Nitric Oxide 15 (2006) 50–57

www.elsevier.com/locate/yniox

Modulation of MCP-1 and iNOS by 50-Hz sinusoidal electromagnetic Weld

Marcella Reale a,¤, Maria Anna De Lutiis b, Antonia Patruno b, Lorenza Speranza b, Mario Felaco b, Alfredo Grilli b, Maria Antonietta Macrì e,f, Silvia Comani c,d, Pio Conti a,

Silvano Di Luzio c,d

a Department of Oncology and Neuroscience, Universita degli Studi G.D’Annunzio, Chieti, Italyb Department of Biomorphology, Universita degli Studi G.D’Annunzio, Chieti, Italy

c Department of Clinical Science and Bioimaging, Universita degli Studi G.D’Annunzio, Chieti, Italyd ITAB—University Foundation G.D’Annunzio, Chieti University, Chieti, Chieti, Italy

e Department of Experimental Medicine and Pathology, Universita degli Studi di Roma La Sapienza, Rome, Italyf INFM CRS-SOFT, Rome, Italy

Received 19 July 2005; revised 14 November 2005Available online 7 February 2006

Abstract

The purpose of this study was to investigate whether overnight exposure to 1 mT–50 Hz extremely low-frequency sinusoidal electro-magnetic Weld (EMF) aVects the expression and production of inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein-1 (MCP-1) in human monocytes. RT-PCR and Western blot analysis demonstrate that EMF exposure aVects the expression of iNOS andMCP-1 in cultured human mononuclear cells at the mRNA level and protein synthesis. Interestingly, the eVects of EMF exposure clearlydiVered with respect to the potentiation and inhibition of iNOS and MCP-1 expression. Whereas iNOS was down-regulated both at themRNA level and at the protein level, MCP-1 was up-regulated. These results provide helpful information regarding the EMF-mediatedmodulation of the inXammatory response in vivo. However, additional studies are necessary to demonstrate that EMF acts as a nonphar-macological inhibitor of NO and inducer of MCP-1 in some diseases where the balance of MCP-1 and NO may be important.© 2005 Elsevier Inc. All rights reserved.

Keywords: Extremely low-frequency electromagnetic Weld; iNOS; MCP-1; Monocytes

Electromagnetic Welds (EMFs)1 are believed to aVectsome enzyme activities, and some proteins are susceptibleto electric and magnetic Weld modulation [1]. In the last20 years, there has been increasing interest in investigatingthe possible eVects of extremely low-frequency (ELF)

* Corresponding author.E-mail address: mreale@unich.it (M. Reale).

1 Abbreviations used: ELF, extremely low frequency; EMFs, electro-magnetic Welds; MCP-1, monocyte chemotactic protein-1; RT-PCR,reverse-transcriptase polymerase chain reaction; WB, Western blot;iNOS, inducible nitric oxide synthase; NBT, nitroblue tetrazolium; LPS,lipopolysaccharide .

1089-8603/$ - see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.niox.2005.11.010

electomagnetic Welds (EMFs) on biological systems and onseveral cell functions [2–13].

ELF-EMF covers the frequency range of 3 Hz–3 kHz,and the most intensely studied frequency is the power fre-quency of 50/60 Hz. The guidelines for occupational expo-sure to power frequency magnetic Welds at 50 Hz are 0.5 mTfor the International Committee on Non-ionizing Radia-tion Protection (ICNIRP) and 0.1 mT for exposures to thegeneral public; 1.6 mT for the National Radiation Protec-tion Board (NRPB), 0.5 mT for the International RadiationProtection Association (IRPA), and 0.1 mT for exposuresto the general public; and 1 mT for the American Confer-ence of Governmental Industrial Hygienists (ACGI) [14].While many reports have demonstrated that ELF

M. Reale et al. / Nitric Oxide 15 (2006) 50–57 51

sinusoidal 50- to 60-Hz magnetic Welds may have adverseeVects on human health, other reports have provided noconclusive and consistent evidence supporting health haz-ards due to exposures to EMF [15,16]. In vitro studies,using ELF Welds (50 Hz), have attempted to resolve thisdebate by determining cell proliferation, apoptosis, expres-sion, and production of many molecules involved in cancerand inXammation. Numerous experimental works haveshown the possibility of modifying and controlling theselective permeability of the cell membrane by transmittingelectromagnetic waves. On these bases, various authorshave noted the modulation of some cell functions, fromionic membrane pumps to many cytoplasmic enzyme reac-tions, including those connected with cell replication[17,18].

