Immune response of mice to 2450MHz microwave radiation: Overview of immunology and empirical studies...

Immune response of mice to 2450-MHz microwave radiation' Overview of immunology and empirical studies of lymphoid splenic cells

Wieslaw Wiktor-Jedrze/czak, A ftab Ahmed, Przemyslaw Czersla', William M. Leach, and Kenneth W. Sell

Cellular Immunology Division, Clinical and Experimental Immunology Department, Naval Medical Research Institute, Bethesda, Maryland 20014 and Division of Biological Effects, Bureau of Radiological Health, Food and Drug Administration,

Rockville, Maryland 2085 7

Male CBA/J mice were exposed to 2450-MHz microwave radiation (each exposure: 30 minutes at an averaged dose rate near 14 mW/g). The mice were tested later to determine effects of the radiation on (1) the relative frequency of T and B cells; (2) the functional capacity of spleen cells from irradiated mice to respond to T- and B-cell-specific membrane stimuli; and (3) the ability to respond to sheep red blood cells and dinitrophenyl-lyslyl-Ficoll. Results demonstrated that microwave radiation has weak stimulatory effects on B- but not T-lymphoid cells in the spleen. Single exposures to radiation produced an increase in the incidence of cells with complement receptor on the cell's surface. Three exposures to radiation induced increases in the total number of splenic cells, in the incidence of immunoglobin-positive cells, and in the incidence of complement receptor-positive cells. The total number of T cells was unaffected by single or triple exposures of mice to the radiation.

1. INTRODUCTION

Rapid increases in the number of devices that emit potentially hazardous radio-frequency radiations prompted us to carry out investigations on effects (harmful or beneficial) that exposure to microwaves may have on the immune system. The immune system produces specific cellular and humoral responses to micro6rganisms and to other intrinsic and extrinsic agents. Among the major elements of the immune system are the lymphoid cells. These cells are highly similar morphologically (relatively small, spherical, with a relatively large nucleus in relation to the cyto- plasm) and are present in the bone marrow, thymus, spleen, peripheral blood, lymph nodes, and Peyer's patches. Despite their morphological similarity, as determined by classical hematological and histologi- cal criteria, the lymphoid cells are composed of a number of functionally distinct subpopulations. All of the subpopulations originate from a common lymph-

the theta isoantigen [Reft and Alien, 1966], whereas B cells possess surface immunoglobuIin (Ig), which is easily detected by membrane immunofiuorescence through the use of a fluorescent-conjugated, anti- immunoglobulin antisera. Further, there is considerable heterogeneity among T cells and B cells. This heterogeneity is based on the observation that there are other markers that are unique to, but are not present on all, T and B cells [Raft et al., 1970]. Thus, a fraction of adult B cells, besides expressing surface Ig, has been shown to express a receptor for activated complement (C'3),which may be detected by its ability to form rosettes with a reagent consisting of antibody and C'3- coated sheep erythrocytes (EAC reagent) as described by Bianco et al. [1970]. Cells of this fraction are not present in neonatal mice. Thus, it can be theorized that stem cells give rise to precursor B cells that, when mature, manifest surface Ig, and then further mature into B cells that express the complement receptor (CR) (Figure 2). After interaction with an antigen, e.g.,

oid stem cell (Figure 1). One of the progeny of the sheep erythrocytes, adult B cells develop into plasma stem cells, which infiltrates, differentiates, and matures cells and produce antibody, which may be quantitated in or under the influence of the thymus, thereafter by a local, hemolysis-in-gel (plaque-formation) tech- recirculating to the peripheral lymphoid tissues, is nique of Jerne and Nordin [1963]. Functionally, T called the thymus-derived lymphoid• (T) cell. This and B cells can be assessed by their ability to respond cell interacts with a foreign antigen by direct cellular to T-cell- and B-cell-specific mitogenic agents. Thus, contact and the immune response that follows is termed T cells preferentially undergo DNA synthesis in response "the cellular immune response." Other descendants of to the mitogens phytohemagglutinin and concanavalin the lymphoid stem cell are present in the bone marrow -- A, and the B cells preferentially undergo DNA synthesis and from the marrow, directly infiltrate peripheral in response to the mitogenic action of bacterial lipopoly- organs such as the spleen, lymph nodes, and Peyer's saccharide and polyinosinic-polycytidylic acid. The patches -- and have been termed the bone-marrow- magnitude of DNA synthesis can be assessed and used derived lymphoid (B) cells. These cells interact with as an indicator both of the presence and of the fre- foreign antigens by secreting specific proteins called immunoglobulins (or antibodies). Thus, the two major subclasses of lymphoid cells that are part of the immune system are the T cells and the B cells.

