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Abstract

Objective

To determine the role of apoptosis inhibition in the prevention of diabetic neuropathy using the antiFas cell surface death receptor (Fas) antagonistic monoclonal antibody ZB4.

Methods

This prospective study enrolled patients with type 2 diabetes with and without neuropathy and a group of healthy controls. The serum concentrations of Fas and Fas ligand (FasL) were measured in all study participants using an enzyme-linked immunosorbent assay. The ability of serum from study participants to induce apoptosis was evaluated in a human neuronal cell line using flow cytometry.

Results

A total of 28 healthy subjects and 57 patients with diabetes were enrolled in the study. Serum Fas concentrations were significantly increased in diabetes patients with and without neuropathy compared with the controls. Cells treated with the serum from diabetes patients with neuropathy had significantly higher rates of early apoptosis compared with cells treated with control serum. Monoclonal antibody ZB4 was able to block serum-induced apoptosis.

Conclusions

Serum-induced apoptosis of a human neuronal cell line appeared to be mediated via Fas, which suggests that targeting and inhibiting Fas might offer a therapeutic target for diabetic neuropathy.

Keywords

Diabetes mellitus neuronal cell apoptosis

Introduction

Diabetes mellitus is a major public health problem that is increasing in incidence. It is associated with long-term complications.¹ Currently, there are approximately 347 million people with diabetes and it affects people in both high- and low-income countries.² Diabetes mellitus is common in the Saudi Arabian population.³ Patients with diabetes are at a high risk of developing microvascular and macrovascular complications.⁴ Diabetic neuropathy is a common microvascular complication and is the most frequent cause of lower limb complications.⁵ Diabetic neuropathy increases morbidity and reduces the quality of life.^6,7 Accumulating evidence suggests that abnormal apoptosis, which is a type of programmed cell death, is involved in the pathogenesis of diabetic neuropathy.⁸ Premature apoptosis may be involved in the pathogenesis of a variety of neurodegenerative disorders.⁸ The results of our previous study suggested that the proapoptotic protein markers, Fas cell surface death receptor (Fas) and Fas ligand (FasL), were involved in the apoptosis of a neuronal cell line,⁹ while Fas blockers can act as antiapoptotic protein markers.¹⁰

Control or modulation of apoptosis could provide a new target for therapy for patients with diabetic neuropathy. To reduce the occurrence and progression of diabetic neuropathy, it is important to identify and investigate the major risk factors associated with this microvascular complication. This study aimed to determine the role of apoptosis inhibition in the prevention of diabetic neuropathy using the antagonistic antiFas monoclonal antibody (human, neutralizing) clone ZB4.

Patients and methods

Patient population

This prospective study enrolled consecutive patients in the Department of Physiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia, between January 2005 and February 2007. Patients with type 2 diabetes were referred from the University Diabetes Centre, College of Medicine, King Saud University. Patients with an established diagnosis of type 2 diabetes according to the American Diabetes Association Guidelines with and without neuropathy were included in the study.¹¹ Patients with type 1 diabetes, autoimmune diseases, nephropathy, proliferative retinopathy or vascular disease, and patients with diabetes being treated with calcium channel blockers, were excluded from the study. Healthy volunteers were enrolled from the local population of Riyadh, Saudi Arabia and formed the control group.

Based on the patient’s clinical history and the results of a nerve conduction study undertaken as previously described,⁹ patients with diabetes were divided into two groups: diabetes patients with neuropathy and diabetes patients without neuropathy. The investigation of Fas-mediated apoptosis was conducted for these two groups as well as on the healthy control subjects. A venous blood sample was collected from each study participant; the serum was collected by centrifugation and stored at −70℃ until analysis.

The study protocol was approved by the Institutional Review Board (no. At-12-85), College of Medicine Research Centre, King Saud University, Riyadh, Saudi Arabia. All study participants provided written informed consent.

Routine biochemical analyses

Routine biochemical analyses were undertaken to measure the serum glucose concentration, glycosylated haemoglobin percentage and serum creatinine concentration using a Siemens Dimension Xpand Plus autoanalyser (Diamond Diagnostics, Holliston, MA, USA).