In mammals, three distinct isoforms of nitric oxide syn-thase (NOS) have been cloned: endothelial (eNOS), neuro-nal (nNOS), and inducible (iNOS) NOS [19]. Eachisoenzyme has been implicated in speciWc diseases that aremediated by excess or in appropriate levels of NO. eNOSand nNOS are constitutive isoforms that can rapidly syn-thesize small amounts of NO following receptor stimula-tion; iNOS is a high-output, Ca2+-independent enzymewhose expression can be induced in a wide range of cellsand tissues by cytokines and other agents. The iNOSinduced by proinXammatory stimuli trigger resident andimmigrant inXammatory cell populations to produce largeamounts of NO for sustained periods of time, until theenzyme is degraded [20,21]. iNOS is mainly regulated on anexpression level, with transcriptional, posttranscriptional,and translational mechanisms involved. The stimuli andconditions that determine iNOS expression are cell and spe-cies speciWc, and often data have been contradictory. Onceexpressed, iNOS does not seem to be subject to any majorregulation of its enzymatic activity. Nitric oxide (NO), pro-duced by iNOS, acts as an important physiological media-tor, protecting and injuring cells. Although highconcentrations of NO can be beneWcial as an antibacterialand antitumor agent, an excess of NO or related nitrogenreactive species, such as peroxynitrite, can be detrimental oreven fatal. Their excessive production following NOSinduction can exert deleterious eVects in many acute inXam-matory responses and chronic diseases [22], and inhibitionof iNOS has great potential therapeutic value, such as inendotoxic shock, where levels of NO vastly exceed normalphysiological levels. Chemokines selectively attracting andactivating immune cells are involved in lymphocyte traYck-ing, angiogenesis, hematopoiesis, allergy, atherogenesis,neurodegenerative disease, and malignancies [23–26]. Che-mokines are used to subvert and divert eVective antiviral/antitumor immunity by amplifying polarized type Th2responses, playing a key role in guiding dendritic celltraYcking, and available information suggests that they areimportant for the localization of these cells in tumors.

Monocyte chemotactic protein-1 (MCP-1), the bestcharacterized member of the �-chemokines, is an importantmediator of monocyte/macrophage recruitment and activa-

tion at the site of chronic inXammation and neoplasia, andis involved in a variety of disease conditions. Chemokinesand iNOS play a central role in various immunological andinXammatory processes, mediating cellular and physiologi-cal responses. NO and MCP-1 reciprocally modulate theirexpression and play an important, but not fully deWnedrole, in the development and progression of many diseases;very little is known about the inXuence of EMF on NO gen-eration. The aim of this study was to investigate the eVect ofEMFs on MCP-1 and iNOS and to examine the alterationin the expression of iNOS and MCP-1 in EMF-exposedhuman monocytes. Our results support the hypothesis thatEMF exposure acts as a modulator of cell activation; thusit is now possible to estimate the eVect of magnetic Welds oninXammatory cells, studying iNOS and chemokines thatplay critical roles in various immunological and inXamma-tory processes.

Experimental procedures

Isolation of human peripheral adherent mononuclear cells

BuVy coats were collected from blood of 10 healthyindividual donors from the Transfusion Blood Bank Ser-vices of Chieti, Italy. All donors (Wve men and Wvewomen; age§ SD, 55.1§ 7.3) gave informed consent andno individuals were taking medications. Primary bloodmononuclear cells were isolated by density-gradient cen-trifugation through Ficoll/Hypaque (Pharmacia), sus-pended (8£ 106 cells/mL) in RPMI-1640 medium with10% heat-inactivated human serum (Sigma), and seededin Xasks. After incubation for 1 h at 37 °C, adherent cellswere detached, resuspended (2£ 106 cells/mL) in amedium supplemented with 10% fetal bovine serum(Sigma), and seeded onto six-well tissue culture plates.The cell viability, determined by trypan blue exclusion,was >99%. The same batch of serum and medium wereused in all experiments. The media and sera were of verylow lipopolysaccharide (LPS) content as determined bytesting the chromogenic assay using limulus amoebocytelysate (minimum detection level 0.1 �g/mL; WhittakerBioproducts, Walkersville, MD, USA).

