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Intracellular oxidative activity and respiratory burst of leukocytes isolated from multiple sclerosis patients
Neurochemistry International 48 (2006) 87–92 www.elsevier.com/locate/neuint
Intracellular oxidative activity and respiratory burst of leukocytes isolated from multiple sclerosis patients G. Ferretti a,*, T. Bacchetti a,1, F. DiLudovico b,2, B. Viti c,3, V.A. Angeleri b,2, M. Danni b,2, L. Provinciali b,2 a
Istituto di Biochimica, Facolta` di Medicina e Chirurgia, Universita` Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy b Clinica Neurologica, Facolta` di Medicina e Chirurgia, Universita` Politecnica delle Marche, Italy c Istituto di Riabilitazione, Villa Adria Santo Stefano, Ancona, Italy Received 24 February 2005; accepted 22 September 2005 Available online 2 November 2005
Abstract Oxidative damage induced by free radicals and reactive oxygen species (ROS) have been suggested to play an important role in the development of autoimmune diseases such as multiple sclerosis (MS) disease and it has been hypothesised that oxidative injury could mediate demyelination and axonal injury in MS subjects. In our study, we compared intracellular oxidative activity and the respiratory burst activity in MS patients (n = 20) and healthy controls (n = 15) using leukocytes as cellular model. At this purpose, intracellular ROS levels were evaluated by fluorometric assay using the 20 -70 -dichlorodihydrofluorescin diacetate probe (H2DCFDA) in untreated or in leukocytes stimulated with phorbol-12-myristate-13-acetate (PMA). Our results demonstrate that the intracellular spontaneous ROS production in leukocytes from MS patients was higher with respect to cells from control subjects ( p < 0.001). PMA addition induced a higher formation of ROS both in leukocytes from MS patients and controls ( p < 0.001). The PMA-induced production of ROS was significantly higher in leukocytes from MS with respect to controls ( p < 0.001). Significant positive correlations were established between intracellular spontaneous or PMA-induced production of ROS in leukocytes isolated from MS patients and the clinical parameters used to evaluate disease disability such as expanded disability status scale (EDSS), brain lesions evaluated by MRI and visual evoked potential (VEP) ( p < 0.001). In conclusion, our results demonstrate higher levels of intracellular ROS in untreated or in PMA-treated leukocytes isolated from MS patients with respect to healthy subjects confirming the role of oxidative stress in multiple sclerosis. # 2005 Elsevier Ltd. All rights reserved. Keywords: 20 -70 -dichlorodihydrofluorescin diacetate (H2DCFDA); Oxidative damage; Leukocytes; Multiple sclerosis; Respiratory burst
1. Introduction Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune attack to myelin antigens (Steinman, 1996). There are increasing evidences that support the role of the oxidative stress in the inflammatory processes and in the pathogenesis of multiple sclerosis (LeVine, 1992; Smith et al., 1999; Gilgun-Sherki et al., 2004). High levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in * Corresponding author. Tel.: +39 071 2204968; fax: +39 071 2204398. E-mail address: [email protected] (G. Ferretti). 1 Tel.: + 39 071 2204968; fax: + 39 071 2204398. 2 Tel.: + 39 071 5964501; fax: + 39 071 887262. 3 Tel.: + 39 071 218951; fax: + 39 071 887150. 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.09.005
MS patients have been demonstrated either directly postmortem in brain (Langemann et al., 1992) or indirectly through the measurements of the products of lipid peroxidation in the CSF (Naidoo and Knapp, 1992; Calabrese et al., 1994, 1998) and plasma (Calabrese et al., 1998; Naidoo and Knapp, 1992; Besler et al., 2002). Moreover, low levels of antioxidant intracellular enzymes and vitamins and a decreased plasma antioxidant capacity have been observed in MS patients with respect to healthy subjects (Jensen et al., 1980; Calabrese et al., 1994; Besler et al., 2002; Besler and Comoglu, 2003). Several studies have demonstrated that monocytes/macrophages play an important role in demyelinating processes and axonal injury in multiple sclerosis (LeVine, 1992; Lassmann, 2003). Such cells are consistently found in close contact with degenerating axons and their presence, probably derived from circulating blood monocytes, at the site of the active MS
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plaques supports their involvement in the disease. It has been hypothesised that the cytotoxic effects of activated monocytes/ macrophages on tissues could be related to the production of ROS such as superoxide and hydrogen peroxides (Klebanoff, 1980; Halliwell, 1982; Fantone and Ward, 1982). In fact, it has been demonstrated that ROS and RNS lead to axonal injury and demyelination (Lassmann, 2003; Smith et al., 1999). Previous studies have shown that the formation of ROS by stimulated peripheral blood mononuclear cells, the ‘‘respiratory burst’’, from MS patients is significantly higher with respect to cells from healthy subjects (Hammann and Hopf, 1986; Fisher et al., 1988; Vladimirova et al., 1999). It has been suggested that blood monocytes in MS patients may be ‘‘primed’’ to produce cytotoxic free radicals when exposed to inflammatory stimuli and therefore, they have an increased ability to mediate host myelin destruction. The aim of the study is to compare the intracellular ROS levels and the respiratory burst activity in leukocytes isolated MS patients (n = 20) and healthy controls (n = 15), by fluorometric assay using the 20 -70 -dichlorodihydrofluorescin diacetate (H2DCFDA) probe and phorbol-12-myristate-13acetate (PMA), as triggering agent. Moreover, to investigate the relationship between biochemical data and disease disability, the correlations between intracellular spontaneous or PMAinduced production of ROS in leukocytes isolated from MS patients and clinical parameters such as expanded disability status scale (EDSS) values, brain lesions and visual evoked potentials (VEPs) were studied. 2. Methods 2.1. Materials Phosphate buffer solution (PBS), dextran, butylated hydroxytoluene (BHT), phorbol-12-myristate-13-acetate and Trypan-blue (0.4%) were purchased from Sigma Chemical Company (St. Louis, MO); 20 -70 -dichlorodihydrofluorescin diacetate was obtained from Molecular Probes Inc. (Eugene, OR).
2.2. Subjects Fifteen healthy (9 females and 6 males; 35.5 15.3 years old) and 20 MS patients (12 females and 8 males; 39.3 10.2 years old) were included in the study. Controls and MS patients were not taking drugs, antioxidants or other medication, which can affect oxidative metabolism. No patients were treated with immunomodulators, immunosoppressors or cortisteroids and they had not clinic relapse within 2 months prior to the sampling. Smokers were excluded from the study. During the time of the study, no controls or patients had clinical or laboratory signs of an acute infection. Lifestyle and dietary habits were not significantly different in MS patients with respect to controls, as demonstrated by a questionnaire aimed to evaluate physical activity (sedentary/exercise life style) and dietary habits of both groups. Informed consent was obtained from each participating subject. The study was approved by the local ethics committee and it was carried out in accordance with the principles of the Declaration of Helsinki as revised in 2000.
