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Cell Biology International (2004) 28, 517521 (Printed in Great Britain)
Effect of stress-induced lipid peroxidation on functions of rat peritoneal macrophages
V Nimet İzgüt‑Uysala*, Ruken Tana, Mehmet Bülbüla and Narin Derinb
aAkdeniz University, Faculty of Medicine, Department of Physiology, 07070 Antalya, Turkey
bAkdeniz University, Faculty of Medicine, Department of Biophysics, 07070 Antalya, Turkey
The aim of the present study was to investigate the effects of stress-induced lipid peroxidation on macrophages' functions. Animals were subjected to 4
Keywords: Lipid peroxidation, Phagocytosis, Chemotaxis, Catalase activity, Vitamin E.
*Corresponding author. Tel.: +090-242-2274483; fax: +090-242-2274483.
Stress is accepted as a response of an organism to external stimuli or changes (Dorshkind and Horseman, 2001; Peng et al., 2000). Numerous studies show that stress can be immunosuppressive. In these studies, stress has also been shown to suppress different immune parameters, e.g. delayed type hypersensitivity, antibody production, NK activity, leukocyte proliferation, skin homograft rejection, and virus-specific T cell activity (Dorshkind and Horseman, 2001; De Castro et al., 2000; Dhabhar, 2000).
It is well known that stress induces formation of reactive oxygen species (ROS) and leads to the oxidative injury in various tissues (De Castro et al., 2000; Nishida et al., 1997; Toleikis and Godin, 1995). Oishi and Machida (2002) have shown that a significant increase in plasma TBARS was observed during and after the stress. The activation of immune cells can be a source of the stress-induced ROS production and antioxidant enzymes in immune cells play an important role in preventing the ROS-induced injury (De Castro et al., 2000; Babior, 2000; Victor et al., 2003). Oxidant–antioxidant balance is critical for immune cell functions because of its protective effect of the maintenance of cell membrane integrity and functionality (Pawlak et al., 1998a, 1998b; Knight, 2000; Celada and Nathan, 1994).
Immune cells are particularly sensitive to oxidative stress because of the presence of polyunsaturated fatty acids in their plasma membranes and production of ROS, which is part of their normal function (Pawlak et al., 1998a; Knight, 2000; Celada and Nathan, 1994). Moreover, membrane-related functions are critical in maintaining normal function of immune cells and their ability to defend against foreign antigens (Biselli et al., 1996; Yuli et al., 1982). These funtions are highly sensitive to ROS (Babior, 2000; Victor et al., 2003).
Macrophages can survive exposure to endogenously generated ROS for a long period and play important roles in phagocytosis-mediated host defence against microbial infection. The role of superoxide anion liberation by the macrophages in the defence against pathologic agents is well known (De Castro et al., 2000; Babior, 2000; Alvarez et al., 1996). However, studies are still required concerning the effect of liberation of oxidizing agents by peritoneal macrophages, and their effects on changes due to stress. The present work was aimed at examining the effect of cold and restraint stress-induced lipid peroxidation on macrophages' functions.
2 Materials and methods
Thirty-two male Wistar rats (200–220
2.2 Preparation of peritoneal macrophages
The animals were anesthetized with diethyl ether. Ten milliliters of Krebs-phosphate buffer solution was injected intraperitoneally and after 3
2.3 Determination of TBARS
Lipid peroxidation was measured by the method of Stocks and Offerman (1972). Samples were adjusted by dilution with the homogenization buffer solution to 2
2.4 Measurement of catalase activity
Intracellular catalase activity was measured according to the method described by Aebi (1987). To measure the intracellular catalase of 2
2.5 Measurement of chemotactic activity
Preparation of zymosan-activated serum (ZAS, 10%) was by the method of Goldstein et al. (1975), involving incubation of rat serum with boiled and washed zymosan particles at 1
The chemotaxis assay was performed by Boyden's method (1962), using a nitrocellulose filter of 8
2.6 Determination of phagocytic activity
One hundred microliters of 1% activated charcoal was added to 100
Data are presented as the means
3.1 TBARS content of macrophages
The changes observed in lipid peroxidation status in peritoneal macrophages are shown in Fig. 1. Cold-restraint stress increased TBARS content of peritoneal macrophages as an index of lipid peroxidation with statistical significance when compared to control rats (196.07
Effects of cold-restraint stress and vitamin E treatment on TBARS production of peritoneal macrophages. Data represent mean
3.2 Catalase activity
We examined the levels of intracellular catalase activity that catalyze H
3.3 Chemotactic activity
Macrophages obtained from stressed group presented lower chemotactic capacity than that of control group with statistical significance (14.6
Effects of cold-restraint stress and vitamin E treatment on chemotactic activity of peritoneal macrophages. Data represent mean
3.4 Phagocytic activity
Fig. 3 shows the phagocytic activity of peritoneal macrophages. The number of the particles phagocyted by peritoneal macrophages from stress group was significantly (p
Effects of cold-restraint stress and vitamin E treatment on phagocytic activity of peritoneal macrophages. Data represent mean
This study was designed to investigate the effect of cold-restraint stress-induced lipid peroxidation on the functions of peritoneal macrophages. For the supplementation of antioxidant activity, vitamin E was used and TBARS as the oxidative stress marker, and catalase activity were investigated. Numerous previous studies (Dorshkind and Horseman, 2001; De Castro et al., 2000; Dhabhar, 2000; Vizi, 1998) along with our data support the hypothesis that stress situations are associated with altered immune functions. We showed that peritoneal macrophages are not resistant to oxidative stress. Cold-restraint stress was found to induce oxidative stress through decreased chemotactic and phagocytic activities, and increased lipid peroxidation, as shown by enhanced TBARS levels.