EMF exposure system

All experiments were performed using a sinusoidal 50-Hz EMF at a Xux density of 1 mT (rms) produced by anelectromagnetic generator (Hewlett–Packard mod. 33120with stability higher than 1% both in the frequency and inthe amplitude). A current Xow of 1.20 A (IeV) passedthrough a 160-turn solenoid coil of length lD0.22 m andradius aD0.06 m. The copper wire thickness was1.25£ 10¡3 m. The generator was connected to a powerampliWer (Denon POA 2800). Cells were located in the cen-tral part of the solenoid, which presented the highest degreeof Weld homogeneity (98%). Magnetic Weld strength anddistribution within the solenoid were measured with a Hall

52 M. Reale et al. / Nitric Oxide 15 (2006) 50–57

eVect probe connected to a Gaussmeter (MG-3D, WalkerScientiWc, Worcester, MA, USA). Geomagnetic Weld inten-sity inside the incubator was about 40 �T; in the same area,the environmental magnetic noise due to power line (50 Hz)was about 7 �T (rms). The duration of cell exposure wasovernight. The experimental setup for exposure to magneticWeld is shown in Fig. 1.

Exposure conditions of cell cultures

Each experiment included two or three subjects. Exper-imental conditions were determined in preliminary experi-ment. Human peripheral adherent mononuclear cells(2£ 106/mL), obtained from each subject, were resus-pended in complete medium (RPMI-1640 supplementedwith 10% heat-inactivated fetal bovine serum, 100 U/mLpenicillin, and 100 �g/mL streptomycin). Cells from eachsubject, prepared following the same experimental proce-dure, were divided in four groups: (1) control cells; (2)cells stimulated with LPS (10 �g/mL); (3) control cellsexposed to EMF; (4) cells stimulated with LPS andexposed to EMF. Cells were seeded onto six-well tissueculture plate and incubated overnight at 37 °C in a humid-iWed atmosphere of 5% CO2. Nonexposed cell groups 1and 2 were placed in an area of the incubator far from thesolenoid, where only environmental magnetic contamina-tion can be detected. The cells of groups 3 and 4 wereplaced in the central area of the solenoid that was placedinside the incubator in order to maintain constant temper-ature and a 5% CO2 atmosphere. The temperature insidethe incubator was monitored by a built-in thermometer(TD 37§ 0.5 °C); a digital thermometer (HD 9216, DeltaOHM, Padua, Italy) was inserted at the sample position toquantify local temperature variations in proximity of cul-tures (�T D 0.2 °C). No signiWcant temperature changesrelated to applied ELF Welds were therefore observed. Atthose temperature values, no hypothesis of thermal eVectson cells can be made, because the interactions of ELFwith biological molecules are known to be of nonthermalnature [27]. The low-level Joule heating is eYciently dissi-pated inside the incubator by the fan system. Cell viabilitywas not inXuenced in nonstimulated nor in LPS-stimu-lated cells by Weld exposure. More than 98% of cells wereviable, as determined by trypan blue dye exclusion at the

Fig. 1. Schematic representation of a typical exposure system. The sole-noid was placed inside the cell incubator. The diameter of the solenoidwas a D 0.06 m and its length was lD 0.22 m. The number of solenoid turnswas 160, and the diameter of the copper wire was 1.3 £ 10¡3 m. B indicatesthe direction of the magnetic Weld.

beginning of the culture, and more than 90% were viablebefore cells were collected.

