0892-6638/92/0006-31 77/$01 .50. FASEB 3177

?magnetic fI4d eflccts on ee11soft1t1*1e of calcium signaling

J 1LLECZW,Rs#{231}ardMedicine nd Radiation Bigpltyeics Dvssion, Lawencc Bei*dey&xkdcy Cahfornia 9472OL USA

immune sten;

abatory, unkvcreity of Cei1ittnia

ABSTRACT During the past decade considerable evi-dence has accumulated demonstrating that nonthermalexposures of cells of the immune system to extremely low-frequency (ELF) electromagnetic fields (< 300 Hz) canelicit cellular changes that might be relevant to in vivoimmune activity. A similar responsiveness to nonionizingelectromagnetic energy in this frequency range has alsobeen documented for tissues of the neuroendocrine andmusculoskeletal system. However, knowledge about theunderlying biological mechanisms by which such fieldscan induce cellular changes is still very limited. It isgenerally believed that the cell membrane andCa2-regulated activity is involved in bioactive ELF fieldcoupling to living systems. This article begins with ashort review of the current state of knowledge concerningthe effects of nonthermal levels of ELF electromagneticfields on the biochemistry and activity of immune cellsand then closely examines new results that suggest a rolefor Ca2 in the induction of these cellular field effects.Based on these findings it is proposed that membrane-mediated Ca2 signaling processes are involved in themediation of field effects on the immune system.-Walleczek, J. Electromagnetic field effects on cells of theimmune system: the role of calcium signaling. FASEB J.6: 3177-3185; 1992.

Key Wonir: immune system signal transduction calcium cellularmechanisms ELF electromagnetic fields

THE BIOLOGICAL EFFECTS OF Low-ENERGy electromagneticfields (EMF5)3 have fascinated scientists for a long time. Butit is only during the past decade that the study of EMF inter-actions with whole organisms or isolated cells has become anincreasingly recognized area of research within the biologicalsciences. The main reasons for this development are: 1) anincreasing number of experimental findings are reportedeach year and possible theoretical explanations of weak EMFbioeffects begin to emerge (for reviews see refs 1-9); 2) evi-dence has accumulated demonstrating the efficacy of low-energy; ELF-pulsed magnetic fields in medical therapy, espe-cially in non-union bone fracture healing, confirming thatunder certain conditions nonionizing electromagneticenergy can influence physiological processes in organisms(10, 11); 3) from the point of view of public health, there isnow a growing demand for the study of possible adversehealth effects from interactions between the human body andEMFs generated, for example, by 50-60 Hz high-voltagetransmission lines, video display terminals, electric blankets,or clinical nuclear magnetic resonance-imaging procedures.Investigations of the possible physiological effects of EMFsare necessary especially because epidemiologists have al-ready found, for example, that an association might exist be-

tween magnetic fields in the home, as assessed indirectly us-ing the Wertheimer-Leeper wiring code (12), and elevatedchildhood leukemia risks (12-15). However, a cause-effectrelationship between EMF exposure and increased cancerrisk in principle cannot be established from such work alone.Therefore, in order to assess the relevance of the epidemio-logical findings, it is critical to understand how weak EMFscan interact with biological activity under controlled labora-tory conditions.

In light of the epidemiological results, it is worthwhile toreview the current state of knowledge regarding the effects ofextremely low-frequency (ELF) EMF signals on the immunesystem. Because the immune system functions as the bodysmain protective mechanism against invasion by pathogensand against tumor formation and growth, EMF-inducedchanges in immune cell biochemistry could affect the organ-isms immune response directly in either a negative or posi-tive manner. The physiological significance of the epidemio-logical results as well as of the reported immunological EMFeffects, however, cannot be fully evaluated until 1) there areconvincing results from whole organism exposure studiesthat can be directly related to the in vitro evidence, and 2)the biological mechanisms by which weak EMFs may inter-act with cells of the immune system begin to be understood.With regard to in vivo exposure effects on the organisms im-mune system, it has already been demonstrated in several in-dependent laboratories that nonthermal ELF EMF intensi-ties can modify blood leukocyte counts (16, 17),inflammatory responses (18, 19), peripheral blood naturalkiller (NK) cell activity (20), and the appearance or weightof primary and secondary lymphoid organs (20, 21). In noneof the cited in vivo studies, however, have experimentalanimals been exposed to ELF EMFs at levels as low as thoselinked by epidemiologists to increased cancer risks. Withrespect to the underlying cellular mechanisms of interactionbetween the immune system and EMFs, one can only specu-late. Nevertheless, there is new evidence from several in-

From the Symposium Recent Advances in Understanding Elec-tromagnetic Energy Interactions With Biological Systems spon-sored by the International Society for Bioelectricity at the 75th An-nual Meeting of the Federation of American Societies forExperimental Biology, April 24, 1991, Atlanta, Georgia.

2Present address: Jan Walleczek, Research Service, Jerry L. Pet-tis Memorial Veterans Hospital, 11201 Benton St., Loma Linda,CA 92357, USA.

3Abbreviations: EMF, electromagnetic field; ELF, extremely lowfrequency; HPBL, human peripheral blood lymphocytes; Con A,concanavalin A; PHA, phytohemagglutinin; LCa2I1, cytosolic freecalcium concentration; J, electrical current density; E, electric fieldintensity; B, magnetic flux density; NK cells, natural killer cells;CTLL-1, murine cytotoxic T cells.

3178 Vol. 6 October 1992 The FASEB Journal WALLECZEK

dependent research groups suggesting that Ca2 signals playa key role in the mediation of EMF effects in cells of the im-mune system.

This review summarizes representative findings of ELFEMF exposure studies using immune cells and to discuss re-cent results suggesting that the observed EMF influences canbe explained at least to some extent by the interaction of anexternal EMF with transmembrane Ca2 signaling events.The immunological effects of in vivo exposures to EMFs andthe effects of field signals oscillating in the radio and micro-wave frequency range will not be addressed. The review firstoutlines biophysical concepts frequently used in the charac-terization and description of EMF bioeffects. Next, it offersa brief overview of representative EMF effects on the bi-ochemistry and in vitro activity of normal and transformedcells of the immune system. Then the role of Ca as a possi-ble mediator of EMF influences on the immune system, inparticular during cell membrane-mediated signal transduc-tion processes, is evaluated. Finally, selected theoretical ap-proaches that seek to identify the physical basis of nonther-mal cellular field effects are mentioned and conclusions aredrawn.