ELISA determination of sFas and sFasL

The serum levels of soluble Fas (sFas) and soluble FasL (sFasL) were measured using an enzyme-linked immunosorbent assay (ELISA) technique and monoclonal antibodies specific for sFas or sFasL, which were precoated onto the individual wells of a microtitre plate. Biological standard solutions and experimental samples were pipetted into the wells and incubated for 2 h at room temperature. The wells were washed four times at 4℃ to wash away any unbound substances using 300 µl of concentrated wash buffer from the sFas and sFasL ELISA kits (pH 7.5) (R&D Systems Europe, Abingdon, UK) diluted with deionized water. An enzyme-linked polyclonal antibody specific for sFasL or sFas was added to the wells and incubated for 2 h at room temperature. Following a wash using the same wash buffer to remove any unbound antibody–enzyme complex, a substrate solution was added to the wells and incubated for 2 h at room temperature. Colour develops in proportion to the amount of sFas or sFasL bound in the initial step. The intensity of the colour was measured at 450 nm microplate reader (ELx800™, Bio-Tek® Instruments, Winooski, VT, USA). All ELISA reagents were purchased from PAA Laboratories (Pasching, Austria) and Sigma-Aldrich (St Louis, MO, USA). sFas and SFasL standard preparations were dissolved in 1 ml of deionized water, which produced stock solutions of 20 000 pg/ml for sFas and 1000 pg/ml for sFasL.

Cell culture of neuronal cell lines

Two neuronal cell lines (human neuroblastoma cell line SH-SY5Y and NIE-115 murine neuroblastoma cell line) were obtained from the European Collection of Cell Cultures (Porton Down, UK). The NIE-115 murine neuroblastoma cell line was used to establish the technique of cell culture and determination of apoptosis. The human neuroblastoma cell line SH-SY5Y was used to investigate the induction and inhibition of apoptosis. Cell culture media and all reagents were purchased from PAA Laboratories and Sigma-Aldrich. The cell culture media was prepared by mixing 75 ml of filtered fetal bovine serum, 146 ml of l-glutamine, 413 ml of incomplete Dulbecco’s modified Eagle’s medium, 5 ml of non-essential amino acids, 5 ml of penicillin/streptomycin and 2.5 ml gentamycin. Cells were cultured continuously in a humidified incubator at 37℃ with 5% CO₂.

Cell count and viability determination

The trypan blue dye exclusion technique was used to determine cell counts and cell viability.¹² The neuronal cell lines were removed from the cell culture plates using a standard trypsinization technique. The cells were centrifuged and the cell pellets washed using complete cell culture medium. The washed cells were resuspended in 10 ml of complete cell culture medium and mixed well. Of this mixture, 50 µl was transferred to a 1.5 ml vial. Trypan blue (50 µl) was added and the cell suspension was mixed well. A drop of cell suspension was transferred to a haemacytometer for counting. The proportion of viable cells was determined using the equation: viable cells (%) = viable cell count/total cell count.

Induction and inhibition of apoptosis in a neuronal cell line

These experiments were undertaken to confirm the role of Fas-mediated apoptosis in the pathophysiological mechanism of diabetic neuropathy. Human serum and agonistic antiFas antibody (i.e. a positive control) were added to the human neuroblastoma cell line SH-SY5Y to induce apoptosis, and an antagonistic antiFas antibody (i.e. a Fas receptor blocker) was added to inhibit apoptosis.

In order to measure the induction of apoptosis by an agonistic antiFas antibody, 1 ml of cell suspension (1 × 10³ cells/ml) of the human neuroblastoma cell line SH-SY5Y was added to each well of a 12-well plate. Cells were allowed to settle and attach for 2–3 h at 37℃. Apoptosis was induced in the SH-SY5Y cell line by adding agonistic antiFas monoclonal antibody CH-11 (FasL; purified immunoglobulin M, clone CH-11; Millipore, Billerica, MA, USA) at concentrations of 100, 500 and 1000 ng/ml and the cells were incubated for a further 24 h at 37℃. The induction of apoptosis were determined by flow cytometry (see later) and 500 ng/ml of agonistic antiFas antibody CH-11 was demonstrated to be the optimum concentration to induce apoptosis in SH-SY5Y cells.

In order to measure the induction of apoptosis by serum from patients with diabetes, 1 ml of cell suspension (2.5 × 10⁵ cells/ml) of the human neuroblastoma cell line SH-SY5Y was added to each well of a 12-well plate. Cells were allowed to settle and attach for 2–3 h at 37℃. Then 500 ng/ml of agonistic antiFas antibody CH-11 was added to the Fas + wells as a positive control, and 150 µl and 300 µl of serum from patients with diabetes (with and without neuropathy, respectively) and healthy control subjects was added to corresponding wells. The cells with either agonistic antiFas antibody CH-11 or human serum were incubated for further 24 h at 37℃. The rate of apoptosis was determined by flow cytometry (see below).