Semiquantitative RT-PCR for iNOS and MCP-1

Semiquantitative reverse-transcriptase polymerasechain reaction (RT-PCR) was used to determine mRNAlevels of the iNOS and MCP-1 in human peripheraladherent mononuclear cells. Total RNA was extractedusing 1 mL ULTRASPEC-RNA (Biotech Laboratories,Houston, TX, USA), as recommended by the manufac-turer. RNA was dissolved in diethyl pyrocarbonate(DEPC)-treated water and quantiWed spectrophotometri-cally at 260 nm. First-strand cDNA was generated by add-ing RNA (1 �g) to a mixture containing 1 mMdeoxynucleoside triphosphates (d-NTP), 1 U/�L RNaseinhibitor, 2.5 U/�L moloney murine leukemia virusreverse transcriptase, 2.5 �M oligo (dT), 5 mM MgCl2,10£PCR buVer in a Wnal volume of 20 �L. Reverse tran-scription was performed at 42 °C for 1 h followed by heatinactivation of reverse transcriptase at 92 °C for 10 min.18S was ampliWed from the same amount of RNA to cor-rect for variation of diVerent samples. PCR ampliWcationwas performed using a Programmable Thermal Control-ler (MJ Research, Watertown MA, USA). The PCR solu-tion contained 10 �L of Wrst-strand cDNA, 4 �L 10£ PCRbuVer, and 2 mM MgCl2. The following primer pairs(MWG-Biotech AG, Milan, Italy) were used: sense5�-CGT AAA GAC CTC TAT GCC AA-3� and antisense5�-AGC CAT GCC AAA TGT CTC AT-3� for iNOS;sense 5�-TAGCAGCCACCTTCATTCC-3� and anti-sense 5�-TTCCCCAAGTCTCTGTATCT-3� for MCP-1;and 18S primers, 0.15 mM of both sense 5�-TAC GGAGCA GCA AAT CCA C-3� and antisense 5�-GAT CAAAGG ACT GCA GCC TG-3�, 2 U Thermophylus acquati-cus (Taq) DNA polymerase (Celbio, Milan, Italy), andwater to a Wnal volume of 50 �l. These samples were over-laid with mineral oil and subjected to 35 cycles at 95 °Cfor 60 s, 60 °C for 60 s, and to 1 cycle at 72 °C for 7 min foriNOS and 40 cycles at 95 °C for 60 s, 58 °C for 60 s, and toone cycle at 72 °C for 7 min for MCP-1. PCR productswere run on 2% agarose gel electrophoresis and photo-graphed after ethidium bromide staining under UV light.Bands on the gel were scanned using a computerized den-sitometric system (Bio-Rad Gel Doc 1000, Bio-Rad,Milan, Italy).

Isolation of nuclei

Cells, resuspended in 10 mM Tris–HCl, pH 7.4, 10 mMNaCl, 2 mM MgCl2, 0.6% Triton X-100, 1.0 mM PMSF,1 mg/mL leupeptin, and aprotinin, were incubated at roomtemperature for 2 min, then cooled on ice for 5 min. AfterWve passages through a 22-G needle, MgCl2 concentrationwas adjusted to 5 mM. Nuclei were obtained by centrifug-ing the suspension at 1200g for 15 min and cytoplasmicfractions consisted of the postnuclear supernatants. Nuclei

M. Reale et al. / Nitric Oxide 15 (2006) 50–57 53

were then harvested in RIPA buVer (1£ PBS, 1% NonidetP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/mLPMSF, 100 mM sodium orthovanadate, and 1 mg/mL leu-peptin and aprotinin).