BIOPHYSICAL BACKGROUND

Nonthermal electromagnetic field effects

EMFs and radiation in the frequency range from 0 Hz toseveral hundred GHz are known to be nonionizing forms ofenergy, because their quantum energy is too low to causephysicochemical or biological effects by ionization ofmolecules. This is illustrated by the fact that even relativelystrong, power frequency (50/60 Hz) EMFs were unable toproduce genotoxic effects in lymphocytes (22-24), whereasappropriate doses of ionizing radiation routinely producechromosomal damages in these cells. This is the major rea-son why biologists and physicists have long held that non-ionizing electromagnetic energy in the ELF, low-frequency,radio, and microwave frequency range can produce detecta-ble effects only via mechanisms involving significant heatingin cells, such as the nonexcitable cells of the immune system.During the past 10 years, however, bioelectromagneticsresearch has shown that nonionizing electromagnetic energycan indeed induce a variety of biological effects not only bythermal interactions but also through interaction mechan-isms that do not involve any macroscopic heating (this meansthat the global temperature rise in the exposed biologicalsample, due to the EMF influence, is generally less than0.001#{176}C;e.g., ref 10). Low-intensity field effects, which ap-parently are not induced by thermal interactions, arereferred to in the literature as athermal, or alternatively,nonthermal field effects.

Biophysical concepts and units

In this section concepts and units frequently used in bioelec-tromagnetics research are briefly described to give the readerunfamiliar with the terminology some useful background: adescription of these concepts and quantities allows compari-son of the field exposure conditions based on parameterssuch as the applied magnetic flux density, electric field inten-sity, signal frequency, and current density between the differ-ent exposure experiments. (For a detailed introduction, seethe book edited by Polk and Postow (25).)

The electric field intensity (E) is given in volts per meter(Vim), where volt is the unit of the electric potential. When

a biological system (cell, tissue, or whole organism) is ex-posed to an electric field, the mobile charges in the systemwill be forced to move in the direction of the induced electricfield lines, hence establishing an electric current measured inamperes (A). The distribution of this current and the direc-tion of its flow are defined by the current density U)quantified in amperes per square meter (A/rn2). In the bioe-lectromagnetics literature, E and J are frequently defined inmV/cm and pA/cm2 instead of the standard units Vim andA/rn2. Therefore, in keeping with this convention the unitsmV/cm and pA/cm2 will be used throughout this article.

If a time-varying magnetic field is applied to an electri-cally conductive material, such as living matter, an electricfield is induced in a direction perpendicular to the magneticfield vector in accord with Faradays law of induction. For anidealized, linearly behaving homogenous system that is ex-posed to a spatially uniform sinusoidal magnetic field, Fara-days law can be stated: E = irrfB, where r is the radius inmeters of a circular conductive path in the magnetic field-exposed object, f is the frequency in Hz, and B is the mag-netic flux density that defines the intensity of the magneticfield (for details, see ref 25). The unit of B is tesla (T). Anolder unit for B is gauss (G), where I T = 10 G. In thisreview, a magnetic field is termed weak if it is of nonthermalintensity and B 0.1 mT (1 G), whereas if B < 0.1 mT itis considered a very weak field. For the experiments describedhere, the magnitude of the applied magnetic fields rangesfrom 0.02 to 22 mT (0.2-220 G). For comparison, the inten-sity of the static earth magnetic field environment is from0.02 to 0.07 mT (0.2-0.7 G). The peak magnetic flux densi-ties used in EMF-faciitated bone healing range from 1 to 16mT (10-160 G; ref 26), and peak flux density values as-sociated with switched gradient magnetic fields in existingand planned clinical NMR imaging systems are on the orderof 0.1 to 10 mT (1-100 G; ref 27).

ELECTROMAGNETIC FIELD EFFECTS ON CELLS OFTHE IMMUNE SYSTEM

Effects from exposures to time-varying magnetic fields

To date at least 10 laboratories have carried out indepen-dent studies with immune cells by investigating possible non-thermal effects of time-varying magnetic fields of intensitiesin the range from 0.1 to 10 mT or more on Ca metabolism,RNA transcript levels, and RNA or DNA synthesis (22, 23,28-36). Of the laboratories, nine have published evidencedemonstrating nonthermal field effects on 1) intracellularfree Ca24 concentration ([Ca24]1) or mitogen-dependent45Ca24 uptake; 2) [3H]uridine uptake or specific gene tran-script levels; and 3) [3H]thymidine uptake or cell cycle ki-netics (see Table 1). Laboratory results reporting the absence of any detectable field effects (22), however, do notnecessarily contradict the positive findings of the otherresearch teams, because these investigators have used mag-netic fields that were about 10- to 30-fold weaker in intensitycompared with the magnetic fields used in the other studies(for comparison, see Table 1: effects on DNA synthesis).

In addition to the studies using magnetic field intensitiesof 0.1 mT (1 G) and more (see Table 1), there are at least tworeports indicating that ELF magnetic fields even at intensi-ties as low as 0.02 mT could possibly affect lymphocyte45Ca uptake (37, 38). Consistent with these data arepreliminary results from this laboratory that have been

TABLE 1. Low-frequency magnetic field exposure effects on cells of the immune system: representative reports from 10 dfferent laboratories

ELECTROMAGNETIC FIELDS AND THE IMMUNE SYSTEM 3179

Description of field effect

Exposure parameters

f, Hz B, mT E, mV/cm t, h Reference

Effects on calcium metabolism70% reduction (P < 0.001) in PHA-dependent45Ca24 uptake in HPBL. No effect on restingcells.

3.0 square5 6.0 n.r. 1.0 28, 29

170% increase (P < 0.01) in Con A-dependent45Ca2 uptake in rat thymic lymphocytes.No effect on resting cells.

28% increase (P < 0.01) in cytosolic free Ca2concentration in HL-60 cells exposed tomagnetic fields generated by an NMR unit

60.0 sine

0-100

22.0

0.1

1.0 (16.0)

n.r.

1.0

0.38

33

34

Effects on RNA transcript levels and RNA synthesis50-100% increase in c-myc, histone H2B,fl-actin RNA-transcript levels in HL-60 cells.

60.0 sine 1.5 n.r. 0.3 32

30-50% increase in 13H]uridine uptake inHL-60 cells.

60.0 sine 1.0 0.034 0.5 36

Effects on DNA synthesis55% reduction (P < 0.01) in [3H]thymidineuptake in PHA-activated HPBL. No effects onresting cells.

3.0 square 6.0 n.r. 72 28, 29

Up to 60% reduction (P < 0.001) in[3H]thymidine uptake PHA-activated HPBL.No effects on resting cells.

3.0 saw tooth 4.5

.

0.23 72 30

No effect on the cell cycle progression ofPHA-activated HPBL.

60.0 sine 0.2 n.r. 69 22

30-60% increase (P < 0.0001) in [3H]thymidineuptake in mitogen-activated HPBL, dependingon mitogen concentration and donor age. Noeffects on resting cells.

50.0 sawtooth 2.5 0.02 66 31

8% increase (P < 0.0001) in cell cycleprogression of PHA-activated HPBL.

50.0 sine 5.0 n.r, 48 23

30% reduction (P < 0.01) in [3H]thymidirieuptake in Con A-activated HPBL. No effectson resting cells.

