutrophil respiratory burst activity was measured using a modification of a previously published method [24]. Briefly, freshly isolated and 6 h cultured neutrophils were resuspended in complete media at a concentration of 1 × 106 cells/ml and preloaded with DHR-123 (1 μmol/l) by incubating the cells with DHR-123 in a waterbath at 37°C for 10 min, with gentle mixing every 5 min, followed by washing with phosphate-buffered saline (PBS) three times and immediate analysis on a fluorescence activated cell sorter (FACScan) flow cytometer (Becton Dickinson). A total of 10 000 events from each sample were collected.

Analysis of spontaneous cell death

Cell death was measured in neutrophils cultured in complete media during two different times: 0 h and 6 h. To determine spontaneous cell death, neutrophils were left in culture with media alone during a period of 6 h at 37°C and 5% CO2. Cells were incubated with any of the following reagents: p38 MAPK inhibitor (20 μM), ERK MAPK inhibitor (50 μM) [25], JNK inhibitor (20 μM) [26], caspase-3 inhibitor (10 μM), caspase-8 inhibitor (20 μM) or catalase/superoxide dismutase (1000 U/ml and 50 U/ml, respectively) [27]. In some experiments, neutrophils were incubated with 500 ng/ml of anti-Fas IgM monoclonal antibody ZB4 [28], capable of blocking the Fas triggered signal.

Determination of cell death using annexin-V and propidium iodide

Annexin-V–FITC staining procedure was conducted following the manufacturer's instructions. Briefly, treated and untreated cells were collected by low-speed centrifugation, washed twice with cold PBS and resuspended in assay buffer at a concentration of 1 × 106 cells/ml; from these suspensions, 100 μl aliquots were incubated with 1 μg of annexin-V–FITC and 10 μg of PI during 15 min at room temperature and analysed immediately by flow cytometry.

Protein immunoblotting

Neutrophils (2 × 106 cells/ml) were lysed in buffer A containing 50 mM TrisHCl, pH 8, 1% Triton X-100, 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 μg/ml leupeptin/aprotinin and 1 mM sodium orthovanadate and incubated on ice for 15 min; lysates were clarified by centrifugation at 14 000 g for 10 min at 4°C. Supernatants containing equivalent amounts of protein (Bradford, Bio-Rad, Hercules, CA, USA), were resuspended in sample buffer heated in a boiling water bath for 3 min, separated by electrophoresis on 10% sodium dodecyl sulphide (SDS) polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, MA, USA) and probed with different antibodies: anti-p38, anti-phospho-p38, anti-caspase-3, anti-caspase-8, anti-ERK, anti-phospho-ERK, anti-JNK and anti-phospho-JNK. Labelled protein bands were detected using enhanced chemiluminescence (SuperSignal, Pierce, Rockford, IL, USA).

Statistical analysis

Data are presented as means ± standard deviation (s.d.). The significant differences between parametric variables obtained from the different experiments were calculated by analysis of variance (anova); P < 0·05 was considered statistically significant.

Results

Subjects

As shown in Table 1, all patients had 200 or more CD4+ cells/ml (684 cells/ml ± 480 s.d.), with a viraemia of 206 537 HIV-1 RNA copies/ml mean value. No history of opportunistic infections was recorded. The mean ages in years (± s.d.) of the study subjects were as follows: 34 ± 10 (range 23–50) for the control group and 31 ± 9 (range 15–40) for HIV patients. All subjects had a Hispanic genetic background.

Table 1.

Characteristics of human immunodeficiency virus (HIV)-infected patients and control group.