Macrophages can survive for a long period to an exposure of exogenously generated reactive oxygen species and play important roles in phagocytosis-mediated defence against microbial infection (De Castro et al., 2000; Babior, 2000; Alvarez et al., 1996). Phagocytosis stimulates respiratory burst, which contains several chemical events including ROS production (De Castro et al., 2000; Babior, 2000; Pawlak et al., 1998a). ROS can function as signaling molecules in regulation of fundamental cell activities such as cell growth and cell adaptation responses, whereas at higher concentrations, ROS can cause cellular injury and death (Lum and Roebuck, 2001). Excess ROS generated by phagocytes may damage biologically important macromolecules. Membrane lipids are particularly vulnerable to peroxidation. Lipid peroxidation damages the cell membrane, with the result that an altered membrane fluidity and cell membrane-related function occur, including chemotaxis and phagocytosis (Peng et al., 2000; Babior, 2000; Victor et al., 2003; Pawlak et al., 1998a, 1998b; Knight, 2000). Immune cell functions are specially linked to reactive oxygen species generation, the oxidant–antioxidant balance is essential for these cells. Intake of antioxidant vitamins resulted in a significant increase in the phagocytic function of polymorphonuclear neutrophils as well as in a significant decrease of lipid peroxides (Del Rio et al., 1998; De La Fuente et al., 2002). It was found that vitamin E possess a high immunomodulating and antioxidant activity under the acute cold-induced stress conditions. Vitamin E is an efficient antioxidant that functions as a “chain breaker” in lipid peroxidation of cell membranes (Uteshev et al., 2001). It is demonstrated that restraint stress induced a decrease in the level of activities of antioxidant enzymes, while the levels of TBARS were found elevated. Antioxidant, especially vitamin E, supplementation enhances cell-mediated immunity (Zaidi and Banu, 2004).
Peritoneal macrophages are sensitive to exogenous H
Phagocytic cells (neutrophils, monocytes, and macrophages) are the first line of defence system of the organism against the infectious agents (Babior, 2000; Alvarez et al., 1996). Macrophages accomplish nonspecific immune function through what is known as phagocytosis. Phagocytic function can be divided into several stages as chemotaxis, ingestion and killing of infectious agents by producing superoxide anion radicals (Babior, 1992; Witko-Sarsat et al., 2000). According to our data, exposure to cold-restraint stress rendered peritoneal macrophages incapable of chemotactic and phagocytic activities. Our findings are also corroborated by demonstration of Victor et al. (2003) that the chemotactic activity in peritoneal macrophages from stressed rats is reduced. The decline in macrophages' functions in stress conditions in our study may be related to the increase of lipid peroxidation in macrophages, which is because vitamin E treatment was found to be effective in preventing the stress-induced decrease of phagocytosis, chemotaxis activities and increase of TBARS levels in this study. The findings of this study indicate that the increase in lipid peroxidation causes the inhibition of chemotaxis and phagocytosis of macrophages.
This work was supported by a grant from the Research Foundation of Akdeniz University (Project number: 99.01.0103.12).
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Received 26 August 2003/19 February 2004; accepted 26 April 2004doi:10.1016/j.cellbi.2004.04.006