Western blot analysis for iNOS, MCP-1, and NF-�B

Determination of iNOS, MCP-1, and proteins wereperformed in three series of protein extracts by Westernblotting. 50 �g cytoplasmatic and 10 �g nuclear proteins,quantiWed, by spectrophotometric assay (HP 8452A, PaloAlto, CA, USA) using Lowry method, from humanperipheral adherent mononuclear cells were separated byelectrophoresis in a 7.5% sodium dodecyl sulfate–poly-acrylamide gel (SDS–PAGE; Bio-Rad, Hercules, CA,USA) and transferred at 4 °C to nitrocellulose membrane(Bio-Rad, Hercules, CA, USA) in glycine–methanolbuVer. Nitrocellulose was then blocked in Tris-buVeredsaline (TBS)–milk and incubated, overnight, with variousprimary antibodies: anti-human iNOS (Santa Cruz Bio-technology, Santa Cruz, CA, USA), anti-human MCP-1,and anti-human nuclear factor-�B (NF-�B) (Santa CruzBiotechnology). The nitrocelluloses were then washed inTBS, incubated with a secondary antibody conjugatedwith alkaline phosphatase for 2 h, washed again, anddeveloped in an alkaline buVer with nitroblue tetrazolium(NBT) as substrate (Alkaline Phosphatase ConjugateSubstrate Kit, Bio-Rad, Hercules, CA, USA). �-Actin(Sigma, 1/10,000) was used as an internal standard. Thedensitometric analysis of Western blots was performedusing a computerized densitometric system (Bio-Rad GelDoc 1000, Milan, Italy).

Citrulline synthesis (NO activity)

The L-arginine to L-citrulline conversion is a standardassay to measure nitric oxide synthase (NOS) activity.BrieXy, 1 �l of radioactive arginine, L-(2,3,4,5)-[3H]argi-nine monohydrochloride (1 �Ci/�L; Amersham, Arling-ton Heights, IL, USA), 5 �L of 10 mM NADPH, 25 �Lreaction buVer 2£ (50 mM Tris–HCl (pH 7.4), 5 �M BH4,2 �M FAD, 2 �M FAM) were added to each cell homoge-nate samples and incubated for 30 min at room tempera-ture. After incubation, the reactions were stopped with400 �L of stop buVer (50 mM Hepes, pH 5.5, 5 mMEDTA) and added the equilibrated resin into each sam-ple. The equilibrated resin bonded unreacted arginine.After centrifugation, the radioactivity corresponding to L-[3H]citrulline was measured with liquid scintillation spec-trometry. Calcium was omitted from these incubations tofavor the determination of the calcium-independent iNOSisoform.

Statistical analysis

Values are expressed as the mean§SEM; each grouphad 10 samples (nD10), each sample was performed in trip-

licate. Data were analyzed using one-way analysis of vari-ance (ANOVA). The level of statistically signiWcantdiVerence was deWned as p < 0.05. The statistical analyseswere performed using the SPSS 10.0.1 for Windows.

Results

Spontaneous and LPS-induced MCP-1 and iNOS mRNA expression and protein production

Fig. 2 shows that unstimulated monocytes spontaneouslyexpress very low levels of MCP-1 and iNOS. Upon stimula-tion with LPS, MCP-1, and iNOS, protein productionincreased signiWcantly (p < 0.05). The increase after LPS stim-ulation was similar for all groups of cells under examination.Mononuclear cells preincubated for 1h with L-NAME(100�M) and then stimulated overnight with LPS expresslower iNOS protein level and higher MCP-1 protein levelthan cells without L-NAME preincubation (p< 0.005).

EVect of electromagnetic exposure on iNOS

The eVect of 50-Hz EMF on monocytes from healthy sub-jects was studied. There was no diVerence in the viability ofthe exposed and unexposed cells (data not shown). An exam-ple of bands seen on 2% gel with RT-PCR products is shownin Fig. 3A as a representative experiment out of similarresults derived, respectively, from each subject. mRNAexpression of iNOS after overnight incubation at 37°C in ahumidiWed atmosphere of 5% CO2 from (1) control cells; (2)cells stimulated with LPS (10�g/mL); (3) control cellsexposed to EMF; and (4) cells stimulated with LPS andexposed to EMF is shown. EMF exposure signiWcantlyreduced the mRNA expression of iNOS in LPS-stimulatedcells (p < 0.01). To determine the eVects of EMF on iNOSprotein, a Western blot was performed (Fig. 3B). The resultsconWrmed those obtained by RT-PCR. Table 1 summarizesthe quantitative densitometry for iNOS of samples collectedfrom control, LPS, control + EMF, and LPS+ EMF. Dataexpressed as integrated optical density (IOD) aremean§SEM values for 10 subjects individually analyzed.