HIV-infected patients Control group
Age (years) 31 ± 9 (range 15–40) 34 ± 10 (range 23–50)
Sex (female/male) 7/19 13/10
CD4 (cells/ml) 684 cells/ml ± 480 s.d. 850 cells/ml ± 333 s.d.
Viral load (copies RNA) 206 537 HIV-1 RNA copies/ml ± 179 483 s.d. n.a.
Length of time HIV-infected (months) 67·9 ± 8·5 n.a.
Clinical status (chronic HIV/AIDS) 26/0 n.a.
HAART (highly active anti-retroviral therapy) 0% n.a.
Granulocytes (%) 54 ± 15 s.d. 69 ± 4·5 s.d.
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n.a.: Not applicable.

Increased superoxide production in HIV-infected patients

As described previously, phagocytes from HIV-infected patients produce altered levels of superoxide, particularly before acquired immunodeficiency syndrome (AIDS) is established [29, 30]. We observed that both freshly isolated and 6 h cultured neutrophils from HIV-infected patients produce increased levels of superoxide when compared with control individuals: 7·3 ± 3·9 versus 2·9 ± 0·8 and 10·1 ± 1·6 versus 4·1 ± 1·5, respectively (Fig. 1) P < 0·05.

Fig. 1.

Fig. 1

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Detection of superoxide production in neutrophils from human immunodeficiency virus-infected patients. Freshly isolated and 6 h cultured neutrophils were preloaded with dihydrorhodamine-123 (1 μmol/l) for 10 min, and analysed immediately by flow cytometry. The figure shows percentages of positive cells expressed in mean values ± standard deviation, *P < 0. 05.

ROS modulate neutrophil death significantly in HIV-infected patients

We studied the effects of these metabolites in neutrophil death by removing them using a combination of SOD and catalase. Spontaneous neutrophil death was reduced significantly in HIV-infected patients upon incubation with catalase/SOD compared to control individuals (27·5 ± 8·5 and 15·5 ± 5·6; P < 0·001 versus 17·82 ± 7·2 and 16·23 ± 3·1) (Fig. 2). Additionally, superoxide production was correlated with spontaneous neutrophil death (r= 0·53, P < 0·05).

Fig. 2.

Fig. 2

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Effect of catalase and superoxide dismutase on spontaneous neutrophil death from human immunodeficiency virus (HIV)-infected patients. Neutrophils were incubated during 6 h with or without catalase and superoxide dismutase, and cell death was measured by annexin-V–fluorescein isothiocyanate and propidium iodide staining. The graphic shows mean values ± standard deviation of spontaneous cell death expressed in percentages from control (left) and HIV-infected patients (right) *P < 0·001.

Caspase-3 is an important mediator of neutrophil death in HIV-infected patients

Neutrophil death was reduced significantly upon inhibition of caspase-3 in neutrophils from HIV-infected patients compared to control individuals (30·1 ± 12·8 and 21·2 ± 8·3 (P < 0·05) versus 16·42 ± 4 and 19·43 ± 5·2) (Fig. 3b). Activation of caspase-3 was therefore analysed at 0 h and 6 h by immunoblotting to detect cleavage of the proenzyme. Our data show hydrolysis of caspase-3 in neutrophils from HIV-infected patients and control individuals at times 0 h and 6 h (Fig. 4), confirming that caspase-3 was hydrolysed significantly in neutrophils from HIV+ patients. Conversely, we observed that inhibition of caspase-8 did not have a significant effect on spontaneous cell death in neutrophils from HIV-infected patients (30·5 ± 17·2 and 26·9 ± 9·2 (P > 0·5) versus 19·55 ± 3·4 and 18·55 ± 4·1) (Fig. 3a).

Fig. 3.

Fig. 3

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Spontaneous cell death in neutrophils from human immunodeficiency virus (HIV)-infected patients, following inhibition of caspase-8 and 3. Neutrophils were incubated during 6 h with or without caspase-8 inhibitor integral equation time domain–Chinese hamster ovary at a concentration of 20 μM (Fig. 3a) or caspase-3 inhibitor (DEVD–CHO) at a concentration of 10 μM (Fig. 3b), and cell death was measured by annexin-V–fluorescein isothiocyanate and propidium iodide staining. Both panels show mean values ± standard deviation of spontaneous cell death expressed in percentages from control (left) and HIV-infected patients (right).