The T cells are identified by the use of a cytotoxic antisera against a unique marker on their surface termed

quency of functional T or B cells. Therefore, at present, several techniques are available that not only permit the enumeration of the total number of T and B cells

but also the frequency and functional capacity of each subpopulation in a given mixture of cells. The purpose of our investigation was to learn whether

209

210 WIKTOR-JEDRZEJCZAK, AHMED, CZERSKI, LEACH, AND SELL

Lymphoid Stem cell

T-cell precursor B-cell precursor [responsible for [responsible for cells involved in clones of cells cellular immunity, involved in anti- helper, suppressor, body production accessory cell functions, without (T-independent) tumor immunity, graft or with (T-dependent) rejection, etc.] T helper cells]

1973; Christman et al., 1974]. In the mouse, a lymphoid organ that is widely used for experimental purposes is the spleen. Itis an organ that contains both categories of differentiated T and B cells in sufficient quantities to allow simultaneous performance of different assays with the same suspension of cells.

In this paper, we shall describe the effect of a single microwave exposure and of three such exposures on (a) the frequency of various subpopulations of T and B cells; (b) the functional capacity of splenic cells from exposed mice to respond to T- and B-cell-specific membrane stimuli; and (c) the ability of microwave- and sham-exposed mice to mount an antibody response to SRBC (which requires the presence of T cells) and to DNP4ys-Ficoll (which does not require the presence of T cells and is therefore T-cell independent).

2. MATERIALS AND METHODS

Fig. 1. Lymphoid stem cells are primitive constituents of the immune system that undergo a series of transformations during maturation. The initial transformation is that to precursor T cell (for thymus) or precursor B cell (historically identified with the bursa of an arian species). During subsequent maturation, T

and B cells perform a diversity of specialized functions.

2.1. Animals.

Highly inbred CBA/J male mice (8-12 weeks of age) were purchased from the Jackson Laboratory, Bar Harbor, ME, and were used in all experiments.

exposure of mice to microwaves has an effect on their immune function by determining, not only the relative frequency of each subpopulation, but also, through further study, to determine the functional capacity of the T and B cells to respond to T-cell-specific and B-cell-specific membrane stimuli after immunization of microwave- and sham-exposed mice against T-cell- dependent and T-cell-independent antigens.

According to contemporary opinion, the immune system is essentially the same among differing mammal- ian species and, therefore, the choice of an experimental animal model in studies involving microwave radiation is dependent mostly on the amenities of the radiation facility. An advanced apparatus, an environmentally controlled waveguide system for exposure of mice, is available at the Bureau of Radiological Health, Food and Drug Administration, Rockville, Maryland [Ho et al.,

Ig-, la-, CR- B-Cell Precursor .............. (No Surface Markers)

Immature B Cell I ................ Ig +, la-, CR-

Immature B Cell 2 ................ Ig +, la +, CR'

Mature B Cell .................... Ig +, la +, CR +

Antibody-Forming (Ig', CR') Plasma Cell ............. (Identified By Plaque Formation)

Fig. 2. Sequential development of surface markers on B4ymph- old cells of the mouse.

2.2. Exposure conditions and protocol.

Each mouse was individually positioned in the waveguide with its head towards the source of radiation while restrained in a plastic polystyrene holder. A mouse was exposed to 2450-MHz microwaves at a forward power of about 0.6 watts. The microwave source was a Hewlett- Packard 8616A signal generator that was coupled to a 491C amplifier; the amplifier was tuned to 2450 MHz + 500 Hz by a coherent synchronizer (Sage Laboratories Model 251). The body mass of all animals was measured before exposure and ranged from 15 to 20 grams. Rec- tal temperatures were determined with a Yellow Springs Model 46 TUC telethermometer before and after exposures. During exposures, an ambient temperature of 25 ß 0.5 øC and a relative humidity of 50 + 5% were main- tained as was a constant flow of air in the waveguide (38 liters/min). The duration of exposure was always 30 minutes and, during exposures, forward, reflected, and transmitted powers were measured with power meters. Animals were exposed either once or three times The averaged dose rate for the single exposures was 1'3 ß 2.5 (SD) mW/g and was associated with a mean aT of rectal temperature of-0.05 K. The averaged dose rates for the animals that were exposed three times were, respectively, for the first, second, and third exposure, 15.6 ß 1.2, 15.5 + 0.9, and 13.3 + 0.6 mW/g, which were associated with mean/XTs of-0.10, --0.20, and +0.05 K. The dose rates were based on the body mass of individual animals (in grains), which was divided by the computer-extrapolated, averaged rate of energy absorption (in watts), as previously described [ Youmans and Ho, 1975; Justesen, 1975; Johnson, 1975 ]. Sham- exposed animals served as controls. They were handled in the same manner as microwave-exposed animals and were kept in the same polystyrene holder in the wave-

guide for 30-minute periods, but were not exposed to radiation.