In order to measure the inhibition of apoptosis by an antagonistic antiFas antibody (i.e. a Fas receptor blocker that inhibits the interaction between FasL and Fas), experiments were undertaken to determine the optimum concentration of antagonistic antiFas antibody to use. The human neuroblastoma cell line SH-SY5Y was cultured as described above for the human serum experiments. Fas receptor blocker ZB4 (antagonistic antiFas monoclonal antibody clone ZB4; MBL, Watertown, MA, USA) was added to the cell culture wells at concentrations of 100, 500, and 1000 ng/ml for 24 h at 37℃; 500 ng/ml of ZB4 was demonstrated to be the optimum concentration to inhibit apoptosis. This concentration was used for all subsequent studies that examined the effect of serum from patients with diabetes and the healthy control subjects.

For all of the experiments described above, the induction and inhibition of apoptosis were determined by flow cytometry (see below).

The measurement of apoptosis by flow cytometry

The neuronal cells were removed from the cell culture plates using a standard trypsinization technique. After incubation, apoptosis was measured using annexin V/propidium iodide staining procedures and flow cytometry following the manufacturer’s instructions (Vybrant® Apoptosis Assay Kit; Salima Trading Corporation, Riyadh, Saudi Arabia). In brief, recombinant annexin V antibodies were added for 15 min at room temperature in the dark. The percentage of apoptosis was analysed by flow cytometry (BD FACScan™ system; Becton, Dickinson and Co., Franklin Lakes, NJ, USA). Each curve showed four quadrants: lower left, lower right, upper right and upper left, which represented the proportion of viable cells, early apoptotic cells, late apoptotic cells and necrotic cells, respectively.

Statistical analyses

The data are summarized as mean ± SD. All statistical analyses were performed using the SPSS® statistical package, version 18.0 (SPSS Inc., Chicago, IL, USA) for Windows®. The correlation between different groups and serum sFas and sFasL concentrations was analysed by one-way analysis of variance. A P-value <0.05 was considered statistically significant.

Results

This study enrolled 28 healthy control subjects (19 male and 9 female) with a mean ± SD age of 34.7 ± 9.4 years (range 23–55 years) and 57 patients with type 2 diabetes (20 male and 37 female) with a mean ± SD age of 46.4 ± 7.5 years (range 13–57 years). Of the patients with type 2 diabetes, 21 were categorized as not having neuropathy and 36 were categorized as having neuropathy. The mean ± SD duration of diabetes in patients with type 2 diabetes with and without neuropathy was 10.1 ± 6.1 years and 9.5 ± 5.7 years, respectively. The mean ± SD serum glucose concentration and the mean ± SD percentage glycosylated haemoglobin in all patients with type 2 diabetes included in this study was 11.68 ± 4.07 mmol/l and 10.22 ± 2.64%, respectively. The mean ± SD serum creatinine concentration in all patients with type 2 diabetes included in this study was 76.62 ± 13.99 mmol/l.

Table 1 presents the serum concentrations of sFas and sFasL in control subjects, diabetes patients with neuropathy and diabetes patients without neuropathy. The serum sFas concentration was significantly increased in diabetes patients without neuropathy compared with the control subjects (P < 0.05). The serum sFas concentration was also significantly increased in diabetes patients with neuropathy compared with the control subjects (P < 0.0001). There were no significant differences in the serum concentrations of sFasL between the three groups.

Table 1.

The serum concentrations of the proapoptotic markers soluble Fas cell surface death receptor (sFas) and soluble Fas ligand (sFasL) in healthy control subjects compared with patients with type 2 diabetes with and without neuropathy.

Groups	sFas concentration (ng/ml)	sFasL concentration (ng/ml)
Control subjects, n = 28	6.49 ± 0.88	0.07 ± 0.03
Diabetes patients without neuropathy, n = 21	8.81 ± 2.26*	0.12 ± 0.03
Diabetes patients with neuropathy, n = 36	9.01 ± 1.54**	0.09 ± 0.02

Data presented as mean ± SD.

P < 0.05, **P < 0.0001, compared with the control subjects; one-way analysis of variance.

