Anti-oxidative capacity in patients with ataxia telangiectasia

Abstract

Highly reactive oxygen species (ROS) are involved in T-cell activation and in the defense against environmental pathogens. An imbalance of ROS generation and detoxifying scavenger enzymes could contribute to the increased susceptibility to cancer and infections in ataxia telangiectasia. We studied oxidative status, i.e. plasma total antioxidant capacity (TEAC), retinol, α-tocopherol, ubiquinol, and the number of activated T cells in 10 patients with ataxia telangiectasia (AT) compared to age-matched healthy controls. As expected, patients showed significantly increased levels of activated human leukocyte antigen-DR and CD45RO expressing T cells. TEAC levels as well as the exogenous antioxidants retinol and α-tocopherol were significantly reduced in patients. In addition, patients showed slightly reduced plasma levels of the endogenous ROS scavenger enzyme ubiquinol (Q10). Although no correlation between number of activated T-cells and antioxidant capacity could be demonstrated, an increase in ROS and a diminished reactive oxygen scavenger capacity may be involved in the disease process of patients with AT.

INTRODUCTION

Ataxia telangiectasia (AT) is an autosomal recessive genetic disorder which is characterized by neurodegeneration with progressive ataxia, variable immunodeficiency, premature ageing and an increased predisposition to cancer. A defect in the ‘cell surveillance network’, caused by the damaged ATM (ataxia telangiectasia mutated) gene product has been proposed to explain the chronic cell loss in the immune system and in the cerebellum in AT [1].

The dysregulation of cell death in AT is reflected in increased spontaneous apoptosis, high sensitivity to radiation and radiomimetic agents [2]. Thus an increase of the production of highly reactive oxygen species (ROS) with damage to cellular macromolecules may contribute to the pleiotropic picture of AT [3].

Excessive production of ROS, which might occur in the course of recurrent infection, is commonly referred to as oxidative stress [4]. Since the majority of patients with AT suffer from recurrent infections, they are exposed to oxidative stress to a high degree. Consequently, patients' lymphocytes are continuously in a state of activation. To prevent cells from oxidative damage, the generation and elimination of free radicals has to be in balance. If not, antioxidant defenses such as reactive-oxygen scavenger enzymes will be overwhelmed by any excess of ROS generation [5].

Reduced levels of naturally occurring antioxidant micronutrients have been reported in immunodeficiency diseases and cancer [6–10]. Clinical trials with dietary retinoic acid, vitamin E, or omega-3 polyunsaturated fatty acids showed immunomodulating effects, e.g. an influence on cytokine production, lymphocyte growth and function, and resulted in a prolonged survival of patients with immunodeficiencies [11–13]. This prompted us to study the antioxidant competence in patients with AT. Our analysis demonstrated significantly diminished plasma levels of total antioxidant capacity (TEAC), retinol and tocopherol, whereas levels of the endogenous scavenger protein ubiquinol were only slightly reduced.

PATIENTS AND METHODS

Patients

Ten patients with AT (median age 13.8 years, range 5–23 years, male:female 4:6) were studied. Clinical characteristics and lymphocyte subsets of patients are shown in Table 1.

Table 1.

Clinical characteristics and lymphocyte subsets of patients with ataxia telangiectasia (AT)

Controls: values expressed as median (bold) and range.

*CD4/CD8 ratio consists of the relative number of CD3/CD4 T-cells in proportion to the relative number of CD3/CD8 T-cells.

NA, not analysed.

Diagnosis was established in accordance with the WHO recommendations [14]. All patients showed increased levels of alpha-fetoprotein and atypical G2 arrest measured by flow cytometry (data not shown). Ethylenediaminetetraacetic acid (EDTA) blood was collected from patients and healthy age-matched controls. All subjects had the same dietary habits and none had taken vitamin supplements or any drugs affecting their lipid metabolism. In addition, none had ever suffered from malabsorption.

The study was approved by the local Human Committee of Ethics and informed consent was obtained from patients and parents before entering the study.