2.3. Statistics.

Only groups of six MW-exposed and six sham-exposed mice could be treated during any given experimental run. Data from each mouse were recorded individually and the arithmetic mean (+ SE)of each determination was calculated with the help of a Wang 700 C calculator. Differences between means were analyzed by Student's t test.

2.4. Suspensions of Cells.

During specified days, as indicated in Section 3, mice were anesthetized with methoxyflurane (Penthrane©, Abbott Laboratories) and were euthanized by cervical dislocation; their spleens were removed under aseptic conditions. Cells were obtained by gentle teasing of the splenic capsule with the aid of a rubber spatula in Hanks Balanced Salt Solution (Microbiological Associates), as described in detail elsewhere [Strong et al., 1975]. Cells in suspension were washed in media and were resuspended in RPMI 1640 supplemented with penicillin (100 units/ml), streptomycin (100/•g/ml), L-glutamine (2mM), 25mm Hepes (N-2-hydroxyethylpiperazine N'-2- ethane sulfonic acid) and 10% heat4nactivated (56 øC, 30 min) fetal bovine serum (FBS) (Microbiological Associates). Cell counts were determined with a Fisher Scientific Co. autocytometer.

2.5. Antisera.

Anti-T-cell-specific sera (anti-theta or anti-Thy 1.2 sera) was prepared according to the method of Reifand Allen [1966]. Fluorescent-conjugated goat antimouse- polyvalent IgG was obtained from Meloy Laboratories.

2.6. Mitogen Assay.

An assay of the ability of the splenic cells to respond in vitro to T- and B-cell-specific mitogenic agents was performed with a microculture system described previously [Strong et al., 1973 ]. Essentially, cells in 100-gl suspensions (8 X 106 cells/ml) were cultured with 100 gl of media (control) or 100 gl of media containing an (optimal) concentration of each mitogenic agent. The T-cell mitogens were phytohemagglutinin-P (PHA-P) obtained from Difco Laboratories, and concanavalin A (con A) obtained from Calbiochem. Pokeweed mitogen (PWM), which induces proliferation of both T and B cells, was purchased from Grand Island Biological Co. Various mitogens that specifically induce B-cell acti-

vation were used in these studies. These consisted of dextran sulphate (DxS), which has been shown previously to stimulate immature B cells [Gronowicz et al., 1974; Gronowicz and Coutinho, 1975; Ahmed et al., 1976], bacterial lipopolysaccharide of E. coli (LPS), and the homoribopolynucleotide, polyinosinic polycytidylic acid (Poly I-C), and purified protein derivative

IMMUNE RESPONSE TO RADIATION 211

(PPD). The DxS was purchased from Pharmacia, (Uppsala, Sweden); LPS from Difco Labs; PPD from Parke Davis; and Poly I'C from P. L. Biochemicals.

Cultures were performed in triplicate and were incu- bated at 37 øC in a humidified atmosphere of 5% CO 2 for a total of 72 hours. Eighteen hours prior to harvest each culture received 1 /•Ci of methyl-3H-thymidine (Schwarz-Mann) in 20 gl of media. Cultures were har- vested using the Multiple Automated Sample Harvester as previously described [Thurman et al., 1973].

2.7. Enumeration of complement-receptor-bea•ng lymphoid cells.

The number of CR+ cells was determined by the technique of Strong et al. [1975]. Briefly described, splenic cells were treated with tds-buffered NH4C1 for red-cell lysis, were washed, and then were resuspended to 5 X 106 cells/ml One ml of the .,suspension was incu- bated with 1 ml o• EAC reagent at •7 C for 45 minutes. After incubation, a drop of the mixture was observed under a microscope and the number of lymphoid cells that formed rosettes (at least five erythrocytes adhering to one lymphocyte was enumerated. A total of 300 or more lymphoid cells was counted and the percentage of CR+ cells was determined. The EAC reagent was made of sheep red blood cells (E) and a 19S fraction of human antisheep red-blood-cell antibody (A); fresh mouse serum was used as a source of complement (C). The EAC reagent also contained 0.01 M EDTA to prevent binding of EAC complexes to granulocytes or macrophages.

2.8. Immunization of mice.

Groups of mice were given an immunization treatment of saline (control), of sheep red blood cells, or of DNP- lys-Ficoll, all intraperitoneally. The SRBC was used as an antigen that requires the presence of functional T cells for the B cells to make antibody. The DNP-lys- Ficoll was used as an antigen that has been characterized previously as not requiring T cells for the B cells to make antibody. Thus, SRBC was used as a T-dependent antigen and DNP-lys-Ficoll was used as a T-independent antigen [Sharon et al., 1975]. After immunization, the mice were divided into two groups; one half was exposed to microwaves and the other half was sham exposed.