Table 2 shows the effect of serum from control subjects and diabetes patients on the rate of apoptosis of the SH-SY5Y human neuroblastoma cell line as determined by flow cytometry. Cells treated with serum from diabetes patients with neuropathy showed similar results to cells treated with 500 ng/ml agonistic antiFas antibody CH-11 that induced apoptosis (i.e. the positive control). Cells treated with serum from diabetes patients with neuropathy had a significantly higher percentage of cells in early apoptosis compared with cells treated with serum from the control subjects (P < 0.05). Cells treated with sera from diabetes patients without neuropathy had a higher percentage of cells in late apoptosis compared with cells treated with serum from the control subjects but the difference was not significant.

Table 2.

The effect of serum from control subjects and patients with type 2 diabetes with and without neuropathy and antagonistic antiFas monoclonal antibody (human, neutralizing) clone ZB4 (ZB4) on the extent of apoptosis induced or inhibited in the SH-SY5Y human neuroblastoma cell line as determined by flow cytometry.

Samples applied to the SH-SY5Y cell line	Viable cells (%)	Early apoptosis (%)	Late apoptosis (%)
Serum from control subjects	81.15 ± 2.62	4.58 ± 1.06†	6.88 ± 1.92
Agonistic antiFas antibody CH-11 (500 ng/ml)	64.58 ± 2.15	18.14 ± 1.11†	15.14 ± 2.68†
Serum from diabetes patients without neuropathy	69.29 ± 9.06	2.41 ± 0.32	11.36 ± 2.12
Serum from diabetes patients with neuropathy	57.24 ± 9.44	24.75 ± 6.53*†	15.98 ± 3.39†
Serum from diabetes patients with neuropathy + ZB4 (500 ng/ml)	76.15 ± 2.72	0.44 ± 0.33	2.99 ± 1.14

Data presented as mean ± SD for different wells of cells treated with serum from individual patients or control subjects (or agonistic antiFas antibody CH-11).

P < 0.01 compared with cells treated with serum from the control subjects; one-way analysis of variance.

†

P < 0.05 compared with cells treated with serum from diabetes patients with neuropathy + ZB4; one-way analysis of variance.

Table 2 also shows the effect of the specific antagonistic antiFas antibody ZB4 on the rate of apoptosis. The mean ± SD percentage of early and late apoptosis of ZB4 was 0.44 ± 0.33% and 2.99 ± 1.14%, respectively. The 500 ng/ml dose of ZB4 was optimal for the inhibition of apoptosis. SH-SY5Y cells treated with serum from diabetes patients with neuropathy + ZB4 had a significantly lower percentage of cells in early apoptosis compared with cells treated with serum from the control subjects, cells treated with agonistic antiFas antibody (i.e. the positive control), and cells treated with serum from diabetes patients with neuropathy (P < 0.05 for all comparisons). SH-SY5Y cells treated with serum from diabetes patients with neuropathy + ZB4 had a significantly lower percentage of cells in late apoptosis compared with cells treated with agonistic antiFas antibody (i.e. the positive control) and cells treated with serum from diabetes patients with neuropathy (P < 0.05 for both comparisons).

Discussion

Diabetic neuropathy is a common microvascular complication of diabetes mellitus. Diabetes mellitus is known to induce apoptosis in various organs.¹³ There is published evidence supporting the role of neuronal cell death in the pathogenesis of diabetic neuropathy.¹⁴ Fas is widely expressed in the nervous system both in neurons and glial cells.¹⁵ There is an upregulation of Fas expression and increased production of sFas and sFasL proteins indicating a sFas/sFasL interaction in patients with diabetic neuropathy.¹⁴

In this present study, serum sFas concentrations showed a significant increase in type 2 diabetes patients with and without neuropathy compared with the healthy control subjects. Previous studies have shown that exposure of Schwann cells to hyperglycaemic conditions in vivo or in vitro can alter their viability, growth, activity and electrophysiological features.¹⁶ These present results were in agreement with another study in which diabetes patients with neuropathy showed significantly higher levels of sFas than control subjects or diabetes patients without neuropathy.¹⁴ Certain endogenous insulin-like growth factors can protect cells against apoptosis by upregulating survival factors.¹⁷ In particular, hyperglycaemia induces caspase 3 activation and morphological changes in Schwann cells consistent with apoptotic death.¹⁸ Overexpression of Bcl-xL, or insulin-like growth factor (IGF)-1, signalling via phosphatidylinositol 3-kinase protects Schwann cells from apoptosis in vitro.¹⁸ Thus, addition of IGF-1 may prevent or ameliorate apoptosis in neuronal cells exposed to serum from patients with diabetic neuropathy.¹⁸

