The Effect of Selenium on Immune Functions of J774.1 Cells

Clin Chem Lab Med 2003; 41(8):1005–1011 © 2003 by Walter de Gruyter · Berlin · New York

Nadia Safir1,2, Albrecht Wendel3, Rachid Saile1 and

Layachi Chabraoui2,4*

1 Laboratory of Biochemistry, Faculté de Sciences Ben Msik,Casablanca, Morocco2 Laboratory of Biochemistry, Hôpital d’Enfants de Rabat,Rabat Instituts, Rabat, Morocco3 Faculty of Biology, University of Konstanz, Konstanz,Germany4 Laboratory of Biochemistry, Faculté de Médecine et dePharmacie, Rabat Instituts, Rabat, Morocco

The J774.1 macrophage cell line was used as a tool to

investigate the influence of selenium on macrophage

function. In vitro selenium supplementation enhanced

phagocytosis, degranulation by the release of �-glu-

curonidase after N-formyl-methionyl-leucyl-phenyl-

alanine (FMLP) or cytochalasin B, and the production

of superoxide anion after phorbol myristate acetate

stimulation of these cells, while the release of nitric ox-

ide was not affected by the selenium status. Selenium

supplementation enhanced significantly (p < 0.05) the

release of tumor necrosis factor (5-fold), interleukin-1

(3-fold) and interleukin-6 (2.5-fold) after 10 µg/ml

lipopolysaccharide stimulation compared to selenium-

deficient cells. Clin Chem Lab Med 2003; 41(8):1005–1011

Key words: Macrophage functions; Cytokine; Nitric ox-ide; Lipopolysaccharide; Lipoxygenase; Leukotriene;Selenium.

Abbreviations: 5HETE, 5-hydroxyeicosatetraenoic acid;5HPETE, 5-hydroperoxyeicosatetraenoic acid; FMLP,N-formyl-methionyl-leucyl-phenylalanine; FCS, fetalcalf serum; GSH, reduced glutathione; GPX, gluta-thione peroxidase; IFNγ, interferon-γ; IL-1, interleukin-1; IL-6, interleukin-6; LPS, lipopolysaccharide; LTD4,leukotriene D4; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide; NO, nitric oxide; O2

–, su-peroxide anion; PH-GPX, phospholipid hydroperoxide-glutathione peroxidase; PMA, phorbol myristateacetate; RPMI, Roswell Park Memorial Institute; Se (–),selenium-deficient cells; Se (+), 0.1 ppm selenium sup-plemented cells; SOD, superoxide dismutase; TNF, tu-mor necrosis factor; TMB, tetramethylbenzidin.

Introduction

Selenium is an essential trace element for animals andman. In humans its deficiency has been firmly associ-ated with cardiomyopathy in children (1), and there isan ongoing discussion about whether selenium intake

is related to cancer incidence in humans (2). Recently,evidence has accumulated to suggest that dietary sele-nium can also influence immune processes. Selenium-deficient cells (Se (–)) exhibit impaired immunologicalfunctions, leading generally to an increase in the sus-ceptibility toward various xenobiotics (3). Our presentknowledge on the metabolic role of selenium is largelybased on the reactions catalyzed by selenium-depen-dent enzymes, i.e., the selenium-containing gluta-thione peroxidases, 5'deiodinase, thioredoxin reduc-tase and various other selenoproteins such as seleniumprotein P. Selenium is incorporated into mammalianproteins in the form of the amino acid selenocysteine,thus forming the redox catalytic center in the active site.Cytosolic glutathione peroxidase (GPX) reduces H2O2