2.9. Antigens

Sheep red blood cells (SRBC)were collected in Alsever's solution, were washed several times in isotonic saline and, finally, were resuspended to give a 10% suspension. The washed SRBC were kept at 4 øC and were never more than four weeks old prior to use. Each mouse received 0.2 ml of the washed SRBC suspension. For the other antigenic treatment, dinitrophenol (East- man Kodak, Rochester, NY) was conjugated to L-lysine and Ficoll (both from Sigma Chemicals). Each mouse was injected with 100/•g of DNP-lys-Ficoll in a volume of 0.5 ml (Figure 3).


B Cell Precursor Ig-, la-, CR-

Dextran Sulfate (5(X),000 D)

Immature B Cell 1

(Ig+, la-, CR-)

Immature B Cell 2

(Ig +, la +, CR-)

E. Coli Lipopolysaccharide (LPS)

Polyinosinic, Polycytidilic Acid (Poly I,C)

Mature B Cell I Purified Protein Derivative (Ig +, la +, CR +) of Tuberculin (PPD)

Antibody-Forming Cell Terminal Cell Not Responsive To Mitogenic Stimulation

Fig. 3. Correlation of maturational stage with specificity of response of B-lymphoid cells to mitogenic stimulation. A mitogen not only induces proliferation of cells but drives the cell

to the indicated stage of maturation.

2.10. Direct plaque-forming-cell {PFCJ assay.

The splenic cells from microwave- and sham-exposed mice were assayed for presence of immunoglobulin-M (IgM) antibody-forming cells by a modification [Mosier, 1969] of Jerne's direct PFC assay [Jerne and Nordin, 1963]. The choice of the indicator cells depended on the type of antigen that was used for immunization,

treated controls, and from microwave-exposed and sham-exposed animals were assayed against both kinds of indicator cells. The data are expressed as the PFC per 106 spleen cells and represent the mean and the standard error of triplicate samples.

3. RESULTS

3.1. Effect of a single microwave exposure of mice on frequency of T- and B-lymphoid-cell subpopulations in the spleen.

Mice in groups of six were individual ly exposed to a single dose of 2450-MHz radiation for 30 minutes in the waveguide. Spleens were removed six days after the exposure. Spleen-cell suspensions were then prepared and assayed for the total number of T cells (by the use of cytotoxic anti-Thy-l.2 sera and C'), and the total number of B cells (by the use of fluorescent antimouse Ig sera). Sham-exposed mice were used as controls. As is seen in Table 2, a single exposure to 2450-MHz microwaves failed to produce any significant increase or decrease in the total number of relative frequency of thymus (1.2-T) + cells or IG+ B cells. Interestingly enough, when the frequency of CR+ cells was determined, there were marked net increases of 24 to 29%.

3.2. Effect of three exposures of mice to microwaves on the frequency of T- and B-lymphoid-cell subpopulations in the spleen.

Groups of six mice were given three, 30-minute doses of 2450-MHz radiation, one dose each on days 0, 3, and 6; six days after the last exposure their spleens were

as is summarized in Table 1. Cells producing IgM anti- removed and assayed for the total number of T cells, SRBC antibody in response to SRBC immunization were B cells, and CR+ cells. Sham-exposed mice served as assayed using SRBC as indicator cells. Cells producing controls. As is seen in Table 3, there was no significant IgM anti-DNP antibody in response to DNP-lys-Ficoll change in the total number of (thy-l.2)+-bearing T were assayed using SRBC conjugated with trinitrophenyl- cells, but there was a significant increase in the total haptenated SRBC as indicator cells. This light haptena- number of Ig-bearing cells and further net increases tion of SRBC was accomplished by the method of (52-64%) in the frequency of CR+ cells as compared Rittenberg and Pratt [1969]. Splenic cells from saline- with values obtained for spleen cells from sham-exposed

mice.

TABLE 1. Antigens and appropriate indicator cells that were used to evaluate effects of 2450-MHz microwave radation on IgM response of mice to T-dependent and T-independent anti-

gens.

Antigenic Treatment Indicator Cells for PFC-Assay

TABLE 2. Effect of a single, 30-minute exposure of CBA/J

mice to microwaves (2450-MHz; •' 13 mW/g) on the fre•.uency of subpopulations of T-and B-lymphoid cells (Mean- SE).

0.5-ml saline i.p. (control) SRBC, TNP-SRBC Type of Frequency of Pos•bve Cells' Surface Marker Cell Detected Sham-Exposed M•crowave-Exposed

0.2-ml 10• SRBC in saline i.p. SRBC

100-mg DNP-lys-Ficoll in 0.5-ml saline i.p. TNP-SRBC

Theta T 37.8 +_ 1.7 34.7 +_ 2.3

Ig B 51.6 _+ 2.9 53.9 ñ 2.7 CR B 22.5 •_ 2.0 30.6 + 1.5'

Abbreviations: SRBC, sheep red blood cells; PFC, plaque-forming cells; TNP-SRBC, SRBC coated with trinitrophenyl sulfonic acid; DNP-lys-FicolI, conjugation of dinitrophenyl with lysine and Ficoll, known to be a T-independent antigen.