Fig. 2. EVect of L-NAME preincubation on iNOS and MCP-1 proteinexpression in LPS-stimulated mononuclear cells. A representative experi-ment of separate experiment (nD 3) was shown. The gels were counter-stained for �-actin to assure the application of equivalent cell extracts.

54 M. Reale et al. / Nitric Oxide 15 (2006) 50–57

iNOS activity

Basal L-[3H]citrulline production from L-[3H] argininewas detectable in homogenates obtained from each sample,and no signiWcant diVerence was observed between unstim-ulated cells exposed, or not, to EMF. iNOS activity wasreduced signiWcantly in LPS-stimulated cells exposed to themagnetic Weld when compared to LPS-stimulated cells notexposed to the magnetic Weld (Fig. 4). This reduced enzymeactivity was related with the reduced iNOS mRNA andimmunoreactive iNOS protein observed above.

EVect of electromagnetic exposure on MCP-1 expression and production

To determine the eVect of EMF at 50 Hz exposure onexpression and production of MCP-1, peripheral bloodadherent mononuclear cells were incubated overnight withor without 10 �g/mL LPS and exposed to EMF. As shownin the representative experiment (Fig.5A), RT-PCR ampliW-cation demonstrates that MCP-1 mRNA is expressed inlarger quantities in both nonstimulated and LPS-stimu-lated cells exposed to EMF. Western blot analysis was per-formed to determine the eVects of EMF on MCP-1 protein(Fig. 5B). The results obtained conWrm the values obtained

Fig. 3. EVect of ELF-EMF on iNOS. (A) RT-PCR shows iNOS mRNAexpression in a representative experiment of separate experiment (n D 10).Ribosomal mRNA (18S) and commercial molecular weight standards (S)were employed as internal and external standards, respectively. (B) West-ern blots are illustrated for iNOS protein extracted from PBMC. Immu-noreactivity for iNOS was identiWed at a position in the gel consistentwith a mass of 130 kDa. The gels were counterstained for �-actin to assurethe application of equivalent cell extracts.

Table 1Quantitative densitometry (IOD § SEM) of iNOS of samples collectedfrom control, LPS, control + EMF, and LPS + EMF

Comparison between LPS and LPS + EMF cells show statistically signiW-cant diVerences (p < 0.005, FD 273.2; n D 10).

iNOS Control LPS Control + EMF LPS + EMF

RT-PCR — 6§ 0.5 — 2 § 0.5Western blotting 2.2 § 0.5 9§ 1 2 § 0.5 4 § 1

by RT-PCR. Table 2 summarizes the quantitative densi-tometry (Western blot and RT-PCR) for MCP-1 of sam-ples collected from control, LPS, control + EMF, andLPS + EMF. A signiWcant correlation was observedbetween MCP-1 and iNOS production in both basal(�D¡0.36, p < 0.005) and LPS-induced cells (�D¡0.59,p < 0.005) exposed to ELF-EMF (Fig.6).

EVect of electromagnetic exposure on NF-�B expression

We performed experiments to obtain evidence that ELF-EMF caused changes on NF-�B expression. Western blotanalysis shows that NF-�B protein expression wasincreased in basal and LPS-stimulated mononuclear cells

Fig. 4. iNOS activity in human peripheral blood mononuclear adherentcells. iNOS activity was reduced in both nonstimulated cells and LPS-stimulated cells—magnetic-Weld exposed. Values are means§ SEM of 10experiments (p < 0.05 for each conditions).

Fig. 5. (A) RT-PCR analysis of MCP-1 mRNA expression in humanmonocytes. Cells were incubated overnight at 95% O2 and 5% CO2 andexposed, or not, to 50-Hz ELF-EMF. Therefore, total RNA wasextracted, and RT-PCR was carried as indicated under “ExperimentalProcedures.” An example of bands seen on 2% gel with RT-PCR productswas shown as a representative experiment out of similar results derivedfrom unstimulated and LPS-stimulated monocytes puriWed from eachhealthy subjects (n D 10). Ribosomal mRNA (18S) and commercialmolecular weight standards (S) were employed as internal and externalstandards, respectively. (B) Western blots are illustrated for MCP-1 pro-tein extracted from PBMC. Immunoreactivity for MCP-1 was identiWedat a position in the gel consistent with a mass of 8.7 kDa. The gels werecounterstained for �-actin to assure the application of equivalent cellextracts.