Lymphocyte phenotyping

Whole blood samples containing EDTA were stained with the following monoclonal antibodies directly labelled with fluorescein-isothiocyanate: CD3 (UCHT-1), CD8 (B9.11), CD19 (J4.119), CD45RO (UCHL-1) from Coulter-Immunotech (Marseille, France), CD4 (RPA-T4, from Pharmingen (CA, USA), and human leukocyte antigen (HLA)-DR PE (L-243, Beckton Dickinson, CA, USA). After staining, erythrocytes were lysed with FACS-lysing solution (Becton Dickinson), washed with PBS/NaAzid 0,1% and fixed with 1% paraformaldehyd. Samples were analysed with a flow cytometer (FACScan, Becton Dickinson) equipped with an air-cooled 488 nm argon-ion laser. On each sample 10000 events were collected and data analysis was performed with LYSIS II software (Becton Dickinson).

Measurement of plasma total antioxidant capacity

The Trolox®-equivalent total antioxidant capacity (TEAC) of plasma was measured according to Miller and colleagues [15]. Metmyoglobin was obtained by adding myoglobin stock solution (400 μm, horse heart muscle, Sigma Chemicals (Deisenhofen, Germany) to an equal volume of freshly prepared 740 μm potassium ferrycyanide. This solution was passed through a G15 Sephadex column, and the metmyoglobin fraction was collected. Visible spectra were analysed with a spectrophotometer (LKB, Pharmacia, Sweden), and absorbance was measured at 490, 560 and 580 nm. After subtracting for background absorbance at 700 nm, the purity of metmyoglobin was estimated by applying the Whitburn equations [16]. 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic-acid) (ABTS; 500 μm, 600 μl), and metmyoglobin (70 μm, 36 μl), 50 μl of Trolox®, and human plasma were mixed. The reaction was initiated by adding 334 μl of hydrogen peroxide (450 μm), and the absorbance was continually recorded at 734 nm by a printer until absorbance values increased significantly. A quantitative relationship exists between the absorbance at 734 nm and the antioxidant concentrations of the added sample or Trolox® standard. The sample procedure was followed with 0.125, 0.25, 0.5, 0.75 and 1.0 mm solutions of Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) replacing the plasma. A standard graph was derived from the amount of Trolox® at the infliction point of each graph. This graph was used to determine TEAC plasma levels.

Measurement of retinol, tocopherol and ubiquinol

Plasma samples were stored at −80°C in the dark and assayed within 6 months of sampling. Plasma levels of vitamin A, vitamin E and ubiquinol (Q10) were measured using high-performance liquid chromatography as described by Vuilleumier et al. [17] and Grossi et al. [18]. Briefly, 250 μl ethanol, 1 ml N-hexane and 50 μl plasma sample were mixed. Vitamins were extracted from plasma by density gradient centrifugation at 800 g, dried under nitrogen (N₂) and resuspended with 60 μl of a mixture of ethanol, dioxan and acetonitrile to equal proportions. 20 μl of the suspension obtained was high-performance liquid chromatography-separated through a LC-18-S column (5 μm, 250 × 4.6 mm; Supelco, Bellefonte, PA, USA). Fractions corresponding to retinol were identified by ultraviolet spectrophotometry at 325 nm after 0–5 min, tocopherol was measured at 292 nm after 5–8 min and ubiquinol was detected at 275 nm after 6 min.

Statistical analysis

Results are expressed in median and range. As our data were assumed to be not normally distributed, they were analysed using the Mann–Whitney U-test for nonparametric values. P < 0.05 was regarded as statistically significant.

RESULTS

Plasma total antioxidant capacity

To study the antioxidant status in our patient population, we analysed the TEAC, which reflects the ROS scavenger activity of a variety of proteins, such as superoxide dismutase, glutathione peroxidase, ubiquinol and antioxidant vitamins [15].

TEAC was significantly reduced in patients (Fig. 1): Controls, median 0.48 mmol/l, range 0.31–0.76 mmol/l; TEAC in AT, median 0.37 mmol/l, range 0.25–0.43 mmol/l (P < 0.05).

Fig. 1.