50.0 sawtooth 10.0 n.r. 72 35

f, signal frequency; B rms magnetic flux density; E, induced electric field intensity; t, exposure duration. Description of wave form. Peakmagnetic flux density. Electric current density, J, [iAJcm2]. Approximate range of the frequency components (B 0.1 mT) generated by theNMR unit. n.r., not reported.

reported in abstract form.4: it was observed that 45Ca up-take in concanavalin A (Con A)-activated rat thyrnic lym-phocytes can be enhanced by 60% (P < 0.01) during 1 h ex-posure to a 0.021 mT, 14.3 Hz magnetic field in the presenceof an 0.021 mT static magnetic field. No effect was detectedin resting cells. Although these reports together provide evi-dence that even very weak fields (B < 0.1 mT) can affect cel-lular Ca2 regulation under certain experimental conditions,these results are mentioned only for the purpose of complete-ness and will not be discussed further. The reason is that noreports of field effects on other relevant cellular parametersin lymphoid cells (e.g., cell proliferation) that could berelated to field-induced changes in Ca regulation have beenpublished for magnetic field intensities as weak as 0.02 mT.Thus, the role of Ca24 signals in mediating immune cell fieldeffects at very weak field intensities cannot be assessed yet.In contrast, for higher field intensities (B 0.1 rnT) thereis now enough information from several studies to allow ex-amination of the potential role of Ca in the induction ofcellular field effects (see Table I).

Effects from the application of electrical currents

Research on the effects of electrical currents applied overagar-bridges to prevent possible side effects from electrodeelectrolysis products in the absence of an applied magneticfield on immune cell biochemistry and activity hasprogressed more slowly. There are currently only four in-dependent reports available (see Table 2): three of the fourreports demonstrate that 60 Hz sinusoidal electrical currentscan modify ornithine decarboxylase activity in field-exposedhuman lymphoma cells (39) or lymphocyte-mediatedcytotoxicity when either the target or the effector cells arefield-exposed before the cytotoxicity assay (40, 41). In con-trast, proliferation of neither phytohemagglutinin (PHA)-

4Abstract E-3-2: Combined DC/AC Magnetic Fields Alter Ca2Metabolism in Activated Rat Thymocytes. J. Walleczek and R. P.Liburdy. 12th Annual Meeting of the Bioelectromagnetics Society,June 1990, San Antonio, Texas.

TABLE 2. Electric field effects from the application of 60 Hz sinusoidal electric currents via electrodes (over agar-bridges) to cultured cells

3180 Vol. 6 October 1992 The FASEB Journal WALLECZEK

Description of field effect

Exposure parameters

ReferenceJ, iA/cm2 E,mV/cm t, h

No effect on cell cycle progression of 30.0 2.4 69 22PHA-activated HPBL.

Threefold increase in ornithine 160.0 10.0 1 39decarboxylase activity in humanlymphoma CEM cells.

53-73% increases in human natural killer 30.0 n.r. 24 40cell-mediated cytolysis of field-exposedtarget cells.

19-25% reduction (P < 0.005) of murine n.r. 1-10.0 48 41T lymphocyte cytotoxicity (CTLL-1 cells).No effect on T lymphocyte proliferationor viability.

rms electric current density; E, rms electric field intensity; t, exposure duration. n.r., not reported.

activated human peripheral blood lymphocytes (HPBL) normurine cytotoxic T cells (CTLL-1) was influenced by 60 Hzsinusoidal electric field intensities of 2.4 and 10.0 mV/cm,respectively (Table 2; refs 22 and 41).

Magnetic field intensity response thresholds

The lower-intensity response thresholds for magnetic fieldeffects on cellular Ca24 metabolism and RNA synthesis havenot been established yet. But a first observation can be madefor EMF effects on the proliferation of HPBL: exposures ofPHA-activated HPBL to 60 Hz, 0.2 mT sinusoidal magneticfields proved ineffective in modifying cell cycle progressionrates (22). On the other hand, a 50 Hz sinusoidal magneticfield with a 25-fold higher intensity (5 mT) slightly butsignificantly (8%, P < 0.0001) increased the proliferationindex of PHA-treated HPBL (23). Thus, the field responsethreshold for altering proliferation rates of HPBL should liebetween 0.2 and 5 mT for power frequency (50/60 Hz) mag-netic fields and 2- to 3-day continuous exposures, assumingno frequency dependence of the response between 50 and 60Hz for this end point (compare refs 22 and 23 in Table 1).In contrast, for nonsinusoidal ELF-pulsed magnetic fields,no lower intensity response threshold has been identified yet:the applied peak magnetic flux densities, ranging from 2.5 to10 mT, all appeared to be effective modulators of lymphocyteDNA synthesis as measured by [3H]thymidine uptake inmitogen-activated lymphocytes (compare refs 28-31, 35 inTable 1).

From the data in Table 1 and Table 2, it is evident that avariety of important metabolic activities of immune cells canbe affected by relatively weak EMFs. It is remarkable that bi-oactive EMF signals can differ considerably from each otherin field intensity, frequency, and wave form, and it is appar-ent that a simple linear relationship between field exposureparameters and the magnitude or quality of an EMFresponse cannot be established (see Table I). For example,EMFs can act as inhibitors or stimulators of cellular activity,and the occurrence of field effects on lymphocytes dependsstrongly on the biological status of the exposed cells (31, 33,42) as well as on the applied EMF exposure parameters (e.g.,refs 35, 43), indicating the involvement of complex interac-tion mechanisms.

CALCIUM SIGNALING AS A POSSIBLE TARGET OFELECTROMAGNETIC FIELD ACTION IN IMMUNECELLS

Electromagnetic field interactions with cellmembrane-mediated signal transduction events

Cells interact with their environment through the cell mem-brane. Among other functions, the cell membrane is respon-sible for the detection and subsequent transduction of exter-nal biochemical or other signals into the cytoplasmic space.The cell membrane is also considered the primary interac-tion site of EMF signals with cellular systems, and it hasbeen proposed that an interference of an EMF withmembrane-mediated signal detection, transduction, or am-plification processes may underlie many biological fieldeffects reported in the literature (1, 2, 44). In particular, themobilization of cellular Ca in response to external EMFsignals or the interference of an EMF with Ca regulatoryprocesses is considered an important target of EMF action.