2.3. Clinical characteristics of MS patients Patients included in the study were clinically defined following Poser standards as relapsing–remitting disability (type RR) (Poser et al., 1983). The basic characteristics of age, expended disability status scale (EDSS) and disease duration of MS patients are summarised in Table 1.
Table 1 Clinical characteristics of healthy subjects and patients affected by multiple sclerosis (MS patients)
Age (years) Disease duration (years) EDSS Brain lesions (number)a VEPs latency (ms) b
Healthy subjects (n = 15)
MS patients (n = 20)
35.5 2.9 – – – –
39.2 2.05 3.02 0.35 2.21 0.23 16.26 3.10 123.03 2.93
Mean S.E.M. are shown. EDSS: expended disability status scale; VEPs: visual evoked potentials. a Data available in 15 MS patients. b Data available in 16 MS patients, the VEPs were performed from both right and left eyes and the worst score was used in the analysis. Magnetic resonance imaging (MRI) and the analysis of the brain lesions were performed at the time of diagnosis to evaluate brain tissue characteristics of patients. Magnetic resonance imaging (MRI) was performed in 15 MS patients by 1 T Siemens scan using head coil before and after a single dose of gadolinium (Gd). The following sequences were used: sagittal T1 SE, 5 mm thickness TR 570, TE 14; axial DP-T2, 5 mm thickness, TR 3400, TE 14–85; coronal FLAIR, 5 mm thickness TR 8800, TE 105; axial T1, 5 mm thickness TR 600, TE 14. Single dose of gadolinium was administered after sagittal T1 SE. Enhancing and non-enhancing lesions were counted. According with previous study an arbitrary scoring system weighted for lesion size was used to estimate total lesion load (Filippi et al., 1995). The analysis showed that all patients had a pattern suggestive of demyelinating disease. No Gd-enhancing lesions were observed in MS patients including in our study. The mean value of total brain lesions evaluated by MRI is reported in Table 1. Furthermore, to evaluate functional disability of patients, visual evoked potentials were studied in 16 MS subjects as reported by Filippi et al. (1995). VEPs were performed from both right and left eyes and the worst score was used in the analysis for each patient. The mean VEPs latency value is reported in Table 1.
2.4. Isolation of leukocytes Five millilitres of blood samples were obtained from 15 controls and 20 MS patients by venipuncture during the periodical inspections after overnight fasting and were collected into sterile tubes containing 2 ml dextran solution (5%). Dextran was added in order to increase the blood viscosity. After 90 min at room temperature, the erythrocytes were sedimented, whereas leukocytes and thrombocytes stay in the plasma fraction. Blood supernatant were carefully transferred into separate tubes and the leukocytes were sedimented in a brief centrifugation step. The plasma supernatant containing >90% of the thrombocytes was discarded and the leukocyte pellet was resuspended in 10 mM phosphate buffer saline, pH 7.4, supplemented with 250 mM glucose (PBSG) (Boyum, 1968). Cell viability was assessed by the Trypan-blue exclusion dye test and was found to be greater than 90%. The levels of leukocytes were not significantly different in controls and in MS patients (0.589 0.061 cells 106/100 ml blood and 0.606 0.058 cells 106/100 ml blood, respectively). A significant positive correlation was observed between the cell number and the protein concentration of leukocytes isolated from healthy and MS patients, evaluated by Bradford assay (Bradford, 1976) (r = 0.92, n = 35, p < 0.001) (Fig. 1), therefore the protein concentration, in the following experiments, was used to quantified cells.
2.5. Measurement of spontaneous reactive oxygen species in leukocytes The intracellular levels of reactive oxygen species in leukocytes were measured by fluorometric assay using the probe 20 -70 -dichlorodihydrofluorescin diacetate (Robinson et al., 1988; Brubacher and Bols, 2001; Myhre et al., 2003). H2DCFDA is a non-polar compound that readily diffuses into the cells where it
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Fig. 1. Correlation between number of cells and protein concentration in leukocytes isolated from blood of healthy ( ) and MS patients (^) (r = 0.92, n = 35, p < 0.001). is enzymatically deacetylated by intracellular esterases to the polar nonfluorescent derivate and thereby trapped within the cells. In the presence of ROS, the probe is oxidized to the fluorescent product DCF. Therefore, DCF fluorescence levels reflect the intracellular concentration of ROS (Brubacher and Bols, 2001). Leukocytes (20 mg proteins) isolated from blood of controls and MS patients resupended in 2 ml PBS-G, pH 7.4, were incubated with 20 mM H2DCFDA at 37 8C for 140 min with continuous shaking. To minimize the H2DCFDA photo-oxidation samples were kept in the dark. DCF fluorescence was measured in a Perkin-Elmer LS50B spectrofluorometer (490 nm excitation and 526 nm emission wavelengths). Results of DCF fluorescence intensity were expressed as arbitrary units (a.u.).
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Fig. 2. Measurement of basal intracellular oxidative activity. Time dependent increase of DCF fluorescence intensity in leukocytes isolated from healthy (^) and MS subjects (&). *p < 0.001 vs. healthy subjects.
subjects showed that DCF fluorescence intensity was significantly higher in leukocytes from MS subjects (Fig. 2). The differences were significant even after 15 min of incubation ( p < 0.001). These results suggest that the intracellular oxidative activity of leukocytes isolated from patients affected by multiple sclerosis is higher with respect to healthy subjects.
2.6. Measurement of respiratory burst activity of leukocytes
3.2. Respiratory burst activity in healthy and MS subjects
The respiratory burst activity of leukocytes was evaluated by fluorometric assay using phorbol-12-myristate-13-acetate as a triggering agent (Hu et al., 1999; Brubacher and Bols, 2001). Leukocytes (20 mg protein) isolated from blood of controls and MS patients, previously incubated with 20 mM H2DCFDA at 37 8C for 15 min, were added 33 ml of stock solution 0.3 mM PMA (final concentration 5 mM) or 0.2% [v/v] dimethyl sulfoxide (DMSO) as a carrier control. DCF fluorescence in leukocytes incubated for different times (0– 120 min) in the absence or in the presence of PMA, was monitored as described above.
The respiratory burst activity of leukocytes isolated from healthy and MS subjects was evaluated using phorbol-12myristate-13-acetate as a triggering agent. Treatment of DCFH2-loaded leukocytes with 20 mM PMA led to an increase in oxidation of the probe both in leukocytes from controls and MS patients (Fig. 3). The increase in DCF fluorescence intensity after PMA stimulation was higher in leukocytes isolated from MS patients with respect to control subjects. The percentage increase in leukocytes reached a maximum after 30 min of incubation with PMA (the percentage increase with respect to unstimulated leukocytes were 100 16% and 165 20% in healthy and MS subjects, respectively, p < 0.001) (Fig. 4). These results suggest that leukocytes in MS patients produce more ROS when exposed to PMA stimulation with respect to healthy subjects.