'Data were determined on spleen-cell suspensions from six sham-exposed and six m•crowave-exposed mice. *P< .01


TABLE 3. Effect of three, 30-minute exposure of CBA/J mice to 2450-MHz microwaves (• 14 mW/g) on the frequency of

subpopulations of T- and B-lymphoid cells (Mean + SE).

Frequency of Posibve Cells' Type of -

Surface Marker Cell Detected Sham-Exposed M•crowave-Exposed

Theta T 35.5 t 1.4 30 9 -+ 1.8 Ig B 48 1 + 1 4 59.4 _+ 2 4* CR B 23.4 + 2 1 36 7 _+ 3 8'

'Data were determined on spleen-cell suspensions from s•x sham-exposed and s•x m•crowave-exposed m•ce. 'P< 05

3.3. Effect of a single exposure of mice to 2450-MHz microwaves on the response of splenic cells in vitro to T-cell mitogens. •

Splenic cells from microwave and sham-exposed mice were cultured in vitro in microculture plates in the presence of media (control) or of various dilutions of either PHA-P or con-A, two plant lectins that induce blastic transformation of T cells but not B cells. As is seen in Table 4, no significant difference (P > 0.05) occurred in the uptake of 3H-TdR in splenic cells of microwave- and sham-exposed mice. The six spleen-cell suspensions from microwave-exposed mice and the six spleen-cell suspensions from sham-exposed mice were .assayed individually.

3.4. Effect of a single exposure of mice to 2450-MHz microwaves on the response of splenic cells in vitro to various B-cell mitogens.

Groups of. six mice were exposed once to microwaves and 3, 6, or 9 days thereafter their spleens were removed. Splenic cells were assayed for ability to undergo DNA synthesis in the presence of a variety of B-cell- specific, mitogenic agents. Splenic cells from sham- exposed mice were used as controls. Various concentrations of each mitogen were used in this assay in efforts to determine'if exposure to microwaves caused a change in the optimal mitogenic dose. For reasons of brevity, data from cells that exhibited incorporation of the largest quantities of 3H-TdR are reported here. As is seen in Table 5, splenic cells from both microwave- exposed and sham-exposed mice responded well to all four mitogens. There was no major difference in the response to the various B-cell mitogens. However, there was a sy_stematic trend toward an increasing incorporation of 3H-TdR by splenic cells fr øm microwave-expos ed mice in response to LPS, Poly I-C, and PPD. Although most of the differences are statistically significant, as evaluated by t test, further experiments are necessary to confirm the validity of the data.

3.5. Effect of three exposures of mice to 2450-MHz radiation on the response to T-cell-specific mitogens.

A grouP of 18 CBA/J mice were exposed for 30 min-

utes to 2450-MHz microwave radiation on days 0, 3, and 6. Sham-exposed mice served as controls. Three, 6, and 9 days after the last exposure, splenic cells from six microwave-exposed and six sham-exposed mice were cultured in vitro with varying concentrations of phytohemagglutinin-P and con A, two plant lectins that specifically induce the activation of T cells but not B cells. As seen in Table 6, no significant differences were noted in the ability of splenic cells from either microwave- or sham-exposed mice to respond to PHA-P or to con A.

3.6. Effect of three exposures of mice to 2450-MHz microwaves on the response of splenic cells to a variety orB-cell mitogens.

Aliquots of the same splenic cells that had been tested for ability to respond to the T-cell-specific mitogens were also tested for their ability to respond to a variety of B-cell mitogens. Again, splenic cells from these mice responded quite well to all of the B-cell-specific mitogens (Table 7) and, similar to the results obtained with cells from mice exposed to a single dose of microwave radiation, the cells showed a slight augmentation ;n their response to LPS and to Poly I-C. The augmentation to LPS is statistically significant, but the biological significance is unknown.

3.7. EffeCt of three exposures of mice to 2450-MHz microwaves on the response to thymus-dependent {SRBC} and thymus-independent {DNP-lys-Ficoll} antigens.

Mice in groups of four were given intraperitoneal immunizing treatments either with 0.2 ml of a 10% suspension of SRBC, with 50/.tg of DNP-lys-Ficoll, or with 0.2 ml of saline (control). After the treatment, they were all exposed individually' for 30 minutes to 2450-MHz radiation on days 1,2, and 3. On the 4th day, their spleens were removed and cells were assayed for ability to form antibody to SRBC or TNP-SRBC by the plaque,formation assay. As is seen in Table 8, while there was a modest but insignificant increase in the response of saline controls (from 713 ß 308 to 1219 ß 274 PFC per spleen) there was a significant decrease in the response of mice immunized with SRBC (from 24,625 + 4288 to 11,000 + 2121 PFC per spleen). Similarly, there was a considerable, if statistically insignificant, decrease in the response of mice immunized with DNP-lys-Ficoll (Table 9) from 24,375 + 4399 PFC per spleen to 17,000 + 5700 PFC per spleen. The decrease wa s not secondary to an increase in numbers of splenic cells since the decrease was also of the same magnitude when data were calculated on the basis of plaque-forming cells per 106 splenic cells.