For lymphocytes, Ca mobilization is among the earliestdetectable events triggered upon binding of a ligand (e.g.,antigen, receptor antibody, mitogenic lectin) to an appropri-ate receptor structure exposed on the outer cell surface. Thecascade of cellular reactions in lymphoid cells subsequent toligand-receptor interaction is best understood for T cells andhas been reviewed extensively (e.g., ref 45). In short, ligand-induced Ca24 mobilization is reflected by an initial rise in[Ca24]1 which is caused by inositol 1,4,5-triphosphate-induced release of Ca24 from intracellular stores and followedby a sustained receptor-mediated Ca24 influx from the ex-tracellular medium. It is known that a perturbation of theseevents with chemical agents such as Ca2 channel blockers,Ca24-specific ionophores, or lowering the extracellular Ca24concentration by using chelators can lead to changes in Ca2membrane fluxes and subsequent modifications in cellularactivity including cell proliferation, secretion, motility, orcytotoxicity (45-48). With regard to EMF effects on the im-mune system, it is proposed that Ca regulaton in lymphoidcells could be similarly affected by appropriate EMF signals,thereby leading to the reported cellular EMF responses, e.g.,on proliferation or cell-mediated cytotoxicity.

ELECTROMAGNETIC FIELDS AND THE IMMUNE SYSTEM 3181

Early evidence for field effects on calcium regulation innonlymphoid systems

Historically the effects of nonionizing, nonthermal EMF sig-nals on Ca2 were first demonstrated using nonlymphoid tis-sues. The pioneering work by Bawin and Adey (49) and Ba-win et al. (50) in the middle 1970s documented that Ca2efflux from chick brain can be measurably altered by ex-posures to an ELF-modulated radio frequency carrier wavein vitro; the unmodulated carrier wave was found to have noeffect. This early finding was subsequently confirmed byBlackman and co-workers (51-53) in an extensive series ofexperiments. During the past decade many other studies in-vestigating the direct or indirect role of Ca2 in biologicalfield effects have followed (for reviews, see refs 1-7). Fieldeffects on Ca2 membrane influx or effiux and on [Ca2]have been reported (compare refs 49-53 and Table 1: effectson calcium metabolism). At this point, however, it is still un-clear how field effects on Ca2 membrane fluxes relate to fieldeffects on [Ca24j1. One important notion emerging fromthese experiments was that ELF signal frequencies below 100Hz seemed to be most effective in modifying Ca24 regulation,and in some cases field responses appeared to be frequency-specific. For example, EMF signals oscillating around acenter frequency of 15 Hz were found to be more powerfulmodulators of Ca2 fluxes than field signals of higher or lowerfrequency, and in some cases this frequency dependence wasfound to be modulated by the static magnetic field environ-ment (37, 53). Nonlinear dependencies on the applied fieldintensity were also reported (49-53).

The establishment of strong experimental evidence for theexistence of field effects on Ca2 metabolism in different bio-logical systems was probably the main contribution of theseearly studies. However, new research is only beginning to ad-dress the task of determining the physiological significance ofEMF-altered Ca2 changes in the various systems.

Current evidence for the role of calcium in the mediationof electromagnetic field effects on lymphocyte DNAsynthesis

Several lines of direct and indirect evidence suggest thatEMF-altered Ca2 regulation is an early trigger of fieldeffects in cells of the immune system. First, the findings thatcorrelate EMF-modified Ca2-mediated signal transductionevents with EMF-altered lymphocyte DNA synthesis will bediscussed. This is followed by an analysis of field effects inrelationship to the kinetics of EMF influences on lymphocytemitogenesis and RNA transcript levels, lymphocyte-mediated cytotoxicity, and cell viability within the context ofCa24 homeostasis.

As outlined in the previous sections, evidence exists thatweak ELF magnetic fields can modify Ca24 regulation inlymphoid cells (for examples, see Table 1). Taking into ac-count the established role of Ca2 in the regulation of lym-phocyte proliferation (45, 46), it is reasonable to hypothesizethat EMF-altered Ca24 regulation might be able to modifylymphocyte DNA synthesis. This view is supported byresults from a number of different experiments. For example,it was shown that EMF signals identical in intensity andpulse frequency to signals that inhibited PHA-induced DNAsynthesis in HPBL (refs 28, 30 listed in Table 1) also in-hibited PHA-dependent Ca2 uptake in these cells (28, 29).Furthermore for nonactivated, resting lymphocytes it wasconsistently found that Ca24 uptake as well as DNA synthesiswas unaffected by all the exposure protocols tested so far (B> 0.1 mT). In contrast, in mitogen-activated cells these same

EMF signals were effective modulators of both Ca2 uptakeand DNA synthesis (Table 1, refs 28-31, 33, 35). These latterresults suggest that activation of transmembrane Ca24 signal-ing is required in order to obtain field effects on both Ca24uptake and DNA synthesis (33).

Another set of data also point to a link between field-induced Ca24 changes with cell proliferation: lymphocytepopulations with a diminished ability to mobilize Ca24 inresponse to mitogen were observed to be significantly moresensitive to EMF influences than cells displaying a normalmitogen response (33). Against this background, the resultsfrom studies investigating the effects of EMFs on mitogen- orionophore-induced DNA synthesis in HPBL are revealing: itwas found that lymphocytes with an impaired responsivenessto mitogen-stimulation responded to the EMF influencemore strongly than fully functioning cells (31). This observa-tion is consistent with the report on EMF-induced Ca2 up-take in cells that are less responsive to Con A stimulation(33). Further, Ca24 ionophore (A23187)-induced [3H]thymi-dine uptake in HPBL at suboptimal ionophore concentra-tions was stimulated several-fold (P < 0.0001) in thepresence of a 3 Hz pulsed magnetic field (B = 5 mT) com-pared with nonexposed controls, whereas at optimal iono-phore concentrations DNA synthesis was reduced by themagnetic field (42). One possible interpretation is that thesuboptimal influx of Ca24 was stimulated by the magneticfield; in contrast, a reduction in ionophore-induced DNAsynthesis was caused by field perturbation of optimal Ca24influx rates. A similar qualitative dependence of the fieldeffect on biological activity was also described by Blank andSoo (54). These researchers found that when the enzymeNa4/K4-ATPase functioned at an optimal rate, the appliedELF EMF reduced the activity of the enzyme, whereas whenNa4/K4-ATPase activity was at suboptimal levels the EMFhad the opposite, namely a stimulatory, effect (54). Themechanisms that might underlie these observations are stillunexplored.

Preliminary data exist from three laboratories using theclassical Ca channel blocker verapamil in lymphocyte fieldexposure studies. If confirmed, these studies would suggestthat verapamil can interfere with the effects of EMFs on Ca24uptake (28, 29), DNA synthesis (55), and the viability ofHPBL (56). For example, a 12% drop in the viability ofHPBL after 60 mm exposure to a 100 mT steady-state mag-netic field was found to be reversed in the presence of ap-propriate concentrations of verapamil in a dose-dependentmanner, suggesting the involvement of altered Ca24 mem-brane permeability in the mediation of EMF effects in lym-phocytes (56). This conclusion must remain tentative,however, as it is unclear whether the verapamil effects arecaused by directly affecting Ca24 membrane permeability, byan interaction with voltage-activated K channels, or possi-bly by nonspecific effects of the drug (57).