2.7. Statistics All experiments were performed in triplicate. The results obtained were shown as mean S.E.M. The differences between the results obtained from MS patients and controls were evaluated by Mann–Whitney U-test. Values were considered significant at p less than 0.05. Linear regression analysis was used to calculate correlation coefficients (r) (Microcal Origin 5.0, OriginLab, Northampton, MA).
3. Results 3.1. Intracellular oxidative activity in healthy and MS subjects The spontaneous production of ROS in leukocytes was monitored over a period of 140 min (Fig. 2). A time dependent increase in the levels of DCF fluorescence intensity was observed both in leukocytes isolated from healthy and MS subjects (Fig. 2). After 15 min of incubation, the levels of DCF fluorescence intensity were significantly higher with respect to the initial values both in cells from healthy and from MS subjects (28.7 7.3 and 76.1 9.4 a.u. in controls and in MS patients, respectively, p < 0.001 versus initial values) (Fig. 2). The comparison between healthy and MS
Fig. 3. Measurement of respiratory burst activity. Time dependent increase of DCF fluorescence intensity in leukocytes isolated from healthy (^) and MS subjects (&) after stimulation with PMA. *p < 0.001 vs. healthy subjects.
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Fig. 4. Percentage increase of DCF fluorescence intensity in leukocytes isolated from healthy (&) and MS subjects ( ) after stimulation with PMA with respect to cells not treated with PMA. *p < 0.001 vs. healthy subjects.
3.3. Correlations To investigate the relationship between oxidative damage and severity of multiple sclerosis diseases, we studied the correlations between the DCF fluorescence intensity levels (after 15 min of incubation) and the percentage increase of DCF fluorescence after PMA addition (after 30 min of incubation) and the clinical parameters used to evaluate disease disability. As reported in Table 2, significant positive correlations were established between in DCF fluorescence and EDSS values, the brain lesions evaluated by MRI and VEPs latencies. Significant positive correlations were also observed between the increase of DCF fluorescence after PMA addition and EDSS values, the brain lesions and VEPs latencies (Table 2). These results suggest a relation between intracellular ROS levels and respiratory burst activity and severity of multiple sclerosis. 4. Discussion In the present study, intracellular ROS levels were measured by fluorescent dye technique using H2DCFDA as probe and leukocytes as cellular model. Leukocytes isolated from MS patients showed an increased spontaneous and PMA-induced DCF fluorescence with respect to controls. These results suggested that MS patients are characterized by elevated intracellular levels of oxidative products and higher ‘‘respiratory burst’’ activity with respect to healthy subjects. The study of DCF fluorescence has been demonstrated to be useful in evaluating the intracellular oxidative stress in different cellular types in normal (LeBel et al., 1992; Brubacher and Bols, 2001; Myhre et al., 2003) or pathological conditions (Hassan et al., 1988; Noritake et al., 1992; Tepel et al., 2000). H2DCFDA diffuses passively through the cellular membrane. Intracellular esterase activity results in the formation of
DCFH2, a non-fluorescent compound. DCFH2 becomes trapped within the cells and susceptible to oxidation, generating a fluorescent product 20 -70 -dichlorofluorescin (DCF). Accumulation of DCF indicates the production of redox-active substances in the cell. It is not clear, however, which oxidative species are responsible for oxidation of DCFH2 to DCF in cells. The probe has been initially described as an assay for measurement of H2O2 in the presence of peroxidase, however recent studies have reported that DCFH2 is sensitive towards oxidation of other reactive compounds such as O2 and reactive nitrogen species (RNS) in particularly ONOO (Myhre et al., 2003). Multiple sclerosis is an autoimmune disease characterized by inflammatory as well as degenerative phenomena. An increased oxidative stress in MS patients with respect to healthy subjects has been previously demonstrated both directly postmortem by measurement of ROS concentration in brain (Langemann et al., 1992) or by the evaluation of the levels of lipid peroxidation products in the CSF (Naidoo and Knapp, 1992; Calabrese et al., 1994, 1998) and in the plasma (Calabrese et al., 1998; Naidoo and Knapp, 1992; Besler et al., 2002). However, it has been suggested that the determination of intracellular ROS may reflect the cellular oxidative stress more directly with respect to measurements of plasmatic lipid peroxidation products (Tepel et al., 2000). The higher intracellular oxidative stress observed in MS patients with respect to control subjects could result from either increased production of ROS or reduced levels of antioxidants. Several studies have demonstrated low levels of antioxidant vitamins and enzymes and a decreased plasma antioxidant capacity in MS patients with respect to healthy subjects (Calabrese et al., 1994; Besler et al., 2002; Besler and Comoglu, 2003). Moreover, a decreased activity of glutathione peroxidase and/or reductase in leukocytes of MS patients has been observed (Jensen et al., 1980). Our results demonstrate a higher increase in DCF fluorescence intensity after treatment with phorbol-12-myristate-13acetate in leukocytes isolated from MS patients with respect to controls. PMA activates NADPH oxidase by enhancing protein kinase C and thus stimulates production of ROS (Hu et al., 1999). These results demonstrate that patients affected by multiple sclerosis showed a higher ‘‘respiratory burst’’ activity with respect to healthy subjects and therefore they could be ‘‘primed’’ to produce cytotoxic free radicals than normal when exposed to inflammatory stimuli. These results confirm previous data obtained from stimulated peripheral blood mononuclear cells using different techniques (Hammann and Hopf, 1986; Fisher et al., 1988; Vladimirova et al., 1999). Leukocytes, used in our experimental conditions, are a mixed population of cells such
Table 2 Correlation coefficients (r) evaluated between intracellular ROS levels (DCF fluorescence intensity levels) or respiratory burst activity (percentage increase of DCF fluorescence intensity after PMA addition) in leukocytes and clinical parameters used to evaluate MS severity in patients
Intracellular ROS levels Respiratory burst activity
EDSS
Brain lesions
VEPs latency
r = 0.78, n = 20, p < 0.001 r = 0.68, n = 20, p < 0.001
r = 0.95, n = 15, p < 0.001 r = 0.78, n = 15, p < 0.001
r = 0.79, n = 16, p < 0.001 r = 0.71, n = 16, p < 0.001
EDSS: expended disability status scale; VEPs: visual evoked potentials.