Furthermore, it has been observed that sera from patients with diabetic neuropathy exerts cytotoxic effects on NIE-115 neuroblastoma cells by activation of the Fas-apoptotic mechanism.¹⁹ This suggests that increased serum sFas levels might appear before clinical symptoms in diabetic patients who later present with diabetic neuropathy.¹⁹ On the basis of these observations, it is possible to speculate that serum sFas levels could be used as early markers of diabetic neuropathy. The flow cytometry results demonstrated that SH-SY5Y cells (a human-derived cell line) were sensitive to the activation of Fas by the agonistic antiFas antibody in a dose-dependent manner. For further confirmation of Fas-mediated apoptosis, human serum containing high levels of Fas was added to the SH-SY5Y cells and the rate of apoptotic cells was assessed by flow cytometry. The results obtained showed that serum from diabetes patients with neuropathy with a high serum level of sFas caused a higher rate of early and late apoptosis compared with serum obtained from control subjects or from diabetes patients without neuropathy. These observations support the hypothesis that serum induces apoptosis in human neuroblastoma cells via a Fas-mediated pathway. Exposure of neuroblastoma cells to serum from diabetes patients with diabetic neuropathy was associated with decreased neurite outgrowth, enhancement of calcium influx and induction of apoptosis.¹⁹ Another study reported that sera from type 2 diabetes patients with neuropathy contained an autoimmune immunoglobulin that induced complement-independent, calcium-dependent apoptosis in neuronal cells.²⁰ The findings of the current study, as well as those of the earlier studies,^20,21 support the hypothesis that serum exerts an apoptotic effect on SH-SY5Y human neuroblastoma cells via a Fas-induced mechanism. The rate of apoptosis induced by the serum from diabetes patients with neuropathy was five times higher that that induced by the serum from healthy control subjects. Furthermore, the rate of apoptosis induced by the serum from diabetes patients with neuropathy was similar to that induced by 500 ng/ml of agonistic antiFas antibody CH-11 (i.e. the positive control), which further suggests that this was Fas-mediated apoptosis. None of the previous studies has investigated the inhibition of Fas-mediated apoptosis in a neuronal cell line. A clearer understanding of the mechanism of inhibition of apoptosis might lead to the development of novel preventive measures for diabetic neuropathy management. Dorsal root ganglia, sympathetic ganglion and Schwann cells are lost or damaged in patients suffering from diabetic neuropathy.^18–21 In vitro and in vivo studies have shown features of apoptosis in these cells.^18–21 It has been observed that high glucose levels inhibit neuron growth and induce apoptosis in cultured sympathetic primary neurons.²¹ These previous findings support the hypothesis that deficient direct insulin signalling may play an important role in the development of diabetic neuropathy.²² It has also been shown that ZB4 notably reduced the apoptosis induced by an agonistic antiFas antibody CH-11.¹⁵ Although preliminary blocking or inhibiting of the Fas/FasL pathway by antagonistic ZB4 antibody might be a promising target for therapeutic intervention in diabetic neuropathy, further animal and preclinical studies, with a larger number of patients, are needed to validate the preliminary data presented here. This could provide the basis for further research towards the identification of inhibitors of Fas-mediated apoptosis.

In conclusion, apoptosis of a human neuronal cell line induced by serum from diabetes patients with neuropathy appeared to be blocked or inhibited by antagonistic antiFas ZB4 antibody. These preliminary findings suggest that the degradation of the neurons in patients with diabetic neuropathy is induced by Fas, so targeting and inhibiting the Fas receptor might offer a therapeutic target for diabetic neuropathy.

Footnotes

Declaration of conflicting interest

None declared.

Funding

The authors thank the Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia for supporting the work (research group project number: RGP-VPP 181).

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Protection of neuronal cell death against diabetes-induced apoptosis by Fas blocker ZB4

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Patients and methods

Patient population

Routine biochemical analyses

ELISA determination of sFas and sFasL

Cell count and viability determination

The measurement of apoptosis by flow cytometry

Statistical analyses

Results

Discussion

Footnotes

Declaration of conflicting interest

Funding

References