4. DISCUSSION

These studies demonstrate that exposure of mice to 2450-MHz microwaves in• an environmentally controlled waveguide has weak stimulatory effects on B- but not


TABLE 4. The effect of a single exposure of micel to 2450-MHz microwaves on response of splenic cells in vitro to T-cell mitogens, PHA and con A.

Mean Uptake 2 of 3H-Thymidine cpm _+SE

Time Following Exposure

Three Days 2 Six Days 2 Nine Days 2

Concentration

Mitogen per Culture 3 Sham Exposed Sham Exposed Sham Exposed

None Media control 2415 _+ 165 2423 + 198 5077 + 325 4563 +434 2118 + 555 2081 _+ 462

PHA-P 0.1% 100158_+ 5671 108051 +3149 99386+6116 108421 + 1312 54385+7773 46551 +482

Con A 0.25 ]jg 208616 _+ 7695 193889 + 9302 218837 + 6503 215914 + 4255 192687 + 25302 185&33 + 15564

•Six animals per group. 272-hr. culture evaluated by the pulse with 1 ]aCi of 3H-TdR 18 hr prior to harvest. 3Optimal concentration.

on T-lymphoid cells of the spleen. A single exposure 2450-MHz radiation induced an increase in the total of mice neither increased the total number of splenic number of splenic cells, in the total frequency of cells nor the frequency of either theta-bearing (T)cells Ig+ (B cells), and further increased the frequency of or Ig-!- (B) lymphoid cells. However, the single expo- CR-!- B cells, but had no significant effect on the total sure caused a highly significant increase in the frequency number of T cells. The results indicate, at least at the of cells possessing complement receptors (CR)on their dosages used, that microwaves act primarily on B- surface, which is a characteristic of mature B cells. lymphoid cells and produce quantitative changes in the In contrast, three 30-minute exposures of mice to absolute frequency of B-cellsubpopulations.

TABLE 5. The effect of a single exposure of mice I to 2450-MHz microwaves on the response of splenic cells in vitro to B-cell mitogens.

Mean Uptake 2 of 3H-Thymidine cpm _+SE

Time following exposure

Three Days 2 Six Days 2 Nine Days 2

Concentration

Mitogen per Culture 3 Sham Exposed Sham Exposed Sham Exposed

None Media control 2415 + 165 2423 + 198 5077 + 325 4563 z 434

DxS 50 •1 g Not performed 12307 _+ 1074 9515 + 1784

LPS 25 ].lg 47496+ 3313 49823 + 1882 72385 + 3299 80151 +4297

Poly I-C 50 ].lg 50523_+ 4950 63577 + 3432' 70851 + 5413 93465 + 3032'

PPD 100 IJ g 36898 _+ 4408 46763 _+ 1275' 34254 + 1156 43406 + 1735'

2118 + 555 2081 + 262

Not performed

60121 --+2252 75119+_ 1844'

48358 _+ 8629 58873 z 3667

53436 _+ 2972 60002 + 1279'

'Six animals per group. 272-hr culture evaluated by a pulse with I •Ci of 3H•TdR 18 hr prior to harvest. 3Optimal concentration 'P < .05

IMMUNE RESPONSE TO RADIATION 21 $

TABLE 6. The effect of three exposures of mice I to 2450-MHz microwaves on the response of splenic cells in vitro to T-cell mitogens, PHA-P and con A.

Mean Uptake 2 of 3H-Thymidine cpm + SE

Time following first exposure

Nine Days 2 Twelve Days 2

Concentration

Mitogen per Culture 3 Sham Exposed Sham Exposed

None Media control 6539 + 1109 5051 + 574 4409 + 795

PHA-P 0.1% 98949 + 7980 108337 + 5930 66848 + 10724

Con A 0.25 1J g 225033 + 38054 237792 + 38645 276672 + 34783

3830 + 796

79035 + 22031

298164 + 32991

•Six animals per group. 272-hr. culture evaluated by a pulse with I 1JCi of 3H-TdR 18 hr prior to harvest. aOptimal concentration.

TABLE 7. The effect of three exposures of micel to 2450-MHz microwaves on response of splenic ceils in vitro to B-cell mitogens, LPS, POLY ! 'C, PPD.