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mononuclear and polymorphonuclear phagocytes, lymphocytes and natural killer cells that were reported to produce molecules of respiratory burst. However, it has been demonstrated that the increase in burst activity in MS subjects with respect to healthy subjects is manly derived from mononuclear cells (Hammann and Hopf, 1986). The production of ROS is necessary for cell activity and proliferation and phagocytic cells produce reactive oxygen species to destroy microbial organisms. However, an increased ROS formation may be harmful to cells and cause functional disabilities. In fact, free radicals, particularly the reactive oxygen or nitrogen intermediates, are involved in inflammatory processes, exacerbating inflammation and exerting tissue damage (Winrow et al., 1993; Cardoso et al., 2004; Celsi et al., 2004). Moreover, it has been suggested that oxygen and nitrogen free radicals generated by macrophages have been implicated, as mediators of demyelination and axonal injury, in multiple sclerosis (Smith et al., 1999; Lassmann, 2003). Generation of ROS by cells leads to the activation of protein tyrosine kinases followed by stimulation of several signalling systems, including mitogen-activated protein kinases, nuclear transcription factors, caspases and intracellular calcium concentration (Chakraborti and Chakraborti, 1998), which regulate the expression of many genes involved in multiple sclerosis such as tumor necrosis factor, iNOS, ICAM-1 and VCAM-1 (Barnes and Karin, 1997). In addition, redox reactions are involved in the activity of matrix metalloproteinases (MMP), which are important to T cell trafficking into the central nervous system (Merrill and Murphy, 1997). The presence of many monocytes/macrophages probably derived from blood monocytes in close contact with degenerating axons and at the sites of acute MS plaques has been described (Fantone and Ward, 1982) and confirms their role in the inflammation and in the demyelinating process in MS. Moreover, it has been shown that treatment with antioxidant such as a-lipoic acid, induces a marked decline in the number of T cells and macrophage/monocytes in spinal cord of animal model of multiple sclerosis with amelioration of inflammation demyelination and axonal loss (Marracci et al., 2002). The correlation between markers of lipid peroxidation or free radical damage and disease severity or relapse time has been investigated previously, contrasting results have been reported by Naidoo and Knapp (1992) and Greco et al. (2004). In previous studies, a correlation between biochemical markers of oxidative damage and degree of disability in MS has been demonstrated in humans and animal models (Ferretti et al., 2005; Greco et al., 1999; Liu et al., 2003). A relationship between biochemical parameters of oxidative damage and clinical parameters has been confirmed in the present study; in fact, significant positive correlations have been established between intracellular ROS levels or respiratory burst activity and the clinical parameters used to evaluate disease disability. EDSS values and brain lesions evaluated by MRI are considered objective measures of multiple sclerosis activity. MRI disease activity parameters have been previously used to monitor several controlled trials of new immunomodulatory therapies in MS (Paty and Li, 1993; Johnson et al., 1995), and
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Barkhof (1999) demonstrated that MRI parameters are predictive of the course of disability in MS patients. Furthermore, visual evoked potentials are used to evaluate indirectly the amount of demyelination and axonal loss in the CNS of patients affected by MS by exploring the functional consequences of the disease process (Filippi et al., 1995). It has to be stressed that the alteration of intracellular oxidative activity of leucocytes and the correlations between biochemical and clinical parameters have been observed in relapsing–remitting MS patients with relative low EDSS and short disease duration. These results are in agreement with previous studies that demonstrated that axonal loss may occur early in the onset of the disease process (Ferguson et al., 1997) and occurs not only in chronic lesions but also in acute lesions and in normal appearing white matter (Fu et al., 1998). Further studies are needed to confirm the relationship between oxidative stress and disease severity and to verify whether the evaluation of markers of oxidative damage in circulating cells in the earlier stage of the disease could represent a predictive factor of MS disease development. In conclusion, our results demonstrate higher levels of intracellular ROS in untreated or in PMA-treated leukocytes isolated from MS patients with respect to healthy subjects. The biochemical parameters are related with MS disease severity. These results confirm the role of oxidative stress in multiple sclerosis and suggest that the higher production of cytotoxic free radicals observed in leukocytes isolated from MS patients, when exposed to inflammatory stimuli, could induce tissue damage and represent an important mechanism of cell injury in autoimmune diseases such as multiple sclerosis. References Barnes, P.-J., Karin, M., 1997. Nuclear factor-{kappa}B—a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336, 1066– 1071. Barkhof, F., 1999. MRI in multiple sclerosis: correlation with expanded disability status scale (EDSS). Mult. Scler. 5, 283–286. Besler, H.-T., Comoglu, S., Okcu, Z., 2002. Serum levels of antioxidant vitamins and lipid peroxidation in multiple sclerosis. Nutr. Neurosci. 5, 215–220. Besler, H.-T., Comoglu, S., 2003. Lipoprotein oxidation, plasma total antioxidant capacity and homocysteine level in patients with multiple sclerosis. Nutr. Neurosci. 6, 189–196. Boyum, A., 1968. Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and ficoll as erythrocyte aggregating agents. Scand. J. Clin. Lab. Invest. Suppl. 97, 31–50. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brubacher, J.-L., Bols, N.-C., 2001. Chemically de-acetylated 20 -70 -dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes. J. Immunol. Methods 251, 81–91. Calabrese, V., Raffaele, R., Cosentino, E., Rizza, V., 1994. Changes in cerebrospinal fluid levels of malondialdehyde and glutathione reductase activity in multiple sclerosis. Int. J. Clin. Pharmacol. Res. 14, 119–123. Calabrese, V., Bella, R., Testa, D., Spadaro, F., Scrofani, A., Rizza, V., Pennisi, G., 1998. Increased cerebrospinal fluid and plasma levels of ultraweak chemiluminescence are associated with changes in the thiol pool and lipidsoluble fluorescence in multiple sclerosis: the pathogenic role of oxidative stress. Drugs Exp. Clin. Res. 24, 125–131.