Fig. 4.

Fig. 4

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Effect of catalase and superoxide dismutase on caspase-3 activation in neutrophils from human immunodeficiency virus (HIV)-infected patients. Cell lysates were prepared from freshly isolated and 6 h cultured polymorphonuclear cells and analysed by immunoblotting for caspase-3. (a) Hydrolysis of caspase-3 in neutrophils from two HIV-infected patients and a control individual at times 0 h and 6 h, with or without catalase and superoxide dismutase treatment. Full-length 32 kDa (pro-casp 3) and cleaved 17 kDa caspase-3 fragment (act-casp 3) are indicated. (b, c) Mean ± standard deviation of optical densities (OD) from immunoblots (n = 5) of cleaved caspase-3, P < 0·05.

We also explored the effect of SOD and catalase on caspase-3 hydrolysis during the times indicated. Figure 4a, c shows that by removing ROS a remarkable reduction of caspase-3 hydrolysis was observed, which correlated with reduction of cell death, P < 0·05 (Fig. 4c).

p38 MAPK plays a key role in neutrophil cell death and survival during HIV infection

To understand the role played by this kinase in death and survival of neutrophils during HIV infection, we used the p38 MAPK inhibitor SB203580 [31] at a concentration of 20 μM [25] during 6 h, and observed a significant increase of cell death in neutrophils from HIV-infected patients compared to control individuals (27·23 ± 4·5 and 39·6 ± 5·83; P < 0·01 versus 18 ± 2·3 and 21 ± 4·4) (Fig. 5). Comparable results were observed following inhibition of ERK as described previously [9] (data not shown). Inhibition of JNK, a kinase involved in tumour necrosis factor (TNF)-mediated cell death, did not decrease the percentage of neutrophil death in any of the individuals studied (data not shown).

Fig. 5.

Fig. 5

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Effect of p38 mitogen activated protein kinase (MAPK) inhibitor in spontaneous neutrophil death from human immunodeficiency virus (HIV)-infected patients. This figure shows mean values ± standard deviation of spontaneous neutrophil death expressed in percentages from control (left) and HIV-infected patients (right), with or without incubation during 6 h with the p38 MAPK inhibitor SB203580, *P < 0·01.

We explored further these findings and found that the p38 MAPK is significantly phosphorylated at time 0 h in neutrophils from HIV-infected patients compared to control individuals (Fig. 6a,b). In order to establish a connection between this result and the increased oxidative stress described in HIV patients [32], neutrophils were treated with SOD and analysed for phospho-p38 MAPK; no differences in p38 phosphorylation were observed (Fig. 6a).

Fig. 6.

Fig. 6

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Effect of catalase and superoxide dismutase on spontaneous p38 mitogen activated protein kinase (MAPK) phosphorylation in neutrophils from human immunodeficiency virus (HIV)-infected patients. Cell lysates were prepared from freshly isolated and 6 h cultured polymorphonuclear cells and analysed by immunoblotting for phospho p38 (top) and p38 MAPK (bottom). (a) Spontaneous phosphorylation of p38 MAPK at 0 h and 6 h of incubation in two HIV-infected patients and a control subject, with or without catalase and superoxide dismutase treatment. (b) Mean ± standard deviation of optical densities from immunoblots of phospho-p38 (n = 5), P < 0·05.