Time dependence of lymphocyte DNA synthesis and therole of calcium

If early magnetic field-induced changes in cellular Ca24homeostasis are indeed participating in the mediation oflater-stage biological effects such as lymphocyte replication,exposure times much shorter than the 66-72 h reported forELF-pulsed fields should be sufficient to alter DNA synthesis(Table 1). This would be expected from the known Ca2 re-quirement of stimulated lymphocytes during early and latestages (> 12 h) of mitogenesis. Although the exact phases inthe cell cycle that are Ca2 dependent have not been clearly

3182 Vol. 6 October 1992 The FASEB lournal WALLECZEK

defined yet, based on present knowledge it is safe to assumethat lymphocyte proliferation is dependent on Ca24 for thefirst 24 h of mitogen-activated proliferation only (46, 47).Thus, it is of interest to review the exposure time depen-dence of field-altered DNA synthesis in relationship to thisCa2 requirement: Cadossi et al. (58) found that after 24 hexposure of PHA-activated HPBL to a 75 Hz pulsed mag-netic field (B = 2 mT), the mitotic index of these cells (asdefined by the number of observed mitosis divided by thenumber of evaluated cells) was increased by 50% comparedwith nonexposed controls. No additional field effect on thecell cycle was seen when the exposure period was extendedto 40 or even 72 h. In agreement with these findings is theobservation that exposures of PHA-activated HPBL res-tricted to the first 24 h of the 72 h incubation period ap-peared to be as effective as subjecting the cells to the fieldinfluence for as long as 66 h (31). There is also informationthat an exposure time of only 6 h, but not 1 h, might besufficient for modifying lymphocyte DNA synthesis (42).However, this result is in contrast to another study reportingthat an exposure period of 12 h was not effective under simi-lar conditions (30). Finally, work studying the effects of 50Hz pulsed magnetic fields (B = 2.5 mT) on surface recep-tor expression revealed that an 18 h exposure period was longenough to enhance the number of interleukin-2 receptors ex-pressed on the cell surface of HPBL by 22% (P < 0.001)compared with controls (59).

In summary, there is evidence that exposure periods ofonly 24 h, or in some cases even less, can induce field effectson DNA synthesis that are similar in magnitude to the effectsfrom much longer exposure times. Thus, the hypothesis thatEMFs interact with Ca24-regulated processes during earlyand late phases in mitogenesis, thereby causing proliferativeeffects at a later stage, appears to be in line with the observedkinetics of field influences on DNA synthesis. Nevertheless,it seems unlikely that an EMF effect on early (< 1 h) ligand-induced Ca signals alone could be sufficient to modify lym-phocyte replication to a similar extent.

The possible role of calcium in the mediation of fieldeffects on RNA transcript levels andlymphocyte-mediated cytotoxicity

Field-induced alterations in cellular Ca2 homeostasis mightagain be important with regard to the effects of magneticfields on RNA transcript levels (see refs 32 and 36 in Table1). For example, it is known that c-myc gene transcription inlymphoid cells can be enhanced 10- to 20-fold upon additionof an Ca24 ionophore within I h (60, 61). Thus, the rathermodest 1.5- to 2-fold increase in c-myc transcript levels meas-ured after field exposure of HL-60 cells (a human promyelo-cytic leukemic cell line) for 20 mm could be explained by aslight field-induced increase in [Ca24]1 alone (ref 32 in Table1). This interpretation is substantiated by the results of Car-son et al. (34), who found that a single 23 mm exposure ofHL-60 cells to weak magnetic fields resulted in a small butsignificant (P < 0.01) rise in [Ca24]1 by 34 10 nM from abasal level of 121 8 nM in the nonexposed control cells asmeasured by spectrofluorimetry (ref 34 in Table 1).

EMF-triggered changes in [Ca24]1 in target or cytotoxiceffector cells after 24 or 48 h field exposures, respectively,could also be responsible for the influence of 60 Hz electricfields on lymphocyte-mediated cytotoxicity (refs 40 and 41 inTable 2). The increased susceptibility of field-exposed targetcells to NK cell-induced cytolysis could be caused by anelevated [Ca24]1 in the target cells after the electric field ex-posure (40); sustained increases in [Ca24]1 are known to be

an important contributing factor to achieving target cell lysisby NK cells (62). Because lethal hit delivery by the effectorcell is also a Ca24-dependent process and can be negativelyaffected by Ca2 channel blockers or in the presence of Ca2chelators (48), it is speculated that reduced lymphocytecytotoxicity after 48 h exposure to 60 Hz electric fields (41)could again be caused by an EMF influence onCa24-regulated activity. However, direct evidence in supportof a role for Ca24 in triggering EMF effects on transcriptlevels or cytotoxicity is not available yet.

Summary of the evidence and a cautionary note

The combined results suggest that interference by an EMFwith the regulation of cellular Ca2 signals represents a plau-sible candidate for the mediation of EMF effects on cells ofthe immune system. The most important evidence in favorof this hypothesis includes correlations between 1) the mag-nitude of EMF-induced Ca2 changes and the cellular activa-tion status and 2) the activation status and extent to whichlymphocyte DNA synthesis is modified by EMF exposures.Furthermore, the observed EMF effects on RNA transcriptlevels, lymphocyte-mediated cytotoxicity, cell viability, andwhat is also important, the kinetics of the EMF influence onDNA synthesis are also consistent with a role for Ca24 inmediating field effects in lymphoid cells.

Currently, only very little can be said about how an EMFacts to modify Ca24 regulation at the molecular level: there aremany possible pathways starting with an interference of thefield with ligand-receptor binding as proposed by Chiabreraet al. (63). Theoretically many different steps in the signaltransduction cascade subsequent to ligand-receptorcoupling, e.g., phosphorylation-dephosporylation events thatact as effectors of selected intracellular responses, could betargets of the field action. No data yet exclude any of thesepossible interactions. For example, some data suggest thatEMF-altered proto-oncogene expression could be involved infield interactions with cell signaling processes (32). The workusing the Ca2 channel antagonist verapamil would indicatethat EMF effects on Ca2 channels could be important ineliciting cellular field responses (28, 29, 55, 56). As pointedout earlier, however, the molecular interaction site of ver-apamil in these studies has to be determined before any firmconclusions can be drawn. Also, an EMF could alter the ac-tivity of the membrane-incorporated Ca24-ATPase responsi-ble for pumping Ca2 out of the cell. Although this possibilityhas not been directly tested, data from two laboratoriesdemonstrate that the activity of another membrane ionpump, Na/K4-ATPase, can be influenced by low-frequencyelectric fields (54, 64). The lowest effective induced currentdensity was 50.0 cA/cm2 at an optimum field frequency of100 Hz; by extrapolation of their measurements, the authorsestimated that a current density as low as 1.0 sA/cm2(E = 0.02 mV/cm under these conditions) would still be ableto alter Na4/K-ATPase activity in their in vitro system (54).