and a large variety of other hydroperoxides to water orthe corresponding alcohols, respectively, with forma-tion of oxidized glutathione, i.e., GSSG (4). The mem-brane-resident enzyme phospholipid hydroperoxide-glutathione peroxidase (PH-GPX) acts preferentially onesterified phospholipid hydroperoxides includingphosphatidylcholine and cholesterol-hydroperoxide(5). These two enzymes deserve special physiologicalinterest because their hydroperoxide substrates are de-rived from arachidonic acid and their products canshunt away intermediates for inflammatory mediatorssuch as leukotrienes. It was found that selenium-defi-cient leukocytes, when stimulated in the presence ofarachidonic acid, produced a 6-fold amount of 5-lipoxy-genase products compared to cells grown in selenium-adequate media (6). In cell homogenates a shift of theproducts in favor of increased 5-hydroperoxyeicosate-traenoic acid (5HPETE) formation was observed in sele-nium-deficient homogenates compared to controlcells. From the kinetics of selenium depletion/repletionexperiments it was concluded that the capacity toreduce 5HPETE to 5-hydroxyeicosatetraenoic acid(5HETE) was due to PH-GPX (6), indicating that bioac-tive selenium is intimately involved in the regulation ofthe production of inflammatory mediators via thelipoxygenase pathway and leukotriene biosynthesis,potent mediators in inflammation and shock.

Macrophages are also potent producers of cysteinylleukotrienes upon stimulation by endotoxins (7). Endo-toxin, i.e., lipopolysaccharide (LPS), is the major im-munoreactive and pathogenic component of the outermembrane of gram-negative bacteria. Macrophagesare identified as mediator cells of endotoxin-inducedhost responses (8). When macrophages are stimulatedby LPS, they also secrete cytokines such as interleu-kin-1 (IL-1) (9), tumor necrosis factor (TNF) (10), inter-leukin-6 (IL-6) (11) and a variety of other interleukinswhose functions have been well characterized (12).Macrophages have further been shown to release nitricoxide (NO), a potent endothelial vasodilator at physio-*E-mail of the corresponding author: [email protected]

The Effect of Selenium on Immune Functions of J774.1 Cells

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1006 Safir et al.: Selenium and macrophage immune functions

logical concentrations and probably a cytotoxic radicalat high concentrations (13). It was demonstrated invivo in mice that LPS-elicited endogenous TNF forma-tion or direct administration of the cytokine producesterminal organ failure (14) and that inhibitors of 5-lipoxygenase activity (15) or inhibitors of leukotrieneD4 synthesis LTD4 (16) can interrupt this sequence andafford protection against LPS by suppression of endo-toxin-induced TNF release.

As selenium regulates the synthesis of lipoxygenasemetabolites and as there is a link between leukotrienebiosynthesis and cytokine production, we investigatedthe effect of selenium in vitro on functional propertiesof the J774.1 murine macrophage cell line, on the re-lease of TNF, IL-1 and IL-6 after LPS stimuli, and on theproduction of NO after LPS and interferon-γ (IFNγ)stimuli.

Materials and Methods

Chemicals

All chemicals were obtained from Sigma Chemical Co. (St.Louis, MO, USA) except the following: fetal calf serum (FCS)was purchased from Serva, Heidelberg, Germany; xanthineoxidase and tetramethylbenzidin (TMB) were obtained fromBoehringer Mannheim (Mannheim, Germany); H2O2 was fromMerck (Darmstadt, Germany); murine recombinant TNF-αwas a gift from Dr. Adolf Boehringer (Austria); Roswell ParkMemorial Institute (RPMI) 1640 medium was from Biochrom(Berlin, Germany); IL-1β-fragment (117–271) was from Hoff-mann-La Roche (Nutley, NJ, USA) and anti-IL-6 antibody wasfrom Pharmingen (Mannheim, Germany).

