Introduction

The immune cell functions are specially linked to reactiveoxygen species (ROS) generation, such as that involved in themicrobicidal activity of phagocytes, cytotoxic activity or thelymphoproliferative response to mitogens.1 However, exces-sive amounts of ROS are harmful for the immune cells,because they can attack cellular components and lead to celldamage or death by oxidizing the membrane lipids, protein,carbohydrates and nucleic acids. To prevent these effects ofROS, they can be neutralized by the complex anti-oxidantsystem that the organisms have developed.2 Thus, anti-oxidants play a vital role in maintaining immune cells in areduced environment and in protecting them from oxidativestress.2 Indeed, the history of the relationship between antiox-idants and immunology began in the early years of the 20thcentury with an appreciation that anti-oxidant nutrient defi-ciencies may cause disease3 and that anti-oxidants have animmunostimulating action.4 However, recent results havethrown doubt on this concept, because a total neutralizationof ROS can block their functional role and higher doses ofanti-oxidants can produce oxidant effects.5,6

Oxidative stress has been increasingly implicated inpathological conditions, such as septic shock,7 and in physi-ological ageing,8 situations in which the anti-oxidant levelsdecrease.8 In septic shock caused by endotoxins, there are

functional and metabolic alterations in cells and tissues,including changes in the immune system, such as a stimulationof phagocytes and pro-inflammatory cytokine production.9

Ageing is associated with a decline of many physiologicalfunctions and changes in the immune function with depressedactivity of T lymphocytes and an increase in several phago-cyte functions, as well as their pro-inflammatory cytokineproduction.2,10 In both situations, the administration of anti-oxidants has been useful for improvements of severalimmune functions.9,11

N-acetylcysteine (NAC) and vitamin E are potent anti-oxidants, the levels of which decrease during oxidativestress.12 Both anti-oxidants inhibit the activation of thenuclear transcription factor NF-κB produced by oxidativestress,13 which could result in a decrease of free radicals andpro-inflammatory cytokine production.5,9 Therefore, theseanti-oxidants have an anti-inflammatory action.14 N-acetyl-cysteine increases the pool of glutathione, which is an impor-tant cellular anti-oxidant, useful to immune cells15 and hasfavourable effects against oxidative stress by endotoxicshock9 and ageing.

Vitamin E is considered the principal anti-oxidant defenceagainst lipid peroxidation in the cell membrane of mammals.Moreover, it modulates the immune cell functions, improvingthem in adults16 and older subjects.11

Taking into account these data and the fact that in previ-ous work we have found that the immunostimulant effect ofanti-oxidants depends on the age and immune state of organ-isms as well as on the kind of immune function studied,9,11 wehypothesize that anti-oxidants, such as NAC and vitamin E,do not exert an indiscriminate stimulating effect on theimmune cell function, but instead are homeostatic factors.This immunomodulatory role of NAC and vitamin E has been

Immunology and Cell Biology (2000) 78, 49–54

Special Feature

Anti-oxidants as modulators of immune function

M DE LA FUENTE AND VM VICTOR

Department of Animal Physiology, Faculty of Biology, Complutense University, Madrid, Spain

Summary In order to confirm the hypothesis of the immunomodulating action of anti-oxidants (bringing backaltered immune function to more optimum values), the possibility that anti-oxidants may be useful in two experi-mental models of altered immune function has been studied. The first is a pathological model, that is, lethal murineendotoxic shock caused by an LPS injection of 100 mg/kg, in which the lymphocytes show increased adherenceand depressed chemotaxis. The injection of N-acetylcysteine (150 mg/kg), which increased both functions incontrol animals, decreased adherence and increased chemotaxis in mice with endotoxic shock. The second is aphysiological model; aged human subjects (70 ± 5-year-old men) who, in their largest segment of population (‘stan-dard’ group) showed an increased lymphocyte adherence and decreased lymphoproliferative response to mitogenscompared with younger adults. The ingestion of vitamin E (200 mg daily for 3 months in this standard group)lowered adherence and stimulated lymphoproliferation. However, a smaller segment of the human population testedshowed ‘non-standard’ values in these lymphocyte functions, that is, very low adherence and very high prolifera-tion. In those subjects, vitamin E showed the opposite effects, namely adherence increase and depressed lympho-proliferation. In both age groups of men, these functions reached adult levels after vitamin E ingestion. These datasuggest that anti-oxidants preserve adequate function of immune cells against homeostatic disturbances such asthose caused by endotoxic shock and ageing.

Key words: ageing, anti-oxidant, endotoxic shock, immune function, lymphocyte.