This effort only represents a first attempt to interpret fieldeffects on the immune system within the framework of Ca24signaling. Obviously there are many open questions, andonly future research can clarify whether EMF-induced alter-ations in Ca24 regulation are indeed causal in the chain ofevents leading to the observed field effects, or if Ca24 changesrepresent effects that are only secondary to more fundamen-tal field-induced molecular changes. If this were the case,EMF-modified Ca24 signals should be considered a suitablemarker only for the early detection of EMF effects in cells ofthe immune system.

ELECTROMAGNETIC FIELDS AND THE IMMUNE SYSTEM 3183

THE SEARCH FOR MECHANISMS: PHYSICALCONSTRAINTS

Reports of biological effects of nonionizing EMFs have beenneglected by biologists for many years because 1) it waswidely assumed that nonthermal levels of EMFs are muchtoo weak to be able to affect cellular activity in anysignificant manner and 2) testable hypotheses explainingsuch field effects were missing. However, careful documenta-tion of selected biological EMF effects in independentlaboratories has initiated a search for the underlying physicalmechanisms. So far, however, no biophysical model can pro-vide a satisfactory explanation of the reported field effects,although some theoretical progress has been made 1) in at-tempts to explain field effects on the basis of long-range,high-cooperativity phenomena in cells (for reviews, see refs2, 6, 8) or 2) in modeling locally restricted magnetic andelectric field influences on ion interactions with cell mem-brane structures (see refs 9, 65-69).

Due to lack of space this discussion will consider only oneapproach that deals exclusively with local field effects at thecell membrane level for very weak electric fields. Weaver andAstumian (9) recently examined the thermodynamic con-straints that would be imposed on EMF-cell membrane in-teractions by the thermal noise associated with randommembrane fluctuations in a single cell. They calculated thatan electric field intensity of about 1 mV/cm would be able toinfluence cells even in the absence of biological amplificationmechanisms. Provided that field effects are frequency-specific and taking into account signal averaging, their com-putations predict that fields as weak as 0.01-0.001 mV/cmcould still significantly affect membrane-associated proteinssuch as receptors, enzymes, or channels without violatingthe thermal noise limit. Although these calculations arebased on a number of as-yet-unconfirmed assumptions, theycan still give a rough estimate of field magnitudes that couldbe detectable by cells. Upon comparison of the computedvalues (E = 0.001-1 mV/cm) with the estimated electric fieldmagnitudes present in the reviewed exposure experiments(E = 0.02-10 mV/cm; see Table 1 and Table 2), it becomesclear that new theoretical developments are already begin-ning to bridge the gap between the experimentally observedEMF sensitivity of biological systems and physical con-straints. It certainly will take many more years of intensiveexperimental and theoretical research, however, before anyof the proposed physical interaction mechanisms can beverified.

CONCLUSIONS

This review has argued that there is now considerable evi-dence in support of the existence of low-frequency EMFeffects in cells of the immune system (for examples, see Table1 and Table 2) and that mechanisms involvingCa2-dependent signal transduction mechanisms are proba-bly participating in the mediation of weak field effects on im-mune cells. The apparent existence of such cellular EMFeffects represents a remarkable discovery for immunology.These in vitro results, together with the immunologicaleffects in organisms observed after in vivo exposures (see be-ginning of article), suggest that the immune system can beinfluenced by ELF EMFs too weak in intensity to actthrough any thermal mechanism. A similar responsivenessto weak EMFs has also been documented, for cells and tissuesof the neuroendocrine and musculoskeletal system, indicat-ing that EMF sensitivity might be a general property of bio-

logical systems (see refs 1-7, 69). Elucidation of the underly-ing cellular and molecular mechanisms that trigger such fieldresponses represents a fascinating challenge to biologists andphysicists alike. From the biologists point of view, the studyof the role of Ca2 signaling processes in the mediation ofEMF effects in biological systems, including the immunesystem, is a promising place to start. Furthermore, the obser-vation that EMF signals can rather rapidly influence cellularCa2 regulation, in combination with recent advances in un-derstanding the molecular details of Ca24 signalingprocesses, establishes lymphoid cells as an excellent modelsystem for studying the fundamental field interactionmechanisms at the cellular and molecular level.

The authors work was supported by the Deutsche Forschungs-gemeinschaft (Wa 680/1-1), the Fetzer Institute and the U.S. Depart-ment of Energy (DE-ACO3-76SF00098). I am grateful to Dr.Thomas F. Budinger for encouragement and to Drs. Jerry L. Phil-lips, Stephen M. Ross, Scott Taylor, and Mark S. Roos for valuablecomments on the manuscript.

REFERENCES

1. Adey, W. R. (1981) Tissue interactions with nonionizing elec-tromagnetic fields. Physiol. Rev. 61, 435-514

2. Adey, W. R., and Lawrence, A. F., eds (1984) Nonlinear Elec-trodynamics in Biological Systems. Plenum, New York

3. Chiabrera, A., Nicolini, C., and Schwan, H. P., eds (1985) Inter-actions Between Electromagnetic Fields and Cells. Plenum, New York

4. Blank, M., and Findl, E., eds (1987) Mechanistic Approaches to In-teractions of Electromagnetic Fields with Living Systems. Plenum, NewYork

5. Marino, A. A., ed (1988) Modern Bioelectricity. Dekker, New York6. Fr#{246}hlich,H., ed (1988) Biological Coherence and Response to External

Stimuli, Springer, Heidelberg, Germany7. Wilson, B. W., Stevens, R. G., and Anderson, L. E., eds (1990)

Extremely Low Frequency Electromagnetic Fields: The Question ofCancer. Batelle, Columbus, Ohio

8. Kaiser, F (1988) Theory of non-linear excitation. In BiologicalCoherence and Response to External Stimuli (Fr#{246}hlich,H., ed) pp.25-48, Springer, Heidelberg, Germany

9. Weaver, J. C., and Astumian, R. D. (1990) The response of liv-ing cells to very weak electric fields: the thermal noise limit.Science 247, 459-462

10. Bassett, C. A. L. (1989) Fundamental and practical aspects oftherapeutic uses of pulsed electromagnetic fields. Crit. Rev. Bio-med. Engin. 17, 451-529

11. Lechner, F., Ascherl, R., Kraus, W., Schmit-Neuerburg, K. P.,St#{252}rmer,K. M., and Bl#{252}mel,G., eds (1989) Elektrostimulation undMagnetfeldeherapie. Anwendun& Ergebnisse und Qualit#{228}tssicherung.Schattauer, Stuttgart and New York

12. Wertheimer, N., and Leeper, E. (1979) Electrical wiringconfigurations and childhood cancer. Am. J. Epideiniol. 109,273-284

13. Savitz, D. A., and Calle, E. E. (1987) Leukemia and occupa-tional exposure to electromagnetic fields: review of epidemio-logical surveys. j Occup. Med. 29, 47-51

14. Nair, I., Morgan, M. G., and Florig, H. K. (1989) BiologicalEffects of Power Frequency Electric and Magnetic Fields. Office of Tech-nology Assessment, Washington, D.C.