Cell culture

The murine macrophage-like cell line J774A.1 was obtainedfrom the American Type Culture Collection and grown on162 cm2 flasks at 37 °C and 5% CO2 in RPMI 1640 medium sup-plemented with 10% heat-inactived FCS, 100 IU/ml penicillinG, 0.1 mg/ml streptomycin and 0.5 µg/ml amphotericin B.Cells were passaged every 3–6 days by diluting a suspensionof the cells 1:10 in fresh medium. As needed, the FCS concen-tration was gradually decreased from 10 to 1% in RPMImedium. One population of cells supplemented with 0.1 ppmselenium (Se (+)) as sodium selenite (Na2SeO3) was main-tained under these conditions, which permitted maximal ex-pression of GPX activity; a corresponding selenium-deficientpopulation was grown without Na2SeO3.

Protein determination

Protein contents were determined using the Lowry methodand human serum albumin as the standard (17). There wasessentially no difference in total protein among the differenttypes of J774.1 as determined to be 0.262±0.045 mg/106 cells.

Estimation of cell number

Estimation of viable cells was performed by the 3-(4,5-di-methylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)reduction assay (18). The blue formazan produced in this as-say by mitochondrial dehydrogenases is proportional to thenumber of viable cells. The murine J774.1 cell lines Se (+) andSe (–) were suspended at a concentration of 2 × 105 cells, and200 µl of each suspension was distributed in 96-well flat-bot-

tom microtiter plates and incubated at 37 °C and 5% CO2.Every day the estimation of viability was made by removingthe culture media, and 200 µl MTT (5 mg/ml); 1/10 (vol/vol))was added for determination of the viability. After incubationfor 4 h at 37 °C in the presence of 5% CO2, the dye was re-moved and cells were lysed by addition of 100 µl of iso-propanol-5% formic acid. The plates were then vigorouslyshaken for 20 min on a shaking plate, at room temperature, toensure homogenous dissolution of formazan granules. Theoptical density of each well was measured on a SLTEAR400 microplate reader (Fa. SLT-Laborinstrumente, Overath,Germany) with a test wavelength of 560 nm and a referencewavelength of 690 nm.

Phagocytosis

J774.1 cells at a concentration of 5 × 105 were allowed to ad-here for 2 hours at 37 °C. Then the medium was changed tomedium containing bead particles (10%) and left for 1 hour foringestion. Particle uptake was measured by fluorescent mi-croscopy in at least 100 cells and defined as the percent ofcells containing three or more bead particles. Bead particleswere sonicated and then washed 3 times with 154 mM NaClbefore use (19). Phagocytic activity was estimated as follows:

Phagocytic activity (%) = [phagocytosed cells/(phagocy-tosed cells + non-phagocytosed cells)] × 100.

β-glucuronidase

Suspended cells, stimulated with 5 µl of N-formyl-methionyl-leucyl-phenylalanine (FMLP) (100 nM) and 5 µl of cytocha-lasin B (10 µg/ml), were incubated with 0.5 ml 3 M acetatebuffer (pH 4.5) and 0.5 ml 3 mM 4-methyllumbelliferyl β-D-glu-curonide in a total volume of 1.5 ml at 37 °C. Incubation wasstopped with glycine buffer (1 M) and the fluorescence wasdetermined at 360/450 nm (20).

Superoxide generation

Production of the superoxide anion (O2–) by cells was mea-

sured as the superoxide dismutase-inhibitable reduction byacetyl ferricytochrome c. Cells were suspended in 100 µl ofHank's balanced salt solution (HBSS) containing acetyl ferri-cytochrome c (150 µmol/l). Superoxide production by cellswas stimulated by the addition of phorbol myristate acetate(PMA) (50 ng/ml). Reduction of acetyl ferricytochrome c wasallowed by the change of absorbance at 550 nm (21).

Cell stimulation

The murine macrophage-like cell line J774A.1 was plated in96-well culture dishes at a concentration of 5 × 105 cells/ml andallowed to adhere at 37 °C in 5% CO2 for 12 h. Thereafter themedium was replaced with fresh medium and cells were acti-vated by LPS (10 µg/ml) from Escherichia coli for cytokinemeasurement. (LPS was suspended in 1% hydroxylamine hy-drochloride).