Discussion

It has been demonstrated that apoptosis could, in part, be responsible for HIV-related neutropenia [33], which could occur either spontaneously [8] or be triggered via death receptors such as Fas/FasL [9]. We have reported previously that spontaneous neutrophil death is not Fas/FasL-induced during HIV infection [9]. Furthermore, it has been demonstrated that neutrophils from Fas (lpr) or FasL (gld) deficient mice show a normal rate of spontaneous death [34]. These observations suggest that the mitochondrial pathway is predominant during spontaneous neutrophil death. In this work we studied further some critical elements known to be engaged during the neutrophil death programme to understand the pathogenesis of neutrophil dysfunction during the course of the disease. Our study involved the measurement of spontaneous cell death by using flow cytometry and Western blot under three basic conditions: incubation with caspase-3 or caspase-8 inhibitors, incubation with p38 or JNK inhibitors and treatment with catalase/SOD. We also measured levels of superoxide production in neutrophils from HIV-infected and control individuals.

Diminished spontaneous cell death was observed in neutrophils from HIV-infected patients treated with caspase-3 inhibitor. Conversely, PCD was not affected in neutrophils treated with caspase 8 inhibitor, suggesting that caspase-8, an initiator of death receptor-triggered cell death, does not account for spontaneous neutrophil cell death during HIV infection, which is supported by the fact that using an antagonist of Fas, which inhibits Fas cross-linking, no changes in spontaneous cell death were observed (data not shown). Our data show for the first time that in the context of HIV infection, caspase-3 is involved in spontaneous neutrophil death, suggesting that this effector caspase is being activated by a mechanism other than the DISC formation during the course of the disease.

Previous [29] and present results show that neutrophils from HIV-infected patients have increased baseline levels of superoxide production, particularly before AIDS is established. Additionally, previous reports have also shown that part of the dysfunction observed in neutrophils from HIV-infected patients is secondary to an altered ROS production [35]. Several studies suggest that ROS affect the intrinsic apoptotic pathway, with mitochondria being the major source and the primary target of these ROS. Oxidative stress leads to the production of ROS that can attack membrane lipid, proteins and deoxynucleic acids resulting in cellular dysfunction and cell death [36]. ROS may oxidize mitochondrial pores that lead to cytochrome c release and caspase 9 activation due to the disruption of the mitochondrial membrane potential [14, 16, 37]. ROS generation may also change the redox status of cells with subsequent effects on specific kinases, phosphatases and transcription factors that alter the sensitivity of the cell to apoptotic stimuli [18, 38, 39]. Thus, neutrophil death during HIV infection could, in part, be a result of excessive production of oxygen-derived species during the development of the disease which activate the intrinsic cell death pathway. This observation is reinforced by: (i) a positive correlation between superoxide production and spontaneous neutrophils cell death was observed; and (ii) using catalase and SOD, known to be oxygen radical scavengers, a significant reduction of spontaneous cell death was observed in neutrophils from HIV-infected patients.

Caspase-3 may also be a target of different intracellular pathways, which could modulate its activity affecting the cell death programme. For instance, caspase-3 has been identified as a downstream target of p38 MAPK in neutrophils, leading to phosphorylation of this caspase, impairing its activity and favouring cell survival [40]. We have observed that inhibition of p38 increased significantly spontaneous neutrophil death in neutrophils from HIV-infected patients, which suggests that p38 is either a target of HIV products, as described for other cells [41, 42], or that activation of this kinase takes place to compensate for the increased spontaneous cell death which may be triggered for several mechanisms, including stress-derived signals.

Taken together, these results suggest that increased oxidative stress could, in part, be responsible for increased spontaneous cells in neutrophils from HIV-infected patients, beginning at early stages of the disease. Within the context of HIV infection, the experimental evidence suggests that the intrinsic pathway seems to be more relevant for spontaneous neutrophil death as a result of increased stress signals derived from the virus. Finally, the data presented here address key mechanisms involved in neutrophil death during HIV infection, contributing to understanding of the pathogenesis of the disease and highlighting possible targets for future therapeutic intervention.

Acknowledgments

This work was supported in part by a grant from FONACIT, code S1-2000000522, F-2000001509 and from the CDCHT-ULA, code M-865-06-07-B. We would also like to thank FUNDESI for referring their patients to our Institution.

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