Correspondence: Prof. M De la Fuente PhD, Departamento deBiología Animal II (Fisiología Animal), Facultad de Ciencias Bio-lógicas, Universidad Complutense, Av. Complutense s/n, E-28040Madrid, Spain. Email: mondelaf@eucmax.sim.ucm.es

Received 15 September 1999; accepted 15 September 1999.

shown in lymphocyte functions in two oxidative stress exper-imental models, endotoxic shock (pathological model) andageing (physiological model), in which these functions arealtered.

Materials and Methods

Pathological model

Animals Female BALB/c mice (Mus musculus; Iffa Credo), aged24 ± 2 weeks, were maintained at a constant temperature 22 ± 2°C insterile conditions inside an aseptic air negative-pressure environmental cabinet (Flufrance, Cachan, France) on a 12 h light/dark cycleand fed Sander Mus (Panlab, Barcelona, Spain) and water ad libitum.The animals used did not show any sign of malignancy or otherpathological processes. Mice were treated according to the guide-lines of the European Community Council Directives 86/6091 EEC.

Experimental protocol Lethal endotoxic shock was induced byintraperitoneal (i.p.) injection of Escherichia coli LPS (055:B5,Sigma, St Louis, MO, USA) at a concentration of 100 mg/kg.17 Eachanimal received this concentration of LPS in a volume of 100 µL and30 min later mice were injected i.p. with 150 mg/kg bodyweight ofN-acetylcysteine (Sigma; LPS + NAC group). Control animals (PBSgroup) received two injections of an equivalent volume of PBS. Ashock control group (LPS group) was injected with LPS and, after 30min, with PBS. The control anti-oxidant animals were injected withPBS, followed by NAC 30 min later (NAC group). All injectionswere carried out between 9.00 and 10.00. Although in previousstudies we have observed that the oestrous cycle phase of the femalemice has no effect on this experimental assay, all females used in thepresent study were in the beginning of dioestrous.

Collection of cells At 2, 4, 12 and 24 h after injection, peritonealsuspensions were obtained by a procedure previously described.18

Briefly, 3 mL Hank’s solution, adjusted to pH 7.4, were injected i.p.and then the abdomen was massaged and the peritoneal exudate cells(PEC), consisting of 60% lymphocytes and 40% macrophages, werecollected, allowing recovery of 90–95% of the injected volume. Lymphocytes were counted and adjusted in Hank’s solution tol × 106 lymphocytes/mL. Cell viability was checked by trypan blueexclusion test and viable cells were over 97%.

Assay of adherence capacity The quantification of substrate adher-ence capacity was carried out by a method previously described.19

Aliquots of 200 µL peritoneal suspension were placed in eppendorftubes. At 10 min of incubation, 10 µL from each sample wasremoved after gently shaking to resuspend the sedimented cells andthe number of non-adhered lymphocytes was determined by count-ing in Neubauer chambers (Blau Brand, Germany) in an opticalmicroscope (40× magnification lens). The adherence index (AI) wascalculated according to the following equation:

Assay of chemotaxis Chemotaxis was evaluated according to amethod consisting basically of the use of chambers with two com-partments separated by a filter with a pore diameter of 3 µm.19

Aliquots of 300 µL peritoneal suspension were deposited in theupper compartment and aliquots of 400 µL of a chemoattractant, f-met-leu-phe (10–8 mol/L), were put into the lower compartment.The chambers were incubated for 3 h and then the filters were fixed

and stained. The chemotaxis index was determined by counting in anoptical microscope (100× magnification lens) the total number oflymphocytes in the lower face of the filter.

Physiological model

Subjects A group of 25 aged men (70 ± 5 years of age) volunteeredfor the present study, which was approved by the Complutense University Human Experimental Ethical Review Committee.Another group of 12 adult men (35 ± 5 years of age) was used asadult controls.

Treatment All older men received a daily supplement of 200 mgvitamin E (Alcala Farma) daily for 3 months. This treatment waschosen on the basis of previous work from our laboratory showingthat this dose was a stimulant of immune function.