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Klebanoff, S.-J., 1980. Oxygen metabolism and the toxic properties of phagocytes. Ann. Intern. Med. 93, 480–489. Langemann, H., Kabiersch, A., Newcombe, J., 1992. Measurement of lowmolecular-weight antioxidants, uric acid, tyrosine and tryptophan in plaques and white matter from patients with multiple sclerosis. Eur. Neurol. 32, 248–252. Lassmann, H., 2003. Axonal injury in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 74, 695–697. LeBel, C.-P., Ischiropoulos, H., Bondy, S.-C., 1992. Evaluation of the probe 20 ,70 -dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5, 227–231. LeVine, S.-M., 1992. The role of reactive oxygen species in the pathogenesis of multiple sclerosis. Med. Hypotheses 39, 271–274. Liu, Y., Zhu, B., Wang, X., Luo, L., Li, P., Paty, D.-W., Cynader, M.-S., 2003. Bilirubin as a potent antioxidant suppresses experimental autoimmune encephalomyelitis: implications for the role of oxidative stress in the development of multiple sclerosis. J. Neuroimmunol. 139, 27–35. Marracci, G.-H., Jones, R.-E., McKeon, G.-P., Bourdette, D.-N., 2002. Alpha lipoic acid inhibits T cell migration into the spinal cord and suppresses and treats experimental autoimmune encephalomyelitis. J. Neuroimmunol. 1131, 104–114. Merrill, J., Murphy, S., 1997. Inflammatory events at the blood brain barrier: regulation of adhesion molecules, cytokines, and chemokines by reactive nitrogen and oxygen species. Brain Behav. Immun. 11, 245– 263. Myhre, O., Andersen, J.-M., Aarnes, H., Fonnum, F., 2003. Evaluation of the probes 20 ,70 -dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem. Pharmacol. 65, 1575–1582. Naidoo, R., Knapp, M.-L., 1992. Studies of lipid peroxidation products in cerebrospinal fluid and serum in multiple sclerosis and other conditions. Clin. Chem. 38, 2449–2454. Noritake, M., Katsura, Y., Shinomiya, N., Kanatani, M., Uwabe, Y., Nagata, N., Tsuru, S., 1992. Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Dissociation between polymorphonuclear leucocytes and monocytes. Clin. Exp. Immunol. 88, 269– 274. Paty, D.-W., Li, D.-K., 1993. Interferon beta-1b is effective in relapsing– remitting multiple sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial. UBC MS/MRI Study Group and the IFNB Multiple Sclerosis Study Group. Neurology 43, 662– 667. Poser, C.-M., Paty, D.-W., Scheinberg, L., McDonald, W.-I., Davis, F.-A., Ebers, G.-C., Johnson, K.-P., Sibley, W.-A., Silberberg, D.-H., Tourtellotte, W.-W., 1983. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann. Neurol. 13, 227–231. Robinson, J.-P., Bruner, L.-H., Bassoe, C.-F., Hudson, J.-L., Ward, P.-A., Phan, S.-H., 1988. Measurement of intracellular fluorescence of human monocytes relative to oxidative metabolism. J. Leukoc. Biol. 43, 304–310. Smith, K.-J., Kapoor, R., Felts, P.-A., 1999. Demyelination: the role of reactive oxygen and nitrogen species. Brain Pathol. 9, 69–92. Steinman, L., 1996. Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell 85, 299–302. Tepel, M., Echelmeyer, M., Orie, N.-N., Zidek, W., 2000. Increased intracellular reactive oxygen species in patients with end-stage renal failure: effect of hemodialysis. Kidney Int. 58, 867–872. Vladimirova, O., Lu, F.-M., Shawver, L., Kalman, B., 1999. The activation of protein kinase C induces higher production of reactive oxygen species by mononuclear cells in patients with multiple sclerosis than in controls. Inflamm. Res. 48, 412–416. Winrow, R., Winyard, P.-G., Morris, C.-J., Blake, D.-R., 1993. Free radicals in inflammation: second messengers and mediators of tissue destruction. Br. Med. Bull. 49, 506–522.
Intracellular oxidative activity and respiratory burst of leukocytes isolated from multiple sclerosis patients G. Ferretti a,*, T. Bacchetti a,1, F. DiLudovico b,2, B. Viti c,3, V.A. Angeleri b,2, M. Danni b,2, L. Provinciali b,2 a
Istituto di Biochimica, Facolta` di Medicina e Chirurgia, Universita` Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy b Clinica Neurologica, Facolta` di Medicina e Chirurgia, Universita` Politecnica delle Marche, Italy c Istituto di Riabilitazione, Villa Adria Santo Stefano, Ancona, Italy Received 24 February 2005; accepted 22 September 2005 Available online 2 November 2005
Abstract Oxidative damage induced by free radicals and reactive oxygen species (ROS) have been suggested to play an important role in the development of autoimmune diseases such as multiple sclerosis (MS) disease and it has been hypothesised that oxidative injury could mediate demyelination and axonal injury in MS subjects. In our study, we compared intracellular oxidative activity and the respiratory burst activity in MS patients (n = 20) and healthy controls (n = 15) using leukocytes as cellular model. At this purpose, intracellular ROS levels were evaluated by fluorometric assay using the 20 -70 -dichlorodihydrofluorescin diacetate probe (H2DCFDA) in untreated or in leukocytes stimulated with phorbol-12-myristate-13-acetate (PMA). Our results demonstrate that the intracellular spontaneous ROS production in leukocytes from MS patients was higher with respect to cells from control subjects ( p < 0.001). PMA addition induced a higher formation of ROS both in leukocytes from MS patients and controls ( p < 0.001). The PMA-induced production of ROS was significantly higher in leukocytes from MS with respect to controls ( p < 0.001). Significant positive correlations were established between intracellular spontaneous or PMA-induced production of ROS in leukocytes isolated from MS patients and the clinical parameters used to evaluate disease disability such as expanded disability status scale (EDSS), brain lesions evaluated by MRI and visual evoked potential (VEP) ( p < 0.001). In conclusion, our results demonstrate higher levels of intracellular ROS in untreated or in PMA-treated leukocytes isolated from MS patients with respect to healthy subjects confirming the role of oxidative stress in multiple sclerosis. # 2005 Elsevier Ltd. All rights reserved. Keywords: 20 -70 -dichlorodihydrofluorescin diacetate (H2DCFDA); Oxidative damage; Leukocytes; Multiple sclerosis; Respiratory burst
1. Introduction Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune attack to myelin antigens (Steinman, 1996). There are increasing evidences that support the role of the oxidative stress in the inflammatory processes and in the pathogenesis of multiple sclerosis (LeVine, 1992; Smith et al., 1999; Gilgun-Sherki et al., 2004). High levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in * Corresponding author. Tel.: +39 071 2204968; fax: +39 071 2204398. E-mail address: [email protected] (G. Ferretti). 1 Tel.: + 39 071 2204968; fax: + 39 071 2204398. 2 Tel.: + 39 071 5964501; fax: + 39 071 887262. 3 Tel.: + 39 071 218951; fax: + 39 071 887150. 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.09.005
MS patients have been demonstrated either directly postmortem in brain (Langemann et al., 1992) or indirectly through the measurements of the products of lipid peroxidation in the CSF (Naidoo and Knapp, 1992; Calabrese et al., 1994, 1998) and plasma (Calabrese et al., 1998; Naidoo and Knapp, 1992; Besler et al., 2002). Moreover, low levels of antioxidant intracellular enzymes and vitamins and a decreased plasma antioxidant capacity have been observed in MS patients with respect to healthy subjects (Jensen et al., 1980; Calabrese et al., 1994; Besler et al., 2002; Besler and Comoglu, 2003). Several studies have demonstrated that monocytes/macrophages play an important role in demyelinating processes and axonal injury in multiple sclerosis (LeVine, 1992; Lassmann, 2003). Such cells are consistently found in close contact with degenerating axons and their presence, probably derived from circulating blood monocytes, at the site of the active MS
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plaques supports their involvement in the disease. It has been hypothesised that the cytotoxic effects of activated monocytes/ macrophages on tissues could be related to the production of ROS such as superoxide and hydrogen peroxides (Klebanoff, 1980; Halliwell, 1982; Fantone and Ward, 1982). In fact, it has been demonstrated that ROS and RNS lead to axonal injury and demyelination (Lassmann, 2003; Smith et al., 1999). Previous studies have shown that the formation of ROS by stimulated peripheral blood mononuclear cells, the ‘‘respiratory burst’’, from MS patients is significantly higher with respect to cells from healthy subjects (Hammann and Hopf, 1986; Fisher et al., 1988; Vladimirova et al., 1999). It has been suggested that blood monocytes in MS patients may be ‘‘primed’’ to produce cytotoxic free radicals when exposed to inflammatory stimuli and therefore, they have an increased ability to mediate host myelin destruction. The aim of the study is to compare the intracellular ROS levels and the respiratory burst activity in leukocytes isolated MS patients (n = 20) and healthy controls (n = 15), by fluorometric assay using the 20 -70 -dichlorodihydrofluorescin diacetate (H2DCFDA) probe and phorbol-12-myristate-13acetate (PMA), as triggering agent. Moreover, to investigate the relationship between biochemical data and disease disability, the correlations between intracellular spontaneous or PMAinduced production of ROS in leukocytes isolated from MS patients and clinical parameters such as expanded disability status scale (EDSS) values, brain lesions and visual evoked potentials (VEPs) were studied. 2. Methods 2.1. Materials Phosphate buffer solution (PBS), dextran, butylated hydroxytoluene (BHT), phorbol-12-myristate-13-acetate and Trypan-blue (0.4%) were purchased from Sigma Chemical Company (St. Louis, MO); 20 -70 -dichlorodihydrofluorescin diacetate was obtained from Molecular Probes Inc. (Eugene, OR).