TNF bioassay

Supernatants were stored at 4 °C and assayed within 48 h ofcollection. TNF was measured using a bioassay performedwith the fibrosarcoma cell line WEHI 164 13 according to Es-pevik and Nissen-Meyer (22). The WEHI cells at a concentra-tion of 2 × 104 cells per 200 µl were incubated with serially di-luted test samples in 96-well flat-bottom microtiter plates andincubated for 18 h at 37 °C and 5% CO2. MTT tetrazolium



Safir et al.: Selenium and macrophage immune functions 1007

((5 mg/ml); 1/10 (vol/vol)) was then added as described above.The titer of TNF is expressed in units per ml and is defined asthe reciprocal of dilution necessary to cause death of 50% ofthe cells. Activities are given as equivalents of murine recom-binant TNF-α (pg per 106 cells).

IL-1 bioassay

IL-1 like activity was measured during a bioassay performedwith the D10G4.1 T cell clone (23).

IL-6 ELISA

Flat microplates were coated by 50 µl of anti mouse α-IL-6 an-tibody for 12 h. After washing, we added 200 µl phosphatebuffered saline (PBS)/ 3% bovine serum albumin (BSA) and in-cubated 2 h at room temperature. After washing 2 times, weadded 50 µl of supernatants for 4 h at room temperature, thenwashed 4 times. Following that was addition of 100 µl of bi-otinylated antibody to mouse α-IL-6 for 45 min, then washingand addition of 100 µl streptavidin peroxidase (SA-POD)(1.8 µl/ml) for 30 min. After washing 8 times, we added 100 µlTMB and stopped the reaction by adding 50 µl of H2SO4. Plateswere read on a SLTEAR 400 microplate reader (Brentside,Huntingdon, UK), with a test wavelength of 450 nm and a ref-erence wavelength of 690 nm. (24).

Nitrite

Production of NO was quantified by measuring the accumula-tion of nitrite in the culture medium, using the Griess reaction(25). The murine macrophage-like cell line J774A.1 was platedin 96-well culture dishes at a concentration of 2.5 × 105 cells/mland allowed to adhere at 37 °C in 5% CO2 for 2 h. Thereafterthe medium was replaced with fresh medium and cells wereactivated by 10 µg/ml LPS and 100 ng/ml IFNγ. After 24 h,200 µl of Griess reagent (1% sulfamylamide and 0.1% N-naph-thylethylendiamine) was added at room temperature for10 min. The absorbance at 560 nm was determined on aSLTEAR 400 microplate reader, with a reference wavelengthof 690 nm. Values were quantified against a sodium nitritestandard curve, which was run on every kind of medium(RPMI + 1% FCS ± 0.1 ppm selenium).

Statistical analysis

We used the Student’s t test with a significance level at α =0.05.

Results

It was important to determine conditions under whichJ774.1 cells can be cultivated under selenium defi-ciency without loss of basic functions such as viability,growth rate and adhesion properties, as well as micro-scopic appearance. Since FCS contained significant

amounts of selenium, a series of culture experimentswas carried out in order to reduce the FCS content untilselenium deficiency was reached without loss of viabil-ity. We found that 1% FCS was a condition under whichselenium was severely depleted after 5 days of culture.Selenium deficiency was established by demonstratinga specific activity of reduced glutathione (GSH) peroxi-dase in the Se (–) cells of 2.05±0.24 mU/mg comparedto 32.8±2.7 mU/mg in the cells grown in the presence of10% FCS.

Selenium concentration response

We then checked the dependence of cellular GSH per-oxidase activity on the selenium concentration at con-centrations of sodium selenite between 0.01 ppm and1 ppm. We found that GSH peroxidase activity reacheda plateau above 0.1 ppm selenium with 210±2.7 mU/mgas shown in Table 1 compared to the activity of GPX inSe (–)cells and the activity of GPX in cells grown in thepresence of 10% FCS. Thus, cells grown under normalconditions of culture (10% FCS) were substantiallyselenoperoxidase-deficient, suggesting that serum-de-rived selenium was only partially available for seleno-peroxidase synthesis.