Immune cell functions Peripheral blood samples were drawn byvein puncture at 9.00–10.00 in heparinized tubes. In the older mengroup, the samples were obtained before (BE) and after (AE) vitaminE ingestion. The adult group was separated into two subgroups of sixsubjects whose blood samples were obtained in parallel with the BEor AE groups. The adherence capacity and the proliferative responseto mitogens of lymphocytes were analysed following methods previ-ously described.20

Adherence lymphocytes assay For adherence capacity measure-ment, 1 mL blood (diluted 1:1 with Hank’s medium) was placed in aPasteur pipette in which 50 mg of nylon fibre was packed to a heightof 1.25 cm. After 10 min, the effluent had drained by gravity. Thepercentage AI was calculated as follows:

Separation of blood lymphocytes and proliferative assay Fromheparinized samples, lymphocytes were obtained by centrifugationat 300 g for 30 min in a density gradient (1.114), using Monopolyresolving medium (Flow Laboratories, McLean, VA, USA). Thecells at the interface, consisting of mononuclear lymphocytes andmonocytes, were harvested and washed twice in RPMI medium(Gibco, Burlington, Ontario, Canada). Cell viability was checkedby the trypan blue exclusion test before and after each assay and inall cases the viability was higher than 95%. The cells of the mononuclear leucocyte suspension were counted and adjusted to 106 lymphocytes/mL RPMI supplemented with gentamicin(1 mg/mL, Gibco) and 10% foetal calf serum (Gibco), previouslyinactivated by heat (30 min at 56°C). Aliquots of 200 µL were dis-pensed into plates of 96 wells (Costar, Cambridge, MA, USA) and20 µL of phytohemagglutinin (PHA, Flow Laboratories) to 20mg/L were added. After 48 h of incubation at 37°C in an atmos-phere of 5% CO

2, 1.85 × 104 Bq/well, [3H]-thymidine (Du Pont,

Boston, MA, USA) was added, followed by another 24 h incuba-tion. The cells were harvested in a semiautomatic harvester andthymidine uptake was measured in a beta counter (LKB, Uppsala,Sweden) for 1 min. The results were expressed as [3H]-thymidineuptake (c.p.m.).

Statistical analysis

The data are the mean ± SD of the values from the number of exper-iments shown in the figures. The normality of the samples was

M De la Fuente and VM Victor50

AI = 100 – ( lymphocytes/mL supernatant

lymphocytes/mL original sample) × 100

AI = 100 – ( lymphocytes/mL of effluent samples

lymphocytes/mL original samples ) × 100

checked by the Kolmogorov-Smirnov test. The data were statisticallyevaluated by the Mann–Whitney U-test for unpaired observations ofnon-parametric data, with P < 0.05 being the minimum significancelevel.

Results

Pathological model

Figure 1 shows the adherence indexes at 10 min of incubationof murine peritoneal lymphocytes from different groups: PBS(controls), LPS (shock controls), NAC (anti-oxidant controls)and LPS + NAC (experimental group). At 2 and 4 h after LPSinjection, the adherence capacity was increased (P < 0.001)with respect to the PBS group. In the NAC group, the adher-ence index was also increased (P < 0.001) at 4, 12 and 24 hafter injection compared with the PBS group. In the LPS +NAC group, a significant increase (P < 0.001) in adherenceat 12 and 24 h after LPS injection was obtained in compari-son with the PBS control. Comparing the results obtained inthe LPS + NAC group with the LPS group, a significantdecrease (P < 0.001) at 2 and 4 h and a significant increase(P < 0.001) at 12 and 24 h after injection was observed. Thechemotaxis indexes of murine peritoneal lymphocytes fromthe PBS, LPS, NAC and LPS + NAC groups at 2, 4, 12 and24 h after injections are shown in Fig. 2. Compared with thePBS group, a highly significant decrease (P < 0.001) wasshown at 2, 4 and 12 h and (P < 0.01) at 24 h after LPS injec-tion (LPS group), whereas after NAC injection (NAC group)a significant increase (P < 0.001) at 12 h was observed. The chemotaxis index in the LPS + NAC group showed a

significant decrease (P < 0.01) at 2 and 4 h compared withthe PBS group and a highly significant increase (P < 0.001)at 2 and 12 h, 4 h (P < 0.01) and 24 h (P < 0.05) comparedwith the LPS group.

Physiological model

The adherence and proliferative responses to the mitogenPHA of blood lymphocytes from aged men before and aftersupplementation of vitamin E, as well as from adult controlmen, are shown in Figs 3 and 4, respectively. Two subgroupswere found in the 25 older men. One subgroup (17 men), thestandard (S) group, was that in which men showed similarvalues to the majority of subjects of their age. The othergroup (eight men), the non-standard (NS) group, showedvalues very different to those expected for their age. Theresults of the adherence index of lymphocytes are shown inFig. 3. The standard group, before ingestion of vitamin E(SBE), showed an adherence index higher (P < 0.05) thanthat of the adult control (AC) group. After ingestion ofvitamin E (SAE), a significant decrease (P < 0.01) wasshown in this index compared with that before vitamin Eingestion, with the values no longer differing from those ofthe adult group. In the non-standard group, the values of theadherence index before vitamin ingestion (NSBE) weresmaller (P < 0.001) than those in the standard and adultcontrol groups. After vitamin E ingestion (NSAE) the adher-ence index increased significantly (P < 0.01) with respect toNSBE, showing similar values to the AC group.