Evaluation of the total plasma antioxidant capacity (TEAC) in patients with ataxia telangiectasia (AT) (n = 10), compared with healthy, age-matched controls. Median values of TEAC (mmol/l) are shown. P < 0.05 was regarded as statistically significant.

Levels of retinol, tocopherol and ubiquinol

As the TEAC was reduced in AT, we analysed individual antioxidants. We chose vitamin A and E as important antioxidants with immunostimulatory effects [4,8,19]. Plasma levels of retinol and α-tocopherol were found to be significantly reduced in AT. (Figs 2 and 3). Retinol in controls, median 597 μg/l, range 344–735 μg/l; retinol in AT, median 435 μg/l, range 195–552 μg/l (P < 0.02). α-Tocopherol in controls, median 12.1 mg/l, range 7.6–15.5 mg/l; α-tocopherol in AT, median 8.1 mg/l, range 2.3–14.3 mg/l (P < 0.05).

Fig. 2.

Plasma levels of retinol (μg/l) in patients with ataxia telangiectasia (AT) (n = 10), compared with healthy, age-matched controls. Median values of retinol are shown. P < 0.05 was regarded as statistically significant.

Fig. 3.

Plasma levels of α-tocopherol (mg/l) in patients with ataxia telangiectasia (AT) (n = 10), compared with healthy, age-matched controls. Median values of α-tocopherol are shown. P < 0.05 was regarded as statistically significant.

Next we analysed ubiquinol (Q10), an important endogenous free radical scavenger enzyme. Levels were not significantly reduced in AT (Fig. 4): Controls, median 0.46 mg/l, range 0.32–0.72 mg/l; AT, median 0.35 mg/l, range 0.29–0.67 mg/l (NS).

Fig. 4.

Plasma levels of ubiquinol (mg/l) in patients with ataxia telangiectasia (AT) (n = 7), compared with healthy, age-matched controls. Median values of ubiquinol are shown. P < 0.05 was regarded as statistically significant.

Increased HLA-DR and CD45RO expression

Patients with AT displayed a significantly increased expression of the T-cell activation marker HLA-DR on CD3⁺ T lymphocytes (Fig. 5). In healthy controls, expression ranged from 4% to 14% (median 9%), whereas in AT expression ranged from 8% to 60% (median 33%) (P < 0.0005). Figure 6 illustrates that CD4/CD45RO expressing cells were also significantly increased in AT: Controls, median 41% (range 20% to 76%); AT, median 92% (range 78% to 98%) (P < 0.00001).

Fig. 5.

Relative percentage of human leukocyte antigen (HLA)-DR expression (%) on peripheral blood lymphocytes of patients with ataxia telangiectasia (AT) (n = 9), compared with healthy, age-matched controls. CD3/HLA-DR expression was determined by using flow cytometry. Median values of CD3/HLA-DR cell subsets are shown. P < 0.05 was regarded as statistically significant.

Fig. 6.

Relative percentage of CD45RO expression (%) on peripheral blood lymphocytes of patients with ataxia telangiectasia (AT) (n = 9), compared with healthy, age-matched controls. CD4/CD45RO expression was determined by using flow cytometry. Median values of CD4/CD45RO cell subsets are shown. P < 0.05 was regarded as statistically significant.

Finally, we analysed the correlation between age, the level of T-cell activation and the antioxidant status of patients. However, no significant associations could be demonstrated between degree of T-cell activation, age and the levels of TEAC, retinol, tocopherol, and ubiquinol, respectively. Age of patients and an increase in numbers of HLA-DR expressing cells seem to be linked. A clear correlation could not be drawn since two older patients with a milder form of disease showed low levels of HLA-DR expressing cells.

DISCUSSION

A number of studies have demonstrated an excess of ROS generation in patients with immunodeficiencies [5,20,21]. ROS are known to be involved in T-cell activation, e.g. in the initiation of antigen-specific immune responses [4,5]. In accordance with prior studies, our patients showed an increase in T-cell activation, which is reflected by an enhanced expression of the activation markers HLA-DR and CD45RO [22,23].