Mean Uptake 2 of 3H-Thymidine cpm + SE

Time following first exposure

Nine Days 2

Concentration

Mitogen per Culture 3 Sham Exposed Sham

Twelve Days 2

Exposed

None Media control

LPS 25 l•g

Poly !-C 50 l•g

PPD 100 l•g

6539 + 1109 5051 + 574 •4409 + 795 3830 + 796

60613 + 4683 61720 + 3266 58641 + 5020 76727 + 5693*

• 73336 + 6521 71356 + 8561 57960 + 7158 70789 + 11314

43160 + 3283 46459 + 4271 41761 + 6692 46128 + 5695

•Six animals per group. •2-hr. culture evaluated by a pulse with I IJCi of 3H-TdR 18 hr prior to harvest. 3Optimal concentration only. *P < .05.

216 WlKTOR-JEDRZEJCZAK, AHMED, CZERSKI, LEACH, AND SELL

TABLE 8. Effect of three exposures to 2450-MHz microwaves on response of mice to a thymus- dependent antigen (SRBC).

Immunizing Treatment

Mean D-PFC/spleen •

Sham-Exposed Microwave-Exposed

Mean D-PFC/106 splenic ceils •


Saline 713 + 308 1219 + 274 ' 5.3 + 2.2 9.7 + 3.0

SRBC 24625 +_ 4258 11000 _+ 2121' 204.4 _+ 22.2 112 _+ 16.8'

•Splenic cells from six mice were assayed in duplicat e for ability to form plaque-forming cells against sheep erythrocytes. Arithmetic means were calculated and from the data obtained the mean of means of direct IgM plaque- forming cells (PFC) per 106 splenic cells (+S.E.) was calculated per group of six mice. *P<.05.

In general, two questions regarding the mechanisms of of microwave radiation produces detectable proliferation these effects should be considered. First, does exposure of cells [Wiktor-Jedrze/czak, unpublished observations]. to microwave radiation result in proliferation of cer- The possibility that B cells may proliferate elsewhere tain subpopulations of B cells? Such an occurrence would be in agreement with the data of Stodolnik- Baranska [1967] and of Czerski [1975]. Second, the same effects might be obtained by stimulation of immature B cells that are already present in the spleen and that subsequently express properties of more highly differentiated cells. A lack of change in the total num-

(e.g., in the bone marrow) from which they may migrate to th e spleen, also seems to be unlikely since no increase was found in the rate of DNA synthesis after short - term (4-hour incubation of bonemarrow cells or peripheral blood lymphocytes in culture [Wiktor-Jedrzejczak, unpublished observations]. There are two major ways of studying the effects of

bet of splenic cells after exposure of mice to micro- microwave radiation on the functional capacity of waves, which was found in an initial pilot study, would lymphoid cells: (1) evaluate the ability of the cells to indicate that the latter possibility was the case :" Further undergo specific activation by antigens, and (2)evaluate experiments were carried out by which to evaluate the the ability of the cells to be stimulated by nonspecific rate of DNA synthesis in splenic cells of mice at various activators -- by mitogens. In any mammal, there are intervals following expOSure to microwaves. Cells were groups of cells, T and B, that are genetically prepro- cultured in vitro for various.periods of time (4 hours gramreed to respond to antigens that possess unique to 7 days) and incorporation of a specific DNA-pre- Structural properties. Such groups of cells are derived cursor (tritiated thymidine) was measured. No differ- originally from single precursors and, therefore, are ences in uptake of thymidine were observed between called "clones." Mitogens can stimulate many clones cells from microwave-irradiated and sham-exposed mice. of lymphoid cells that have similar general characteris- It was concluded that neither a single nor a triple dose tics apart from their specific antigenic reactivities

TABLE 9. Effect of three exposures to 2450-MHz microwaves on response of mice toa thymus - independent antigen (DNP-lys-Ficoli).

Immuni•.ing Treatment

Mean D-PFC/spleen'


Mean D-PFC/106 splenic cells •


Saline 713 _+ 308 1219 + 274 5.3 _+ 2.2 9.7 _+ 3.0

DNP-lys-Ficoll 24375 + 4399 17000 _+ 5700 163 + 23 152 _+ 42

•Splenic cells from six mice were assayed in duplicate for ability to form PFC against sheep erythrocytes. Arithmetic means were calculated and from the data obtained the mean of means of direct IgM plaque-forming cells (PFC) per 106 splenic cells (+S.E.) was calculated per group of six mice.