The results of the lymphoproliferation capacity are shownin Fig. 4. In the standard group, where values of lympho-

Anti-oxidants and immune function 51

Figure 1 Adherence indexes (AI) of murine peritoneal lympho-cytes. (h), phosphate saline buffer (control group); (j), N-acetylcysteine (NAC) injection (anti-oxidant control group;150 mg/kg); ( ), lipopolysaccharide injection (100 mg/kg;shock control group); ( ), LPS injection (100 mg/kg) and NAC(150 mg/kg) 30 min after. The cells, in all cases, were obtained at2, 4, 12 and 24 h after injection. Each column represents themean ± SD of eight values corresponding to eight animals, eachvalue being the mean of duplicate assays. ***P < 0.001 withrespect to the corresponding values in the PBS group.†††P < 0.001 with respect to the LPS group.

Figure 2 Chemotaxis (number of lymphocytes/filter) ofmurine peritoneal lymphocytes. (h), phosphate saline buffer(control group); (j), N-acetylcysteine (NAC) injection (anti-oxidant control group; 150 mg/kg); ( ), lipopolysaccharideinjection (100 mg/kg); ( ), LPS injection (100 mg/kg) and NAC(150 mg/kg) 30 min after. The cells, in all cases, were obtained at2, 4, 12 and 24 h after injection. Each column represents themean ± SD of eight values corresponding to eight animals, eachvalue being the mean of duplicate assays. ***P < 0.001 and**P < 0.01 with respect to the corresponding values in the PBSgroup. †††P < 0.001, ††P < 0.01 and †P < 0.05 with respect to theLPS group.

proliferation before vitamin E ingestion were significantlydecreased (P < 0.001) compared with those in lymphocytesfrom the AC group, the ingestion of vitamin E increased thisproliferation significantly (P < 0.001), showing valuessimilar to those of the AC group. In the NSBE group, inwhich the blood lymphocytes showed an increased prolifera-tion with higher values (P < 0.001) than in the AC and SBEgroups, the ingestion of vitamin E (NSAE) produced a significant decrement (P < 0.01) of this capacity, with pro-liferation values similar to those of the AC group.

Discussion

The present study shows the beneficial effects in vivo of theanti-oxidants NAC and vitamin E on the initial functions ofthe immune response of lymphocytes, such as adherence totissues, migration directed to the antigen focus (chemotaxis)and proliferative response to mitogens, in the two endotoxicshock and ageing models of oxidative stress.

During endotoxic shock, lymphocytes show a dysfunctionexpressed as increased adherence to tissues and depressedchemotaxis. These effects could be due to the increase ofTNF-α and ROS caused by LPS stimulation,9 whichenhances the expression of adhesion molecules,21 or to theproduction of migratory inhibitory factor (MIF) by LPS.22

N-acetylcysteine has anti-oxidant and anti-inflammatoryactions that neutralize ROS production and inhibit the gener-ation of TNF-α through NF-κB.13 Thus, it decreases theadherence index at 2 and 4 h, just when the levels of TNF-αare increased in this endotoxic shock model.9 Chemotaxis

in lymphocytes from mice injected with LPS was increasedafter administration of NAC, which may be due to theinhibitory effect of NAC on TNF-α synthesis, whichdecreases MIF production. However, NAC increases theadherence and chemotaxis of lymphocytes in control animals,showing its immunostimulant action. This favourable effectof NAC on lymphocyte functions has been already found byother authors23 and it may be due not only to its anti-oxidantrole, but also to some specific metabolic actions such asthiolic compound.24 Thus, depending on the state of lympho-cytes, NAC can act by increasing or decreasing their func-tions in a way similar to the response of macrophages.9

Lymphocytes from aged men show an increased adher-ence and a depressed lymphoproliferative response to mito-gens compared with adult values in the majority of thispopulation, which was denoted the S group. This S groupshowed the typical age-related decline of T cell functions,mainly the lymphoproliferative response, as well as theincrease in adherence capacity.2,10 In previous work, we havefound that lymphocyte chemotaxis does not change withageing in this group. Adherence of immune cells increaseswith age, possibly as a consequence of chronic oxidativestress.2 Another less abundant population segment, called theNS group, showed a different behaviour in these lymphocytefunctions with values more similar to those found in adultmen, although the adherence was smaller and the prolifera-tion higher than those from adults. Thus, as it has alreadybeen pointed out,25 ageing is associated with a reduction in many immune responses in most, but not all, elderly individuals.