Several mechanisms have been invoked to explain the continuous state of cell activation in patients with immunodeficiencies. For example, chronic infections of the upper and lower respiratory tract result in a high inflammatory turnover. Alternatively, increased levels of activated cells may compensate for the deficient T-cell dependent B-cell activation seen in AT [22,24]. Whether the increased activation of lymphocytes in AT is the consequence or the cause of an excess of ROS is still unclear. It has been hypothesized that antioxidant endogenous reactive-oxygen scavenger enzymes may be overwhelmed by an excess of ROS [5]. Monitoring the antioxidant status in AT, we have demonstrated decreased ability to counteract oxidative stress. The relative contribution of each antioxidant to TEAC values may not define its importance in vivo, since each may be effective at specific sites and perform special functions [15]. Therefore, we analysed the deficiencies in our patients in more detail: There were significantly decreased levels of retinol and α-tocopherol, whereas ubiquinol levels were not significantly reduced. The clinical relevance of slightly reduced levels of ubiquinol in patients with AT is questionable. However, the reduced levels of antioxidants in patients with AT are not due to reduced food absorption, which is the case for patients with cystic fibrosis or other diseases with maldigestion.

Apart from its antioxidant capacities, vitamin E has important immunostimulatory and antiageing properties [25]. High doses of α-tocopherol stimulate the activity of cytotoxic cells, natural killer (NK) cells, phagocytosis of macrophages, and mitogen responsiveness [19]. Furthermore, it is believed that downregulation of prostaglandin E₂ (PGE₂) by vitamin E serves to increase the production of interleukin (IL)-2 and interferon (IFN)-γ. Accordingly, vitamin E deficiency might contribute to the high susceptibility to neoplastic disorders in patients with immunodeficiency [3]. Therefore it is of particular interest that several studies on hereditary ataxias demonstrate a close association of primary or secondary vitamin E deficiency with severe cerebellar symptoms [26,27], as patients with AT suffer from progressive neurodegeneration and an increased frequency of malignancies [3,24]. In contrast to the results published by Battisti et al. [28], our AT patients displayed significantly reduced levels of α-tocopherol, which might help to explain not only the development of cerebellar ataxia and premature ageing, but also the characteristic sensitivity to ionizing radiation, DNA-strand breaks, and the high rate of spontaneous apoptosis [3,22,24]. Indeed, autopsies of the central nervous system in AT patients have shown lipid peroxidation products in neurones, Schwann cells and dorsal ganglion cells [29]. Vitamin E protects cells from programmed cell death (apoptosis) by increasing Bcl-2 protein levels, but also independently of Bcl-2, possibly by regulation of the protein kinase C (PKC) pathway [30,31]. Another inhibitor of apoptosis via PKC is vitamin A [32]. Like vitamin E, retinoids have antitumour effects, costimulate T-lymphocyte activation and enhance the production of IL-2 and IFN-γ [33,34]. Therefore it is significant that retinol plasma levels were reduced in our patients. Vitamin A-deficient children resemble our patients in showing an increased susceptibility to infection together with a lower immunoglobulin G production, as well as higher proportions of CD45RO T-cells [19,35].

Interestingly, patients with AT displayed lower values of antioxidant capacity than patients with variable immunodeficiency (manuscript in preparation). It can be hypothesized that the ATM gene product might be an upstream sensor that is activated by oxidative damage, initiating pathways responsible for protecting cells from such damages [3,36]. The absence of functional ATM in AT cells would then result in a state of increased sensitivity to oxidative stress [36], and, in addition, might be responsible for the characteristic neurodegeneration, immunodeficiency, premature ageing, development of bronchiectasis and an extremely high incidence of malignancies in adolescence [3,27].

In patients with HIV infection, high doses of vitamin E along with a nutritionally adequate diet have been shown to enhance in vitro and in vivo antibody production, phagocytosis, lymphoproliferative responses, and resistance to infectious diseases [8,19]. Our data suggest that treatment with vitamin E and A would be worthy of trial in the therapy of patients with AT.