Effect of L929 cell-conditioned medium on antioxidant capacity in RAW264.7 cells

Effect of L929 cell-conditioned medium on antioxidant capacity in RAW264.7 cells

Pang, Z J

Abstract: Previously, we found that macrophage colony-stimulating factor (M-CSF) could reduce tert-butyl hydroperoxide (tbOOH)-induced oxidative injury in monocytes/macrophages. In order to find the mechanism by which M-CSF achieves this, we investigate the effect of M-CSF on the antioxidant system in murine macrophage RAW264.7 cells, using L929 cell-conditioned medium (L929-CM) as the source of M-CSF. The results show that L929-CM increased selenium-dependent glutathione peroxidase, non-selenium-dependent glutathione peroxidase, and catalase activities; and it increased reduced glutathione (GSH) but decreased lipoperoxide content in RAW264.7 cells. L929-CM-treated cells maintained their antioxidant capacity (including antioxidant enzyme activities and GSH content) when challenged with tbOOH. We conclude that M-CSF from L929-CM reduces oxidative injury in RAW264.7 cells by increasing antioxidant enzyme activity and improving redox status. Key words: Antioxidants. Cell line, RAW264.7. Enzymes. Macrophage colony-stimulating factor. Oxidative stress.

Introduction

Macrophage colony-stimulating factor (M-CSF) is a macrophage-specific growth factor secreted by monocytes, macrophages, endothelial cells, smooth-muscle cells and fibroblasts. It is highly expressed in the L929 mouse cell line, and L929 cell-conditioned medium (L929-CM) has been used as a source of M-CSF in several studies.1-3 M-CSF plays an important role in the treatment of tumours,4 infection,5,6 and atherosclerosis;7 and we have found that M-CSF can protect monocytes/macrophages from oxidative injury.8,9

Cells possess a wide range of interlinked antioxidant defence mechanisms to protect against damage by reactive oxygen species (ROS). Amongst these mechanisms, the antioxidant enzymes catalase (CAT; EC 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1), glutathione peroxidase (GPx; EC 1.11.1.9) and glutathione S-transferase (GST; EC 2.5.1.18) play an important role in scavenging ROS produced in cells.

Here, to shed further light on the mechanism by which M-CSF reduces oxidative injury in monocytes/macrophages, we investigate the effect of L929-CM on antioxidant enzyme activities in murine macrophage RAW264.7 cells.

Materials and methods

Materials

RPMI-1640, tert-butyl hydroperoxide (tbOOH), reduced glutathione (GSH), pyrogallol, PMSF (phenylmethylsulphonyl fluoride), aprotinin, DTNB (5,5′-[dithiobis {2-nitrobenzoic acid}]), cumene hydroperoxide (cumene-OOH), oxidised glutathione (GSSG), flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Si-ji-qing (Hangzhou, China). All other chemicals used were of analytical grade.

Preparation of L929 cell-conditioned medium

L929 cells were obtained from the Department of Immunology, First Military Medical University, and cultured in RPMI-1640 (supplemented with 10% FBS) in 5% CO2 in humidified air at 37 deg C. The medium was replaced with FBS-free RPMI-1640 medium, five to seven days before the conditioned medium was collected. The L929-CM collected was centrifuged at 1000 xg for 10 min, and the supernatant sterilised by filtration through a 0.22 (mu)m filter.

The proliferative activity assay of L929-CM was performed according to a method reported elsewhere.10 Briefly, mouse bone-marrow cells were cultured in a semi-solid system consisting of 30% FBS, 0.45 mmol/L GSH, 0.3% agar, and 20% L929– CM, with a 10^sup 5^ cells/mL seeding density of mouse bone-marrow cells. After seven days, the number of granulocyte/macrophage colonies (CFU-GM) was calculated, with a mass containing more than 50 cells regarded as a colony. The proliferative activity of the L929-CM prepared in this study was approximately 500 CFU-GM/mL.

Cell lysis

RAW264.7 cells treated with L929-CM (25% [v/v] in the culture medium for 24 h) were harvested by centrifugation at 1000 xg for 10 min, and the cell pellet washed with phosphate-buffered saline (PBS). Lysis buffer (800 gL) (50 mmol/L Tris-HCl [pH8.0]; 150 mmol/L NaCl; 0.02% NaN^sub 3^; 100 (mu)g/mL PMSF; 1 (mu)g/mL aprotinin; 1% Triton X-100) was added to each tube. After incubation at 4 deg C for 30 min, the lysates were centrifuged at 12 000 xg for 30 min at 4 deg C. Supernatant was collected for antioxidant enzyme activity assay, and the protein content determined using Lowry’s method,11 using trypsin as a standard.