M. Reale et al. / Nitric Oxide 15 (2006) 50–57 55

exposed overnight to ELF-EMF (Fig. 7). Comparisonbetween basal and LPS with basal + EMF and LPS + EMFcells show statistically signiWcant diVerences (p < 0.005).

Discussion

Since the immune system plays a primary role in the con-trol of many diseases and tumor growth, many laboratorieshave investigated the inXuence of ELF-EMFs in bloodmononuclear cells, various cellular components, and cellu-

Table 2Quantitative densitometry (IOD § SEM) of MCP-1 of samples collectedfrom control, LPS, control + EMF, and LPS + EMF

Comparison between LPS and LPS + EMF cells show statistically signiW-cant diVerences (p < 0.005, F D 3567.7; n D 10).

MCP-1 Control LPS Control + EMF LPS + EMF

RT-PCR 0.8§ 0.09 3 § 0.12 2 § 0.2 8 § 0.2Western blotting 1 § 0.09 3 § 0.16 3 § 0.2 9 § 0.09

Fig. 6. Relationship of MCP-1 and iNOS syntheses by human monocytes,NO-treated or LPS-treated and exposed overnight to 50-Hz EMF.

Fig. 7. Cells were incubated overnight at 95% O2 and 5% CO2 andexposed, or not, to 50-Hz ELF-EMF. Western blotting analysis of NF-�Bnuclear expression from PBMC was performed. Immunoreactivity forNF-�B was identiWed at a position in the gel consistent with a mass of65 kD.

lar processes; other studies have examined EMF eVects onspeciWc gene expression and signal transduction pathways,but the experimental data obtained are currently controver-sial [28–30].

The recruitment of monocytes/macrophages to inXam-matory sites and neoplastic tissues and their activationtherein is crucial to the success of an immune reaction, inpart, because cell migration is intimately related to leuko-cyte function. Upon activation, monocytes/macrophagesacquire microbicidal/tumoricidal capacity increasing theproduction of chemokines, peroxidases, cytolytic proteases,and NO. NO, produced by iNOS, is a free radical that wasoriginally reported as a physiologic mediator in the vascu-lar, neuronal, and immunological systems. Although highconcentration of NO can be beneWcial as an antibacterialand antitumor agent and a relative deWciency of NO isresponsible for some clinical conditions, an excess of NOcan be fatal and lead to cell injury; so the activity of iNOShas detrimental eVects on oligodendrocyte, cells responsiblefor the myelination of neuron in the CNS [31–33]. Recently,data are accumulating on a protective eVect of high-outputNO synthesis and—equally important—on a gene regula-tory function that helps to mount a protective stressresponse and simultaneously aids in down-regulating theproinXammatory response, but these Wndings appear tocontrast the often observed sustained iNOS expression dur-ing chronic inXammatory diseases and during chronic Th1-like reactivity [34]. The roles of NO in the pathophysiologyof disease are still being deWned, but there is a growing evi-dence that the neutralization of iNOS activity may have atherapeutic value [35,36]. Early studies have focused on thepotential toxicity of the ensuing high-output NO-synthesisserving as a means to eliminate pathogens or tumor cells,but the expression of the iNOS, contributing to local tissuedestruction during chronic inXammation, is one of thedirect consequences of an inXammatory process. Since theirdiscovery, chemokines have emerged as important regula-tors of leukocyte traYcking, and MCP-1, one of the best-studied � chemokines, is known to exert multiple eVects ontarget cells, such as increased cytosolic calcium levels,superoxide anion production, lysosomal enzyme release,production of antiinXammatory cytokines and adhesionmolecules in monocytes. MCP-1 is involved in the induc-tion of polarized type Th2 responses and in the enhance-ment of IL-4 production. A possible feedback loop for Th2activation would be the production of IL-4 and IL-13 byTh2, which stimulates MCP-1 production and leads to fur-ther recruitment of Th2 cells [37]. NO increases the abilityof monocytes to respond to chemotactic agents more eVec-tively, and it is considered to be one of the principal eVectormolecules involved in macrophage-mediated cytotoxicity[38].