Selenium replenishment

Replenishment of the medium with sodium selenite(Na2SeO3) for periods of time ranging from 15 min to5 days was also studied; 0.1 ppm selenium returned en-zyme activity to 50% of that of selenium-repleted cellsor Se (+) by 24 h and to 85% by 7 days.

In order to minimize any potential cytotoxic effects athigh concentrations, J774.1-deficient cells were grownin 1% FCS without selenium and J774.1-supplementedcells were grown in 1% FCS with 0.1 ppm selenium.

Having thus examined the relationship between se-lenium supply and GSH peroxidase activity in J774.1cells, we then investigated the consequences of sele-nium deficiency on macrophage functions such asphagocytosis, respiratory burst capacity and bacterici-dal activity.

Phagocytic activity, degranulation and superoxideproduction

The phagocytic activity was significantly enhanced byselenium supplementation. We found that selenium-supplemented cells had a phagocytic activity of about89±2% compared to selenium-deficient cells, whichwas about 45±4% as shown in Table 2. These data sug-

Table 1 GPX status of J774.1 macrophage cells cultured under control, selenium-deficient and selenium-supplemented conditions.

Parameter Se-deficient Se-supplemented Control cellscells cells (10% FCS)

GPX activity (mU/mg proteins) 2.05±0.24 210±2.7* 32.8±2.7

*p < 0.0001 compared to Se-deficient cells and to control cells.




gest that selenium deprivation might modulate theability of macrophages to phagocyte via the lipoxyge-nase pathway. In fact, it was shown that macrophagefunctions are mainly modulated by the lipoxygenaseproducts (26).

The release of β-D-glucuronidase was more pro-nounced in Se (+) than in Se (–) after stimulation withFMLP and cytochalasin B as shown in Table 2.

PMA stimulation had no effect on selenium-deficientcells because the amount of O2

– released after 90 minwas the same with PMA or with PMA plus superoxidedismutase (SOD). However, in selenium-supplementedcells, stimulation with PMA allowed more release ofO2

–. This latter was absolutely dismutated by SOD asshown in Table 2.

Cytokine release

Since TNF is known to be a central mediator elicited byLPS, we determined whether TNF is affected by sele-nium supplementation. No significant production ofTNF by non-stimulated J774.1 macrophages was seen,showing that the cells were resting under basal condi-tions. The time course of LPS-induced production ofTNF in Se (+) and Se (–) showed that the maximum ofTNF release was at 5 h after stimulation, and Se (+) re-leased a 5-fold amount of TNF compared to Se (–). Thesignificant difference in TNF release found in this studyas shown in Figure 1 suggests that secretion of TNFfrom J774 macrophages exposed to 10 µg/ml LPS invitro is enhanced upon selenium supplementation.

The time course of 10 µg/ml LPS induced productionalso of IL-1 and IL-6 in Se (+) and Se (–), the maximumrelease of these cytokines was at 24 h after 10 µg/mlLPS stimulation. IL-1 release was about 3 times greaterin Se (+) compared to Se (–) as shown in Figure 2. TheIL-6 release was about 2.5 times greater in Se (+) com-pared to Se (–) as shown in Figure 3.

Nitric oxide production

The effect of selenium deficiency on NO productionwas also investigated. The production of NO by non-stimulated J774.1 macrophages was undetectable, andLPS failed to produce measurable amounts of NO un-der conditions in this study. Incubation of cells with

10 µg/ml LPS and 100 ng/ml IFNγ produced a time-de-pendent increase in the release of NO in the medium,reaching a plateau after 24 h. There was no significantdifference in NO release between Se (+) (23±0.2 µMNO/106) and Se (–) (20±0.2 µM NO/106; data notshown).