M De la Fuente and VM Victor52

Figure 3 Adherence index (AI) of human blood lymphocytesfrom old men (70 ± 5 years old) with standard (S; the more fre-quent values in this age) or non-standard (NS; less frequent valuein this age) values of lymphocyte function, before (SBE andNSBE) and after (SAE and NSAE) daily ingestion of 200 mgvitamin E for 3 months, as well as from adult control (AC) men(35 ± 5 years old). Each bar represents the mean ± SD of 12 (ACgroup), 17 (S group) or eight (NS group) subjects, each valuebeing the mean of duplicate assays. **P < 0.01 with respect to thecorresponding values before vitamin E ingestion. †††P < 0.001with respect to the corresponding value in the standard group.#P < 0.05, ###P < 0.001 with respect to AC group values.

Figure 4 Proliferation, in response to the mitogen PHA, ofhuman blood lymphocytes from old men (70 ± 5 years old) withstandard (S; the more frequent values in that age) or non-standard(NS; less frequent values in that age) values of lymphocyte func-tion, before (SBE and NSBE) and after (SAE and NSAE) dailyingestion of 200 mg vitamin E for 3 months, as well as from adultcontrol (AC) men (35 ± 5 years old). Each bar represents themean ± SD of 12 (AC group), 17 (S group) or eight (NS group)subjects, each value being the mean of duplicate assays.***P < 0.001 and **P < 0.01 with respect to the correspondingvalues before vitamin E ingestion. †††P < 0.001 with respect to thecorresponding value in the standard group ###P < 0.001 withrespect to AC group values.

If the oxidant/anti-oxidant balance is an important deter-minant of immune cell function, including the control ofsignal transduction and gene expression, optimal levels ofanti-oxidants will be needed for maintenance of immuneresponse especially in ageing.8 Thus, vitamin E supplementa-tion, specifically the intake of 200 mg/day, has been shown toimprove immune function in aged subjects.8,11 This anti-oxidant is necessary for improving immune function in theadult and even more necessary in old age, when vitamin Erequirements may be greater compared with those of adultgroups.25 In the present study, following administration of adaily dose of 200 mg of vitamin E during 3 months to bothgroups of subjects, a decrease in the SAE group and anincrease in the NSAE group of adherence were observed,whereas an increase in the SAE group and a decrease in theNSAE were seen in proliferation. The ingestion of vitamin Ebrought the values of adherence and proliferation to levelsmore similar to those found in adult controls. Because thepresence of multiple intracellular signalling deficienciescould be the cause of the impaired proliferative response of Tcells with ageing, a condition in which oxidative stress seemsto play an important role,2,10 vitamin E could regulate thislymphocyte function through its control on ROS levels, induc-tion of transcription factors such as NF-κB, phosphorylationof proteins or other molecular mechanisms. Another possiblemechanism could be the inhibitory effect of anti-oxidants onthe apoptosis process, which is a cause of the decline of func-tional T cells with ageing. Moreover, because there are datasupporting the idea that immune function in ageing is similarto that in inflammatory conditions and the anti-oxidants alsohave anti-inflammatory effects, they may act in this way onimmune functions.2 It has been found that vitamin E acts inreducing prostaglandin production by macrophages, whichcontributes to the age-associated decrease in T proliferation.8

Because the differences in the rate of ageing among individ-uals, noted in age-related changes such as those in intra-cellular signal transduction, vary among subjects of the samechronological age,2 the effect of anti-oxidants could also varyin the various groups of subjects.

In summary, the earlier data suggest that anti-oxidants,such as NAC and vitamin E, do not exert an indiscriminatestimulating effect on immune system against disturbanceslike those caused by endotoxic shock and ageing. Instead,they show an immunoregulatory effect, increasing or depress-ing immune functions depending on the cell state and bring-ing back these altered functions to optimum levels. In orderfor the immune system to function optimally and to maintainin vivo homeostasis, the anti-oxidant defence system has tosustain an adequate balance between oxidants and anti-oxidants in the organism, as has been recently pointed out.2

Acknowledgements

This work was supported by FIS (97/2078) and Comunidadde Madrid (08.5/0015/1997) grants.

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