2.2. Subjects Fifteen healthy (9 females and 6 males; 35.5 15.3 years old) and 20 MS patients (12 females and 8 males; 39.3 10.2 years old) were included in the study. Controls and MS patients were not taking drugs, antioxidants or other medication, which can affect oxidative metabolism. No patients were treated with immunomodulators, immunosoppressors or cortisteroids and they had not clinic relapse within 2 months prior to the sampling. Smokers were excluded from the study. During the time of the study, no controls or patients had clinical or laboratory signs of an acute infection. Lifestyle and dietary habits were not significantly different in MS patients with respect to controls, as demonstrated by a questionnaire aimed to evaluate physical activity (sedentary/exercise life style) and dietary habits of both groups. Informed consent was obtained from each participating subject. The study was approved by the local ethics committee and it was carried out in accordance with the principles of the Declaration of Helsinki as revised in 2000.
2.3. Clinical characteristics of MS patients Patients included in the study were clinically defined following Poser standards as relapsing–remitting disability (type RR) (Poser et al., 1983). The basic characteristics of age, expended disability status scale (EDSS) and disease duration of MS patients are summarised in Table 1.
Table 1 Clinical characteristics of healthy subjects and patients affected by multiple sclerosis (MS patients)
Age (years) Disease duration (years) EDSS Brain lesions (number)a VEPs latency (ms) b
Healthy subjects (n = 15)
MS patients (n = 20)
35.5 2.9 – – – –
39.2 2.05 3.02 0.35 2.21 0.23 16.26 3.10 123.03 2.93
Mean S.E.M. are shown. EDSS: expended disability status scale; VEPs: visual evoked potentials. a Data available in 15 MS patients. b Data available in 16 MS patients, the VEPs were performed from both right and left eyes and the worst score was used in the analysis. Magnetic resonance imaging (MRI) and the analysis of the brain lesions were performed at the time of diagnosis to evaluate brain tissue characteristics of patients. Magnetic resonance imaging (MRI) was performed in 15 MS patients by 1 T Siemens scan using head coil before and after a single dose of gadolinium (Gd). The following sequences were used: sagittal T1 SE, 5 mm thickness TR 570, TE 14; axial DP-T2, 5 mm thickness, TR 3400, TE 14–85; coronal FLAIR, 5 mm thickness TR 8800, TE 105; axial T1, 5 mm thickness TR 600, TE 14. Single dose of gadolinium was administered after sagittal T1 SE. Enhancing and non-enhancing lesions were counted. According with previous study an arbitrary scoring system weighted for lesion size was used to estimate total lesion load (Filippi et al., 1995). The analysis showed that all patients had a pattern suggestive of demyelinating disease. No Gd-enhancing lesions were observed in MS patients including in our study. The mean value of total brain lesions evaluated by MRI is reported in Table 1. Furthermore, to evaluate functional disability of patients, visual evoked potentials were studied in 16 MS subjects as reported by Filippi et al. (1995). VEPs were performed from both right and left eyes and the worst score was used in the analysis for each patient. The mean VEPs latency value is reported in Table 1.
2.4. Isolation of leukocytes Five millilitres of blood samples were obtained from 15 controls and 20 MS patients by venipuncture during the periodical inspections after overnight fasting and were collected into sterile tubes containing 2 ml dextran solution (5%). Dextran was added in order to increase the blood viscosity. After 90 min at room temperature, the erythrocytes were sedimented, whereas leukocytes and thrombocytes stay in the plasma fraction. Blood supernatant were carefully transferred into separate tubes and the leukocytes were sedimented in a brief centrifugation step. The plasma supernatant containing >90% of the thrombocytes was discarded and the leukocyte pellet was resuspended in 10 mM phosphate buffer saline, pH 7.4, supplemented with 250 mM glucose (PBSG) (Boyum, 1968). Cell viability was assessed by the Trypan-blue exclusion dye test and was found to be greater than 90%. The levels of leukocytes were not significantly different in controls and in MS patients (0.589 0.061 cells 106/100 ml blood and 0.606 0.058 cells 106/100 ml blood, respectively). A significant positive correlation was observed between the cell number and the protein concentration of leukocytes isolated from healthy and MS patients, evaluated by Bradford assay (Bradford, 1976) (r = 0.92, n = 35, p < 0.001) (Fig. 1), therefore the protein concentration, in the following experiments, was used to quantified cells.
2.5. Measurement of spontaneous reactive oxygen species in leukocytes The intracellular levels of reactive oxygen species in leukocytes were measured by fluorometric assay using the probe 20 -70 -dichlorodihydrofluorescin diacetate (Robinson et al., 1988; Brubacher and Bols, 2001; Myhre et al., 2003). H2DCFDA is a non-polar compound that readily diffuses into the cells where it
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Fig. 1. Correlation between number of cells and protein concentration in leukocytes isolated from blood of healthy ( ) and MS patients (^) (r = 0.92, n = 35, p < 0.001). is enzymatically deacetylated by intracellular esterases to the polar nonfluorescent derivate and thereby trapped within the cells. In the presence of ROS, the probe is oxidized to the fluorescent product DCF. Therefore, DCF fluorescence levels reflect the intracellular concentration of ROS (Brubacher and Bols, 2001). Leukocytes (20 mg proteins) isolated from blood of controls and MS patients resupended in 2 ml PBS-G, pH 7.4, were incubated with 20 mM H2DCFDA at 37 8C for 140 min with continuous shaking. To minimize the H2DCFDA photo-oxidation samples were kept in the dark. DCF fluorescence was measured in a Perkin-Elmer LS50B spectrofluorometer (490 nm excitation and 526 nm emission wavelengths). Results of DCF fluorescence intensity were expressed as arbitrary units (a.u.).
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Fig. 2. Measurement of basal intracellular oxidative activity. Time dependent increase of DCF fluorescence intensity in leukocytes isolated from healthy (^) and MS subjects (&). *p < 0.001 vs. healthy subjects.
subjects showed that DCF fluorescence intensity was significantly higher in leukocytes from MS subjects (Fig. 2). The differences were significant even after 15 min of incubation ( p < 0.001). These results suggest that the intracellular oxidative activity of leukocytes isolated from patients affected by multiple sclerosis is higher with respect to healthy subjects.