Discussion

The results of this study show that J774.1 macro-phages provide an in vitro system for the study of rela-tionships between selenium availability and macro-phage functions. The concentration/response curve forthe Se vs. GSH peroxidase activity demonstrates theknown dependence of the enzyme on selenium avail-ability. Cells were also found to be substantially se-lenoperoxidase-deficient when grown in the presenceof 10% FCS, suggesting that serum-derived seleniumwas only partially available for selenoperoxidase syn-thesis in vitro, as previously observed by Weitzel andWendel (6). We also observed no difference in growthrate in our defined medium between Se (–) and Se (+).J774.1 macrophages grown for 2–3 weeks in the pres-ence of serum without selenium supplementation typi-cally had <10% of the glutathione peroxidase activity ofselenium-supplemented cells. A plateau of GSH perox-idase activity was reached at 0.1 ppm of selenium after48 h repletion. The temporal pattern of the return ofGSH peroxidase activity after selenium replenishmentof selenium-deficient cells suggests that seleniumstimulates de novo protein synthesis. We also ob-served that the decrease in FCS supplementation from10% to 1% had no influence on cytokine production(data not shown).

However, in agreement with previous literature (3),J774.1-deficient cells showed alterations in their capac-ities for phagocytosis and degranulation. This findingmay be interpreted in terms of a partial perturbation ofthe integrity of cellular membranes in selenium-defi-cient cells due to peroxidative damage. However, it isalso possible that a link exists between phagocytosis,lipoxygenase products and selenium status after LPSstimulation.

The significant increase in O2– production with no ef-

fect on NO production in Se (+) compared to Se (–) in-

Table 2 Effect of selenium on phagocytic activity, degranulation and superoxide production in J774.1 macrophage-deficient cells or 0.1 ppm Se-supplemented cells.

Parameter Se-deficient cells Se-supplemented cells

Phagocytic activity (%) 44.7±3.9 89.1±2.1*β-D-glucuronidase (fluorescence) 60±5.2 140±8.9*β-D-glucuronidase + FLMP + cytB (fluorescence) 140±10.2 170 ±7.5**Superoxide production + PMA (nmol 02

–/mg proteins) 1.50±0.14 1.93±0.07**Superoxide production + SOD (nmol 02

–/mg proteins) 1.50±0.12 1.50±0.14

cytB: cytochalasin B. Data are expressed as mean ± SEM oftriplicate determination. Culture conditions for each parame-ter are described in Materials and Methods. * p < 0.001 com-

pared to Se-deficient cells, ** p < 0.05 compared to Se-defi-cient cells.




dicates that adequate selenium supply is necessary formicrobicidal activity of macrophages. The lack of effectof selenium in NO production in our system is in agree-ment with other studies suggesting that products ofthe cyclo-oxygenase pathway, but not leukotriene(lipoxygenase pathway), modulate NO production andconsequently macrophage tumoricidal activity inJ774.1 cells (27).

Our major finding is that selenium supplementationin vitro results in a significant increase in cytokine pro-duction like TNF, IL-1 and IL-6, i.e., major components ofthe mediator responses to LPS during infection. TNF isan early cytokine secreted from macrophages in re-sponse to LPS stimulation and leads to the productionof IL-1 (a synergist of TNF) and IL-6, as reported by oth-ers (28). The weak response to LPS stimulation of sele-

Figure 1 Effect of selenium on TNF-α release 5 h after LPSstimulation in J774.1 macrophage-deficient cells and cells

supplemented with 0.1 ppm selenium. *p < 0.0001 comparedto selenium-deficient cells.