Previous studies suggest that magnetic Welds areinvolved in the alteration of NO production, probablyrelated to a constitutive form of NOS rather than to aninducible form [39,40]. To understand the eVect of low-fre-quency EMF on iNOS and MCP-1, we exposed human

56 M. Reale et al. / Nitric Oxide 15 (2006) 50–57

monocytes to magnetic Welds of 50 Hz, and after an over-night incubation, iNOS and MCP-1 mRNA expressionswere analyzed by RT-PCR and protein production byWestern blotting. The eVects on iNOS gene expressionclearly diVered with respect to the potentiation of MCP-1expression. In our results, we report that, in LPS-stimulatedcells exposed to 50-Hz EMF, iNOS was down-regulated atthe mRNA and protein level, whereas MCP-1 was up-regu-lated, as shown independently by RT-PCR and Westernblot analysis. The changes in MCP-1 and iNOS mRNAexpression and protein production are interesting becausethese molecules are critical throughout the development ofthe inXammatory response. Regulation of the inXammatoryresponse involves production of cytokines and chemokinesas well as reactive oxygen and nitrogen intermediates; thereis increasing evidence that NO could be involved in the up-regulation of cytokine [34]. The process of tissue regenera-tion after injury involves inXammation that is driven bygrowth factors, cytokines and chemokines, released in thearea of injury. In response to nonspeciWc stress, MCP-1mRNA expression increases, subsequently a protein kinaseC (PKC)-dependent mechanism leads to the activation ofiNOS and release of NO [41]. Extensive evidence suggeststhat locally produced NO regulates the expression of MCP-1 and that NO is directly involved in the speciWc up-regula-tion of MCP-1 [42,43].

A variety of diVerent mechanisms for the eVects of EMFon cells have been proposed by a number of investigators.The cell membrane plays an essential role in mediating sig-nal transduction events, and it has been suggested to be theprimary interaction site of EMF signals [44]. Calcium ionXux [44,45], activity of PKC, and electrons moving duringredox reactions have been proposed to explain magneticWeld eVects [46,47].

The present study shows that iNOS and MCP-1 expres-sion are both modiWed after exposure to ELF-EMF, andthese modiWcations are related to each other and are med-iated by the increased NF-�B protein expression. Ourresults are in accordance with previous Wndings that showthe inhibition of nitric oxide generation by endothelialcells increases MCP-1 mRNA levels through increasedredox-sensitive transcription NF-�B-binding activity [41].Our observations provide additional evidence in favor ofthe hypothesis that NO-mediated modulation of MCP-1expression occurs through a redox-sensitive mechanism.To understand better the relationship between iNOS andMCP-1, we performed supplementary experiments incu-bating mononuclear cells with an iNOS inhibitor (L-NAME, 100 �M).The results obtained show a signiWcantreduction of iNOS and an increase of MCP-1 levels, andthese eVects are consistent with iNOS and MCP-1 levelmodiWcations observed in mononuclear cells exposed toELF-EMF.

In summary, EMF may represent a nonpharmacologi-cal inhibitor of NO and an inducer of MCP-1, which acti-vates one of these molecules and lead to inhibition of theother and vice versa, establishing a mechanism that pro-

tects cells from excess stimulation and contributes to theregulation of cell homeostasis. We hypothesize that it maybe important to assess the potential application of EMFin some diseases where the balance of MCP-1 and NOmay be important to regulate the inXammatory response.Future in vivo studies will hopefully shed light on theeVects of EMF on iNOS, MCP-1, and other inXammatorymediators.

Acknowledgments

Authors are grateful to Mirella Salvatore for helpful dis-cussion, and to Renato Barbacane for the preparation ofthis manuscript.

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