Superoxide dismutase activity assay

Total SOD activity was determined using the pyrogallol autoxidation method, with the absorbance (A) value used as a measure of total SOD activity.12 The rate of pyrogallol autoxidation was normalised as 0.07 A/min, and a 50%70 reduction of pyrogallol autoxidation by SOD defined as one unit. Results were expressed as Wing protein.

Glutathione peroxidase activity assay

GPx activity in the cell lysate was determined using a DTNB method. 13 Briefly, 1.5 mmollL H2O2 was used as substrate when selenium-dependent GPx (SeGPx) activity was measured and 9 mmol/L cumene-OOH used when total GPx activity was measured. The difference between SeGPx and total GPx activities represented the non-selenium-dependent GPx (nonSeGPx) activity. One unit was defined as a 1 (mu)mol reduction of the substrate GSH by GPx. All results were expressed as U/mg protein.

Reduced glutathione assay

In the following experiments, RAW264.7 cells were divided into four groups: control; L929-CM treated; control, plus tbOOH; and L929-CM treated plus tbOOH.

Cells in L929-CM-treated groups were pretreated with 25%70 L929-CM (v/v, in the culture medium) for 24 h, using FBS-free RPMI-1640 medium instead of L929-CM as the control. Cells in the two groups receiving tbOOH treatment were incubated with 10^sup -4^ mol/L tbOOH for a further 12 h. Cells were lysed as described above.

GSH content was measured according to a method described elsewhere.14 Briefly, cell lysate (0.1 mL), 0. 1 mol/L phosphate buffer (4.4 mL [pH 8.01), and DTNB solution (0.5 mL) (40 mg DTNB and 1 g sodium citrite in 100 mL H2O) were added to each tube, using H2O instead of DTNB solution as a control. After mixing thoroughly, A^sub 412^ was determined within five minutes. GSH content was determined by reference to a GSH standard curve.

Catalase activity analysis

CAT activity was measured according to a method described elsewhere.15 Briefly, following preparation of the cell lysates, CAT activity was measured within 30 min. To each tube was added 0.015 mmol/L H2O2 (2.8 mL) and sample (0.2 mL), using an equal volume of H2O instead of H2O2 as the control. A^sub 240^ was measured immediately (A^sup 0^) and after 30 sec (A^sup 30^). CAT activity was calculated as follows: CAT (K/g) = (1150/b) lg (A^sup 0^/A^sup 30^), where ‘b’ represents the concentration of protein.

Glutathione reductase activity assay

Glutathione reductase (GR) activity was measured according to the method of Beutler.16 Sample (0.1 mL) was mixed with 0.1 mol/L phosphate buffer (2 mL [pH 7.2]), 80 mmol/L EDTA (0.05 mL), 50 mmol/L GSSG (0.1 mL), and 0.25 mmol/L FAD (0.1 mL). After 30-min incubation at 37 deg C, 4 mmol/L NADPH (0.1 mL) was added to each tube and A34 measured (A’). After a further five-minute incubation at 37 deg C, the reaction was terminated by immersing the tubes in ice-cold water, and A^sub 340^ measured again (A^sup 2^). GR activity was calculated as: GR (U/g) = 78.78 x (A^sup -1^-A^sup 2^)fb, where ‘b’ represents the concentration of protein. One unit was defined as the amount of GR consumed per (mu)mol NADPH within one minute at 37 deg C, where the protein concentration was 1 g/L.

Lipoperoxide content analysis

Lipoperoxide content of cells was measured according to the method described by Fletcher et al.17 Briefly, cell lysate (0.5 mL) was mixed with chlorform/methanol (2:1 [v/v]; 6 mL), incubated at 45 deg C for 1 min, and H2O (1 volume) was added. After mixing thoroughly, it was centrifuged at 3000 xg for 5 min. The bottom layer was collected, added to methanol (0.3 mL), and mixed. Fluorimetric analysis was performed using an RF-540 fluorescence spectrometer at excitation and emission wavelengths of 375 and 450 nm, respectively.

Statistical analysis

Data were expressed as mean (+/- standard deviation [SD]). Statistical differences in the data were analysed by Student’s t-test. P

Results

Effect on glutathione peroxidase and superoxide dismutase activities

RAW264.7 cells were incubated in culture medium containing 25% (v/v) L929-CM (FBS-free RPMI- 1640 medium used as the control) for 24 h prior to cells lysis. GPx activity analysis showed that L929-CM treatment increased SeGPx (P

Effect on reduced glutathione and catalase activity Following 24-h exposure to L929-CM, levels of GSH (Figure 1) and CAT (Figure 2) in RAW264.7 cells were increased (P

Effect on glutathione reductase activity

Before incubation with tbOOH, no significant difference (P >0.1) in GR activity was seen between the RAW264.7 cell group exposed to L929-CM, and the control group (Figure 3). However, after subsequent exposure to 10^sup -4^ mol/L tbOOH for 12 h, GR activity in the control group was reduced considerably, whilst activity in the L929-CM group was significantly higher than in this non-treated group (P