15. London, S. J., Thomas, D. C., Bowman, J. D., Sobel, E.,Cheng, T-C., and Peters, J. M. (1991) Exposure to residentialelectric and magnetic fields and risk to childhood leukemia. Am.J. Epidemiol. 134, 923-937

16. Oroza, M. A., Calcicedo, L., Sanchez-Franco, F., and Rivas, L.(1987) Hormonal, hematological and serum chemistry effects ofweak pulsed electromagnetic fields on rats. J. Bioelectr. 6,139-151

17. Stuchly, M. A., Ruddick, J., Villeneuve, D., Robinson, K.,Reed, B., Lecuyer, D. W., Tan, K., and Wong, J. (1988) Terato-

3184 Vol. 6 October 1992 The FASEB Journal WALLECZEK

logical assessment of exposure to time-varying magnetic field.Teratology 38, 461-466

18. Zecca, L., Dal Conte, G., Furia, G., and Ferrario, P. (1985) Theeffect of alternating magnetic fields on experimental inflamma-tion in the rat. Bioelectrochem. Bioenerg. 14, 39-43

19. Fischer, G., Sametz, W., and Juan, H. (1987) Influence of amagnetic field on the development of the carrageenan-inducedrat paw edema. Med. KIm. 82, 566-570

20. McLean,J. R. N., Stuchly, M. A., Mitchel, R. E.J., Wilkinson,D., Yang, H., Goddard, M., Lecuyer, D. W., Schunk, M., Cal-lary, E., and Morrison, D. (1991) Cancer promotion in a mouse-skin model by a 60-Hz magnetic field: II. Tumor developmentand immune response. Bioelectromagnetics 12, 273-287

21. Bellossi, A., Moulinoux, J. P., and Quemener, V. (1989) Effectof a pulsed magnetic field on healthy mice: a study of weight ofthe thymus. In Viva 3, 29-32

22. Cohen, M. M., Kunska, A., Astemborski, J. A., McCulloch,D., and Paskewitz, D. A. (1986) Effect of low-level, 60-Hz elec-tromagnetic fields on human lymphoid cells: I. Mitotic rate andchromosome breakage in human peripheral lymphocytes. Bio-electromagnetics 7, 415-423

23. Rosenthal, M., and Obe, G. (1989) Effects of 50-Hertz elec-tromagnetic fields on proliferation and chromosomal alterationsin human peripheral lymphocytes untreated or pretreated withchemical mutagens. Mutat. Res. 210, 329-335

24. Livingstone, G. K., Witt, K. L., Gandhi, 0. P., Chatterjee, I.,and Roti Roti, J. L. (1991) Reproductive integrity of mam-malian cells exposed to power frequency electromagnetic fields.Environ. Mol. Mutagen. 17, 49-58

25. Polk, C., and Postow, E., eds (1986) CRC Handbook of BiologicalEffects of Electromagnetic Fields. CRC, Boca Raton, Florida

26. Stuchly, M. A. (1990) Applications of time-varying magneticfields in medicine. Crit. Rev. Biomed. Engin. 18, 89-124

27. Budinger, Tb. F., Fischer, H., Hentschel, D., Reinfelder, H. -E.,and Schmitt, F. (1991) Physiological effects of fast oscillatingmagnetic field gradients. j Comp. Assist. Tomogr. 15, 909-914

28. Conti, P., Gigante, G. E., Alesse, E., Cifone, M. G., Fieschi, C.,Reale, M., and Angeletti, P. U. (1985) A role for calcium in theeffect of very low frequency electromagnetic field on the blasto-genesis of human lymphocytes. FEBS Lett. 181, 28-32

29. Conti, P., Gigante, G. E., Cifone, M. G., Alesse, E., Fieschi, C.,and Angeletti, P. U. (1985) Effect of electromagnetic field on twocalcium dependent biological systems. j Bioelectr. 4, 227-236

30. Mooney, N. A., Smith, R., and Watson, B. W. (1986) Effect ofextremely-low-frequency pulsed magnetic fields on the mito-genie response of peripheral blood mononuclear cells. Bioelec-tromagnetics 7, 387-394

31. Cossarizza, A., Monti, D., Bersani, F., Cantini, M., Cadossi,R., Sacchi, A., and Franceschi, C. (1989) Extremely low fre-quency pulsed electromagnetic fields increase cell proliferationin lymphocytes from young and aged subjects. Biochem. Biophys.Res. Commun. 160, 692-698

32. Goodman, R., Wei, L.-X., Xu,J.-C., and Henderson, A. (1989)Exposure of human cells to low-frequency electromagnetic fieldsresults in quantitative changes in transcripts. Biochim. Bio-phys. Acta 1009, 216-220

33. Walleczek, J., and Liburdy, R. P. (1990) Nonthermal 60-Hzsinusoidal magnetic-field exposure enhances 45Ca2 uptake inrat thymocytes: dependence on mitogen activation. FEBS Lett.271, 157-160

34. Carson,J.J. L., Prato, F. S., Drost, D.J., Diesbourg, L. D., andDixon, S. J. (1990) Time-varying magnetic fields increase cyto-solic free Ca2 in HL-60 cells. Am. j Physiol. 259, 0687-692

35. Petrini, M., Polidori, R., Ambrogi, F., Vaglini, F, Zaniol, P.,Ronca, G., and Conte, A. (1990) Effects of different low-frequency electromagnetic fields on lymphocyte activation: atwhich cellular level?]. Bioelectr 9, 159-166

36. Greene, J. J., Skowronski, W. J., Mullins, J. M., Nardone,R. M., Penafiel, M., and Meister, R. (1991) Delineation of elec-tric and magnetic field effects of extremely low frequency elec-tromagnetic radiation on transcription. Biochem. Biophys. Res.Commun. 174, 742-749

37. Rozek, R. J., Sherman, M. L., Liboff, A. R., McLeod, B. R.,

and Smith, S. D. (1987) Nifedipine is an antagonist to cyclotronresonance enhancement of 45Ca2 incorporation in human lym-phocytes. Cell Calcium 8, 413-427

38. Lyle, D. B., Wang, X., Ayotte, R. D., Sheppard, A. R., andAdey, W. R. (1991) Calcium uptake by leukemic and normal T-lymphocytes exposed to low frequency magnetic fields. Bioelec-tromagnetics 12, 145-156

39. Byus, C. V., Pieper, S. E., and Adey, W. R. (1987) The effectsof low energy 60-Hz environmental electromagnetic fields uponthe growth-related enzyme ornithine decarboxylase. Carcinogene-sic 8, 1385-1389

40. Phillips, J. L. (1986) Transferrin receptors and natural killer celllysis: a study using Cob 205 cells exposed to 60-Hz electromag-netic fields. Immunol. Lett. 13, 295-299