Figure 2 Effect of selenium on IL-1β release after 24 h LPSstimulation on J774.1 macrophage-deficient cells and cells


Figure 3 Effect of selenium on IL-6 release after 24 h LPSstimulation on J774.1 macrophage-deficient cells and cells





nium-deficient cells is in our mind an important func-tional loss of immune competence of these cells, whichadds on top of the previously reported attenuation5HPETE production and thus leukotriene biosynthesis(29). An alternative explanation for the alterations ofTNF release in Se (–)is a modulation of the proteolyticcleavage of the membrane-bound TNF precursor whichis in agreement with our observation of an increased re-lease of proteolytic enzymes under these conditions.

TNF release has been shown to be an advantage inprotection from infection. Together with an increasedsuperoxide production necessary to eliminate bacteria,the organism needs an enhanced protection potentialagainst reactive oxygen during host defense, which infact can only be provided when sufficient selenium isbioavailable. Furthermore, the concomitant enhance-ment of IL-6 production by adequate selenium couldprevent continuous self-amplification of macrophageactivation, potentially leading to excessive and hencedetrimental TNF release.

In conclusion, this study proposes that seleniumsupplementation increases the efficiency of the im-mune response by modulating macrophage proper-ties, particularly enhancing phagocytosis, productionof anion superoxide, degranulation and cytokine pro-duction. It is therefore interesting to extend these stud-ies to the activity of macrophages other than underresting conditions, e.g., to cells from septic or tumor-bearing patients.

Acknowledgements

This work was supported by grant We 686/20-1 of theDeutsche Forschungsgemeinschaft (A.W.) and the DeutscheAkademische Austauschdienst (N.S.). Statistical analysis wasperformed with the aid of LBRCE (Laboratoire de Biostatis-tiques de Recherche Clinique et d’Epidémiologie) of the Fac-ulty of Medicine and Pharmacy of Rabat.

References

1. Burk RF. Biological activity of selenium. Annu Rev Nutr1983; 3:53–70.

2. Schrauzer GN. Selenium and cancer. In: Neve J, Favier A,editors. Selenium in biology and medicine, Berlin: Walterde Gruyter and Co, 1989:251–61.

3. Peretz A. Selenium in inflammation and immunity. In: NeveJ, Favier A, editors. Selenium in biology and medicine.Berlin: Walter de Gruyter and Co, 1988:235–46.

4. Wendel A. Glutathione peroxidase. Methods Enzymol 1981;77:325–33.

5. Ursini F, Maiorino M, Gregolin C. The selenoenzyme phos-pholipid hydroperoxide glutathione peroxidase. BiochimBiophys Acta 1985; 839:62–70.

6. Weitzel F, Wendel A. Selenoenzymes regulate the activity ofleukocyte lipoxygenase via the peroxide tone. J Biol Chem1993; 268:6288–92.

7. Lüderitz T, Brandenburg K, Seydel U, Roth A, Galanos C, Ri-etschel ET. Structural and physicochemical requirements ofendotoxins for the activation of arachidonic acid metabo-lism in mouse peritoneal macrophages in vitro. Eur JBiochem 1989; 179:11–6.

8. Rosenstreich DL, Vogel SN. Central role of macrophages inthe host response to endotoxin. In: Schlessinger D, editor.Microbiology-1980. Washington, DC: American Society forMicrobiology, 1980:11–5.

9. Cavaillon JM, Haeffner-Cavaillon N. Signals involved in in-terleukin-1 synthesis and release by lipopolysaccharide-stimulated monocytes/macrophages. Cytokine 1990; 2:313–29.

10. Beutler B, Mahoney J, Le Trang N, Pekala P, Cerami A. Pu-rification of cachectin, a lipoprotein lipase-suppressinghormone secreted by endotoxin-induced RAW 264.7 cells.J Exp Med 1985; 161:984–95.

11. Hirano T, Akira S, Tagat T, Kihimoto T. Biological and clini-cal aspect of interleukin 6. Immunol Today 1990; 11:443–9.