Effect on lipoperoxide content

Following 24-h exposure to L929-CM, lipoperoxide content in the RAW264.7 cells was reduced (P

Discussion

M-CSF is a homodimeric glycoprotein that has several isoforms, each with a distinct molecular size and derived from a different transcript. Complimentary DNA (cDNA) encoding distinct M-CSF types have been cloned.18-22 M-CSF is secreted in an autocrine or paracrine manner and exerts its effect through stimulation of a specific receptor. M-CSF is vital to monocyte/macrophage differentiation, proliferation, activation and survival. Previously, we showed that M-CSF helped mouse peritoneal macrophages survive oxidative injury caused by the oxidant tbOOH.8

A wide range of eukaryocyte antioxidant enzymes, including CAT, SOD and GPx, play an important role in scavenging ROS produced in cells. The seleniumdependent type of GPx, SeGPx, acts against HZO, and lipid peroxides by catalysing the reaction of GSH with these compounds, and is considered to be the most important antioxidant enzyme. The functions of non– SeGPx are similar to those of SeGPx, with the exception that non-SeGPx cannot catalyse the reduction of H2O2.

Three forms of SOD – copper-zinc SOD (CuZnSOD), manganese SOD (MnSOD) and extracellular SOD (EC-SOD) – exist in cells and tissues.23 Evidence suggests that both CuZnSOD and MnSOD are important in cell defence against oxygen toxicity; however, only MnSOD is induced in response to stimuli such as tumour necrosis factor (TNF). Furthermore, reports in recent years have indicated that MnSOD is a new type of tumour suppressor gene.24-26 CAT can catalyse the conversion of HZO, into H20 and O2; GR can reduce GSSG to GSH (which can be used again by GPx); and all these antioxidant enzymes cooperate in defending cells against oxidative injury.

In order to clarify the mechanisms whereby M-CSF reduces tbOOH-induced oxidative injury to monocytes/macrophages, we investigated the effect of L929-CM on the antioxidant system in RAW264.7 cells. Exposure to L929-CM resulted both in increased GSH levels and the ability to maintain high levels when the cells were challenged with tbOOH. In addition, L929-CM increased total SOD, SeGPx, non– SeGPx and CAT activities.

After exposure to tbOOH, RAW264.7 cells treated with L929-CM maintained higher levels of catalase and GR activity, compared with the control group not exposed to L929-CM. Overall, the results were interpreted to mean that L929-CM could enhance antioxidant enzyme activities in RAW264.7 cells, and may be the reason why M-CSF protects monocytes/macrophages from oxidative injury.

Unfortunately, experimental numbers for each data point in the present study were small (n = 5) and, together with the use of relatively imprecise manual techniques, it is difficult to be confident in the statistical significance of the data presented. Larger experimental numbers or more precise analytical techniques might be applied to increase the statistical significance of the data.

Growth factors in L929-CM are predominantly M-CSF, with a small amount of granulocyte/ macrophage colony stimulating factor (GM-CSF).27 The presence of GM-CSF augments the effect of MCSF on monocytes and macrophages,28 and has a role in the maintenance of macrophage function.29 21 in a previous study,8 we observed the same protective effect with L929-CM on macrophages as that seen using recombinant human M-CSF (rhM-CSF) in protecting mouse peritoneal macrophages – an effect blocked by anti-hM-CSF monoclonal antibody. Therefore, the protective effect of L929-CM on macrophages under oxidative stress was predominantly due to M-CSF. In the present study, we suggest that M-CSF is mainly responsible for the protective effect afforded by L929– CM on RAW264.7 cells, and that a small amount of GM-CSF present augmented this effect.

Cells of the mononuclear phagocyte system play a critical role in maintaining the balance between health and disease in conditions such as infertility, osteopetrosis, osteoporosis, atherosclerosis, uraemia and Alzheimer’s disease; and altered M-CSF expression is associated closely with these pathological processes.3″ In addition, in the pathogenesis of many monocyte/macrophage-associated degenerative diseases (e.g. atherosclerosis and Alzheimer’s disease), ROS and ROS-induced oxidative injury are of key importance. The effects of M-CSF and ROS on monocytes/macrophages, and the reciprocity between them, are significant.

This study was supported by a grant from the Natural Science Foundation of China (No. 39670197) and by Hong Kong University CRGC grants (Nos. 102002 10 and 10200199).

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Z.J. PANG, Y. CHEN* and F.Q. XING

Department of Obstetrics and Gynaecology, Nanfang Hospital, Guangzhou 510515, and *Research Laboratory of Free Radical Medicine, The Fist Military Medical University, Guangzhou 510515, People’s Repulic of China

(Accepted 22 March 2001)

Correspondence to: Zhan-Jun Pang. E-mail: pangzhan@263.net

Copyright Royal Society of Medicine Press Ltd. 2001

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