41. Lyle, D. B., Ayotte, R. D., Sheppard, A. R., and Adey, W. R.(1988) Suppression of T-lymphocyte cytotoxicity following ex-posure to 60-Hz sinusoidal electric fields. Bioelectromagnetics 9,303-313

42. Conti, P., Gigante, G. E., Cifone, M. G., Alesse, E., Fieschi, C.,Bologna, M., and Angeletti, P. U. (1986) Mitogen dose-dependent effect of weak pulsed electromagnetic field on lym-phocyte blastogenesis. FEBS Lett. 199, 130-134

43. Conti, P., Gigante, G. E., Cifone, M. G., Alesse, E., lanni, G.,Reale, M., and Angeletti, P. U. (1983) Reduced mitogenicstimulation of human lymphocytes by extremely low frequencyelectromagnetic fields. FEBS Lett. 162, 156-160

44. Adey, W. R. (1988) Cell membranes: the electromagnetic en-vironment and cancer promotion. Neurochem. Res. 13, 671-677

45. Weiss, A., and Imboden,J. B. (1987) Cell surface molecules andearly events involved in human T lymphocyte activation. Adv.Immunol. 41, 1-38

46. Lichtman, A. H., Segel, G. B., and Lichtman, M. A. (1983) Therole of calcium in lymphocyte proliferation (an interpretivereview). Blood 61, 413-422

47. Harris, D. T., Kozumbo, W. J., Testa, J. E., Cerutti, P. A., andCerottini, J. -C. (1988) Molecular mechanisms involved in T cellactivation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-inducedproliferation of cloned cytotoxic T lymphocytes. J. Immunol.140, 921-927

48. Howell, D. M., and Martz, E. (1988) Low calcium concentra-tions support killing by some but not all cytolytic T lympho-cytes, and reveal inhibition of a postconjugation step by calciumantagonists. j Immunol. 140, 1982-1988

49. Bawin, S. M., and Adey, W. R. (1976) Sensitivity of calciumbinding in cerebral tissue to weak environmental electric fieldsoscillating at low frequency. Proc. NatL Acad. Sci. USA 73,1999-2003

50. Bawin, S. M., Adey, W. R., and Sabbot, I. M. (1978) Ionic fac-tors in release of 45Ca from chicken cerebral tissue by elec-tromagnetic fields. Proc. NatI. Acad. Sci. USA 75, 6314-6318

51. Blackman, C. F., Benarie, S. G., Kinney, L. S., Joines, W. T.,and House, D. E. (1982) Effects of ELF fields on calcium-ionefflux from brain tissue in vitro. Radiat. Res. 92, 510-520

52. Blackman, C. F., Benane, S. G., House, D. E., andJoines, W. T(1985) Effects of ELF (1-120 Hz) and modulated (50 Hz) RFfields on the efflux of calcium ions from brain tissue in vitro. Bio-electromagnetics 6, 1-11

53. Blackman, C. F, Benane, S. G., Rabinowitz, J. R., House, D. E.,and Joines, W. T. (1985) A role for the magnetic field in theradiation-induced efflux of calcium ions from brain tissue invitro. Bioelectromagnetmcs 6, 327-337

54. Blank, M., and Soo, L. (1990) Ion activation of the Na,K-ATPase in alternating currents. Bioelectrochem. Bioenerg. 24, 51-61

55. Cadossi, R., Emilia, G., Ceccherelli, G., and Torelli, G. (1988)Lymphocytes and pulsing magnetic fields. In Modern Bioelectricity(Marino, A. A., ed.) pp. 451-496, Dekker, New York

56. Papatheofanis, F J. (1990) Use of calcium channel antagonistsas magnetoprotective agents. Radiat. Res. 122, 24-28

57. Gardner, P. (1990) Patch clamp studies of lymphocyte activa-tion. Annu. Rev. Immunol. 8, 231-252

58. Cadossi, R., Emilia, G., Torelli, G., Ceccherelli, G., Ferrari, S.,and Ruggieri, P. (1985) The effect of low-frequency pulsing dcc-

ELECTROMAGNETIC FIELDS AND THE IMMUNE SYSTEM iic

tromagnetic fields on the response of human normal lympho-cytes to phytohemagglutinin (PHA). Bloelectrochem. Bioenerg. 14,115-119

59. Cossarizza, A., Monti, D., Bersani, F., Paganelli, R., Montag-nani, G., Cadossi, R., Cantini, M., and Franceschi, C. (1989)Extremely low frequency pulsed electromagnetic fields increaseinterleukin-2 (IL-2) utilization and IL-2 receptor expression inmitogen stimulated human lymphocytes from old subjects.FEBS Lett. 248, 141-144

60. Grausz, J. D., Fradelezi, D., Dautry, F, Monier, R., and Lehn,P. (1986) Modulation of c-fos and c-myc mRNA levels in normalhuman lymphocytes by calcium ionophores A23187 and phor-bol esters. Eur.]. ImmunoL 16, 1217-1221

61. Lindsten, T., June, C. H., and Thompson, C. B. (1988) Multi-ple mechanisms regulate c-myc gene expression during normalT cell activation. EMBOJ 7, 2787-2794

62. Poenie, M., Tsien, R. Y., and Schmitt-Verhulst, A.-M. (1987)Sequential activation and lethal hit measured by [Ca2]1 in in-dividual cytolytic T cells and targets. EMBOJ 6, 2223-2232

63. Chiabrera, A., Grattarola, M., and Viviani, R. (1984) Interac-tion between electromagnetic fields and cells: microelec-trophoretic effect on ligands and surface receptors. Bioelectromag-

netics 5, 173-19164. Serpersu, E. H., and Tsong, T. Y. (1984) Activation of electro-

genie Rb transport of (Na/K)-ATPase by an electric field. jBiol. Chem. 259, 7155-7162

65. Liboff, A. R. (1985) Cyclotron resonance in membrane trans-port. In Interactions Between Electromagnetic Fields and Cells(Chiabrera, A., Nicolini, C., Schwan, H. P., eds) pp. 281-296,Plenum, London

66. Blank, M. (1987) The surface compartment model: a theory ofion transport focused on ionic processes in the electric doublelayers at membrane protein surfaces. Biochim. Biophys. Acta 906,277-294

67. Tsong, T Y. (1990) Electrical modulation of membrane pro-teins: enforced conformational oscillations and biologicalenergy and signal transduction. Annu. Rev. Biophys. Chem. 19,83-106

68. Lednev, V. V. (1991) Possible mechanisms for the influence ofweak magnetic fields on biological systems. Bioelectrornagnetics12, 71-75

69. Grundler, W., Kaiser, F, Keilmann, F, and Walleczek,J. (1992)Mechanisms of electromagnetic interaction with cellular sys-tems. Naturwissenschaften In press