12. Cavaillon JM. Les cytokines, 2nd ed. Paris: Masson Paris,1996:501–22.

13. Hibbs JB Jr, Taintor RR, Vavrin Z, Rachlin EM. Nitric oxide:a cytotoxic activated macrophage effector molecule.Biochem Biophys Res Commun 1988; 157:87–94.

14. Tiegs G, Wolter M, Wendel A. Tumor necrosis factor is aterminal mediator in galactosamine/endotoxin-inducedhepatitis in mice. Biochem Pharmacol 1989; 38:627–31.

15. Schade UF, Ernst M, Reincke M, Wolter DT. Lipoxygenaseinhibitors suppress formation of tumor necrosis factor invitro and in vivo. Biochem Biophys Res Comm 1989;159:748–54.

16. Tiegs G, Wendel A. Leukotriene-mediated liver injury.Biochem Pharmacol 1988; 37:2569–73.

17. Lowry OH, Rosebrough NJ, Farr AL, Randall JR. Proteinmeasurement with the folin- phenol reagent. J Biol Chem1951; 193:265–75.

18. Yamamoto C, Takemoto H, Kuno K, Yamamoto D, TsuburaA, Kamata K, et al. Cycloprodigiosin hydrochrloride, a newH(+)/Cl(–) symporter induces apoptosis in human and rathepatocellular cancer cell lines in vitro and inhibits thegrowth of hepatocellular carcinoma xenografts in nudemice. Hepatology 1999; 30:894–902.

19. Wright SD. Special methods for the study of receptor-me-diated phagocytosis. Methods Enzymol 1986; 132:204–21.

20. Stahl PD, Fishman WH. β-D-Glucuronidase. In: BergmeyerHU, editor. Methods of enzymatic analysis, 3rd ed. Wein-heim: Verlag Chemie, 1984:246-56.

21. Newburger PE, Chovaniec ME, Cohen HJ. Activity and ac-tivation of the granulocyte superoxide-generating system.Blood 1980; 55:85–92.

22. Espevik T, Nissen-Meyer J. A highly sensitive cell lineWEHI 164 clone 13, for measuring cytotoxic factor/tumornecrosis factor from human monocytes. J Immunol Meth-ods 1986; 95:99–105.

23. Beuschuer HU, Günther C, Röllinghoff M. IL-1β is secretedby activated murine macrophages as biologically inactiveprecursor. J Immunol 1990; 144:2179–83.

24. Gantner F, Leist M, Kusters S, Vogt K, Volk HD, Tiegs G. Tcell-stimulus-induced crosstalk between lymphocytes andliver macrophages results in augmented cytokine release.Exp Cell Res 1996; 229:137–46.

25. Di Rosa M, Radomski M, Carnuccio R, Moncada S. Gluco-corticoids inhibit the induction of nitric oxide synthase inmacrophages. Biochem Biophys Rev Commun 1990,172:1246-52.

26. Schade UF, Burmeister I, Engel R. The role of lipoxyge-nases in the activation of macrophages by lipopoly-sacharide. Prog Clin Biol Res 1988; 272:125–34.

27. Marotta P, Sautebin L, Di Rosa M. Modulation of the induc-tion of nitric oxide synthase by eicosanoids in the murinemacrophage cell line J774. Br J Pharmacol 1992; 107:640–1.




28. Vassali P. The pathophysiology of tumor necrosis factor.Annu Rev Immunol 1992; 10:411-52.

29. Wendel A. Biochemical functions of selenium. Phospho-rus, Sulfur and Silicon, 1992; 67:405–15.

Received 9 February 2003, revised 5 May 2003, accepted 5 May 2003

Corresponding author: Prof. Dr. Layachi Chabraoui,Laboratory of Biochemistry, Faculté de Médecine et dePharmacie, BP. 6203 Rabat Instituts, MoroccoPhone: +212 (0) 37 771300, Fax: +212 (0) 37 773701, E-mail: [email protected]



The Effect of Selenium on Immune Functions of J774.1 Cells

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