2.6. Measurement of respiratory burst activity of leukocytes
3.2. Respiratory burst activity in healthy and MS subjects
The respiratory burst activity of leukocytes was evaluated by fluorometric assay using phorbol-12-myristate-13-acetate as a triggering agent (Hu et al., 1999; Brubacher and Bols, 2001). Leukocytes (20 mg protein) isolated from blood of controls and MS patients, previously incubated with 20 mM H2DCFDA at 37 8C for 15 min, were added 33 ml of stock solution 0.3 mM PMA (final concentration 5 mM) or 0.2% [v/v] dimethyl sulfoxide (DMSO) as a carrier control. DCF fluorescence in leukocytes incubated for different times (0– 120 min) in the absence or in the presence of PMA, was monitored as described above.
The respiratory burst activity of leukocytes isolated from healthy and MS subjects was evaluated using phorbol-12myristate-13-acetate as a triggering agent. Treatment of DCFH2-loaded leukocytes with 20 mM PMA led to an increase in oxidation of the probe both in leukocytes from controls and MS patients (Fig. 3). The increase in DCF fluorescence intensity after PMA stimulation was higher in leukocytes isolated from MS patients with respect to control subjects. The percentage increase in leukocytes reached a maximum after 30 min of incubation with PMA (the percentage increase with respect to unstimulated leukocytes were 100 16% and 165 20% in healthy and MS subjects, respectively, p < 0.001) (Fig. 4). These results suggest that leukocytes in MS patients produce more ROS when exposed to PMA stimulation with respect to healthy subjects.
2.7. Statistics All experiments were performed in triplicate. The results obtained were shown as mean S.E.M. The differences between the results obtained from MS patients and controls were evaluated by Mann–Whitney U-test. Values were considered significant at p less than 0.05. Linear regression analysis was used to calculate correlation coefficients (r) (Microcal Origin 5.0, OriginLab, Northampton, MA).
3. Results 3.1. Intracellular oxidative activity in healthy and MS subjects The spontaneous production of ROS in leukocytes was monitored over a period of 140 min (Fig. 2). A time dependent increase in the levels of DCF fluorescence intensity was observed both in leukocytes isolated from healthy and MS subjects (Fig. 2). After 15 min of incubation, the levels of DCF fluorescence intensity were significantly higher with respect to the initial values both in cells from healthy and from MS subjects (28.7 7.3 and 76.1 9.4 a.u. in controls and in MS patients, respectively, p < 0.001 versus initial values) (Fig. 2). The comparison between healthy and MS
Fig. 3. Measurement of respiratory burst activity. Time dependent increase of DCF fluorescence intensity in leukocytes isolated from healthy (^) and MS subjects (&) after stimulation with PMA. *p < 0.001 vs. healthy subjects.
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Fig. 4. Percentage increase of DCF fluorescence intensity in leukocytes isolated from healthy (&) and MS subjects ( ) after stimulation with PMA with respect to cells not treated with PMA. *p < 0.001 vs. healthy subjects.
3.3. Correlations To investigate the relationship between oxidative damage and severity of multiple sclerosis diseases, we studied the correlations between the DCF fluorescence intensity levels (after 15 min of incubation) and the percentage increase of DCF fluorescence after PMA addition (after 30 min of incubation) and the clinical parameters used to evaluate disease disability. As reported in Table 2, significant positive correlations were established between in DCF fluorescence and EDSS values, the brain lesions evaluated by MRI and VEPs latencies. Significant positive correlations were also observed between the increase of DCF fluorescence after PMA addition and EDSS values, the brain lesions and VEPs latencies (Table 2). These results suggest a relation between intracellular ROS levels and respiratory burst activity and severity of multiple sclerosis. 4. Discussion In the present study, intracellular ROS levels were measured by fluorescent dye technique using H2DCFDA as probe and leukocytes as cellular model. Leukocytes isolated from MS patients showed an increased spontaneous and PMA-induced DCF fluorescence with respect to controls. These results suggested that MS patients are characterized by elevated intracellular levels of oxidative products and higher ‘‘respiratory burst’’ activity with respect to healthy subjects. The study of DCF fluorescence has been demonstrated to be useful in evaluating the intracellular oxidative stress in different cellular types in normal (LeBel et al., 1992; Brubacher and Bols, 2001; Myhre et al., 2003) or pathological conditions (Hassan et al., 1988; Noritake et al., 1992; Tepel et al., 2000). H2DCFDA diffuses passively through the cellular membrane. Intracellular esterase activity results in the formation of
DCFH2, a non-fluorescent compound. DCFH2 becomes trapped within the cells and susceptible to oxidation, generating a fluorescent product 20 -70 -dichlorofluorescin (DCF). Accumulation of DCF indicates the production of redox-active substances in the cell. It is not clear, however, which oxidative species are responsible for oxidation of DCFH2 to DCF in cells. The probe has been initially described as an assay for measurement of H2O2 in the presence of peroxidase, however recent studies have reported that DCFH2 is sensitive towards oxidation of other reactive compounds such as O2 and reactive nitrogen species (RNS) in particularly ONOO (Myhre et al., 2003). Multiple sclerosis is an autoimmune disease characterized by inflammatory as well as degenerative phenomena. An increased oxidative stress in MS patients with respect to healthy subjects has been previously demonstrated both directly postmortem by measurement of ROS concentration in brain (Langemann et al., 1992) or by the evaluation of the levels of lipid peroxidation products in the CSF (Naidoo and Knapp, 1992; Calabrese et al., 1994, 1998) and in the plasma (Calabrese et al., 1998; Naidoo and Knapp, 1992; Besler et al., 2002). However, it has been suggested that the determination of intracellular ROS may reflect the cellular oxidative stress more directly with respect to measurements of plasmatic lipid peroxidation products (Tepel et al., 2000). The higher intracellular oxidative stress observed in MS patients with respect to control subjects could result from either increased production of ROS or reduced levels of antioxidants. Several studies have demonstrated low levels of antioxidant vitamins and enzymes and a decreased plasma antioxidant capacity in MS patients with respect to healthy subjects (Calabrese et al., 1994; Besler et al., 2002; Besler and Comoglu, 2003). Moreover, a decreased activity of glutathione peroxidase and/or reductase in leukocytes of MS patients has been observed (Jensen et al., 1980). Our results demonstrate a higher increase in DCF fluorescence intensity after treatment with phorbol-12-myristate-13acetate in leukocytes isolated from MS patients with respect to controls. PMA activates NADPH oxidase by enhancing protein kinase C and thus stimulates production of ROS (Hu et al., 1999). These results demonstrate that patients affected by multiple sclerosis showed a higher ‘‘respiratory burst’’ activity with respect to healthy subjects and therefore they could be ‘‘primed’’ to produce cytotoxic free radicals than normal when exposed to inflammatory stimuli. These results confirm previous data obtained from stimulated peripheral blood mononuclear cells using different techniques (Hammann and Hopf, 1986; Fisher et al., 1988; Vladimirova et al., 1999). Leukocytes, used in our experimental conditions, are a mixed population of cells such
Table 2 Correlation coefficients (r) evaluated between intracellular ROS levels (DCF fluorescence intensity levels) or respiratory burst activity (percentage increase of DCF fluorescence intensity after PMA addition) in leukocytes and clinical parameters used to evaluate MS severity in patients
Intracellular ROS levels Respiratory burst activity
EDSS
Brain lesions
VEPs latency
r = 0.78, n = 20, p < 0.001 r = 0.68, n = 20, p < 0.001
r = 0.95, n = 15, p < 0.001 r = 0.78, n = 15, p < 0.001
r = 0.79, n = 16, p < 0.001 r = 0.71, n = 16, p < 0.001
EDSS: expended disability status scale; VEPs: visual evoked potentials.
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mononuclear and polymorphonuclear phagocytes, lymphocytes and natural killer cells that were reported to produce molecules of respiratory burst. However, it has been demonstrated that the increase in burst activity in MS subjects with respect to healthy subjects is manly derived from mononuclear cells (Hammann and Hopf, 1986). The production of ROS is necessary for cell activity and proliferation and phagocytic cells produce reactive oxygen species to destroy microbial organisms. However, an increased ROS formation may be harmful to cells and cause functional disabilities. In fact, free radicals, particularly the reactive oxygen or nitrogen intermediates, are involved in inflammatory processes, exacerbating inflammation and exerting tissue damage (Winrow et al., 1993; Cardoso et al., 2004; Celsi et al., 2004). Moreover, it has been suggested that oxygen and nitrogen free radicals generated by macrophages have been implicated, as mediators of demyelination and axonal injury, in multiple sclerosis (Smith et al., 1999; Lassmann, 2003). Generation of ROS by cells leads to the activation of protein tyrosine kinases followed by stimulation of several signalling systems, including mitogen-activated protein kinases, nuclear transcription factors, caspases and intracellular calcium concentration (Chakraborti and Chakraborti, 1998), which regulate the expression of many genes involved in multiple sclerosis such as tumor necrosis factor, iNOS, ICAM-1 and VCAM-1 (Barnes and Karin, 1997). In addition, redox reactions are involved in the activity of matrix metalloproteinases (MMP), which are important to T cell trafficking into the central nervous system (Merrill and Murphy, 1997). The presence of many monocytes/macrophages probably derived from blood monocytes in close contact with degenerating axons and at the sites of acute MS plaques has been described (Fantone and Ward, 1982) and confirms their role in the inflammation and in the demyelinating process in MS. Moreover, it has been shown that treatment with antioxidant such as a-lipoic acid, induces a marked decline in the number of T cells and macrophage/monocytes in spinal cord of animal model of multiple sclerosis with amelioration of inflammation demyelination and axonal loss (Marracci et al., 2002). The correlation between markers of lipid peroxidation or free radical damage and disease severity or relapse time has been investigated previously, contrasting results have been reported by Naidoo and Knapp (1992) and Greco et al. (2004). In previous studies, a correlation between biochemical markers of oxidative damage and degree of disability in MS has been demonstrated in humans and animal models (Ferretti et al., 2005; Greco et al., 1999; Liu et al., 2003). A relationship between biochemical parameters of oxidative damage and clinical parameters has been confirmed in the present study; in fact, significant positive correlations have been established between intracellular ROS levels or respiratory burst activity and the clinical parameters used to evaluate disease disability. EDSS values and brain lesions evaluated by MRI are considered objective measures of multiple sclerosis activity. MRI disease activity parameters have been previously used to monitor several controlled trials of new immunomodulatory therapies in MS (Paty and Li, 1993; Johnson et al., 1995), and
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Barkhof (1999) demonstrated that MRI parameters are predictive of the course of disability in MS patients. Furthermore, visual evoked potentials are used to evaluate indirectly the amount of demyelination and axonal loss in the CNS of patients affected by MS by exploring the functional consequences of the disease process (Filippi et al., 1995). It has to be stressed that the alteration of intracellular oxidative activity of leucocytes and the correlations between biochemical and clinical parameters have been observed in relapsing–remitting MS patients with relative low EDSS and short disease duration. These results are in agreement with previous studies that demonstrated that axonal loss may occur early in the onset of the disease process (Ferguson et al., 1997) and occurs not only in chronic lesions but also in acute lesions and in normal appearing white matter (Fu et al., 1998). Further studies are needed to confirm the relationship between oxidative stress and disease severity and to verify whether the evaluation of markers of oxidative damage in circulating cells in the earlier stage of the disease could represent a predictive factor of MS disease development. In conclusion, our results demonstrate higher levels of intracellular ROS in untreated or in PMA-treated leukocytes isolated from MS patients with respect to healthy subjects. The biochemical parameters are related with MS disease severity. These results confirm the role of oxidative stress in multiple sclerosis and suggest that the higher production of cytotoxic free radicals observed in leukocytes isolated from MS patients, when exposed to inflammatory stimuli, could induce tissue damage and represent an important mechanism of cell injury in autoimmune diseases such as multiple sclerosis. References Barnes, P.-J., Karin, M., 1997. Nuclear factor-{kappa}B—a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336, 1066– 1071. Barkhof, F., 1999. MRI in multiple sclerosis: correlation with expanded disability status scale (EDSS). Mult. Scler. 5, 283–286. Besler, H.-T., Comoglu, S., Okcu, Z., 2002. Serum levels of antioxidant vitamins and lipid peroxidation in multiple sclerosis. Nutr. Neurosci. 5, 215–220. Besler, H.-T., Comoglu, S., 2003. Lipoprotein oxidation, plasma total antioxidant capacity and homocysteine level in patients with multiple sclerosis. Nutr. Neurosci. 6, 189–196. Boyum, A., 1968. Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and ficoll as erythrocyte aggregating agents. Scand. J. Clin. Lab. Invest. Suppl. 97, 31–50. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brubacher, J.-L., Bols, N.-C., 2001. Chemically de-acetylated 20 -70 -dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes. J. Immunol. Methods 251, 81–91. Calabrese, V., Raffaele, R., Cosentino, E., Rizza, V., 1994. Changes in cerebrospinal fluid levels of malondialdehyde and glutathione reductase activity in multiple sclerosis. Int. J. Clin. Pharmacol. Res. 14, 119–123. Calabrese, V., Bella, R., Testa, D., Spadaro, F., Scrofani, A., Rizza, V., Pennisi, G., 1998. Increased cerebrospinal fluid and plasma levels of ultraweak chemiluminescence are associated with changes in the thiol pool and lipidsoluble fluorescence in multiple sclerosis: the pathogenic role of oxidative stress. Drugs Exp. Clin. Res. 24, 125–131.
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