[Nowell, 1976]. Moreover, some routinely used mitogens stimulate clones of T-cells only, while others only stimulate clones of B cells. For example, phytohemagglutinin (PHA) and concanavalin A (con A) only activate clones of T-lymphoid cells [Greaves and Bauminger, 1972; Andersson et al., 1972]. On the other hand, dextran sulfate, the lipopolysaccharide of E. coli (LPS), polyinosinic polycytidylic acid (poly I-C), and purified protein derivative of tuberculin (PPD) only stimulate B cells [Gronowicz and Coutinho, 1975; $cher et al., 1973]. There are also mitogens such as pokeweed (PWM) that stimulate both T-and B-lymphoid cells [Janossy and Greaves, 1971]. Further, there are indica- tions that difference subpopulations of T or B cells are stimulated by difference T-and B-cell-specific mitogens. These differences correlate with the functional (maturational) level of the cells. For example, con A was found primarily to stimulate T-helper cells [Hirst et al., 1975]; that is, cells that help B cells "recognize" certain antigens. In the case of B-cell-specific mitogens, a good correlation exists between stage of maturation of the B cells and their response to a given mitogen (Figure 3). Dextran sulfate stimulates precursors of B cells [Grono- wicz and Coutinho, 1975]; LPS and Poly I-C stimulate immature B cells [Gronowicz and Coutinho, 1975; Ahmed et al., 1976]; and PPD stimulates mature B cells [Gronowicz and Coutinho, 1975]. These mitogens stimulate cells at a given stage not only to proliferate but also to make the transition to the next maturational

stage. The process whereby B cells from many different clones are stimulated nonspecifically is called "B-cell polyclonal activation" [Gronowicz and Coutinho, 1975]. The ability of lymphoid cells to undergo mitogenic stimulation is, therefore, a measure of the functional capacity of subpopulations of lymphoid cells. The assays for mitogenic activity are partly automated [Strong et al., 1973], which allows simultaneous performance of several assays at various concentrations of cells and mitogens. The "mitogen assays" are useful tools, therefore, in the screening of functional changes in lymphoid cells that are produced by various factors; the assays were used by us to characterize the effects of microwave radiation on the function of lymphoid cells.

Neither a single exposure nor a triple exposure of mice to microwaves affected the response of their splenic cells to the T-cell-specific mitogens. Also, the response to PWM, which stimulates both T and B cells, was unchanged. The response to the B-precursor-cell-specific mitogen, dextran sulfate, was unaltered, while the response to the B-cell-specific mitogens LPS and Poly I-C was significantly increased, both after single and triple exposures. The response to the mature B-cell mitogen PPD was also significantly increased after a single exposure. The results were clearly correlated with previously discussed changes in the proportion of cells that bear appropriate surface markers. Microwave irradiation did not stimulate lymphoid cells to proliferate; the irradiation probably increased the number of cells that are responsive to stimulation by the various B-cell mitogens. The radiation apparently acted as a polyclonal B-cell activator without mitogenic proper-

ties. The data obtained on mitogens also provided further evidence that the microwaves did not affect the T4ymphoid cells.

Another important approach to the study of the effects of microwaves on the function of the immune

system is the evaluation of T- and B-cell responses to specific antigens. Studies of the specific response require the choice of an antigen, the response to which can serve as an example of the immune response to a given category of antigens. For this purpose, one must distin- guish between antigens that activate appropriate clones of T cells and antigens that stimulate antibody-producing B cells. The structural properties of an antigen determine the clone of T- and B-lymphoid cells with which it will interact [Feldmann et al., 1975 ; Coutinho, 1975]. The main structural criterion for the selection of the type of clone of responsive cells is the difference between the structure of the antigens that are recognized by lymphoid cells as foreign and the structure of autologous antigens of the organism. In essence, B cells by themselves respond to antigens that are clearly different structurally from autologous antigens, e.g., bacterial antigens. The T cells, on the other hand, are able to recognize antigens that are highly similar to, yet different from, those normally occurring in the body, e.g., antigens of another animal of the same species or antigens of autologous, malignant tumors. There are also antigens that B and T cells respond to cooperatively. As already mentioned, an antigen must be highly different from an autologous antigen in order to be recognized as foreign for B cells. In the case of antigens where structural differences are of the inter- mediate type, B cells recognize a part of them as foreign. The other part is recognized as foreign by a special subpopulation of T cells, called "T-helper cells," and these T-helper cells, in turn, specifically activate maturation of B cells and lead to the production of antibody that is specific to the antigen. The type of antigens that require help of T cells to engender a B-cell response are called "T-dependent," while other antigens, for which B cells do not require T-helper cells, are called "T- independent."

In order to determine if exposure of mice to microwaves induces a specific effect on their ability to respond to thymus-dependent and thymus-independent antigens, groups of mice were immunized with a well- known and established thymus-dependent antigen, sheep red blood cells (SRBC) and a well-known and docu- mented thymus-independent antigen (DNP-lys-Ficoll). The studies indicate that while three small doses of

microwave radiation produce polyclonal stimulation of B-lymphoid cells in the spleen of exposed mice, they may simultaneously decrease the number of antibody- forming cells that are induced specifically by injection of thymus-dependent or thymus-independent antigens.

The apparent discrepancy between the two effects may be explained as follows: injection of antigen produces a stimulus for activation of a specific clone of B cells which, in the course of the reaction to antigen, generates antibody-producing plasma cells. The magnitude of the response depends to some extent on the


number of available antigen-reactive B cells. Microwaves can be extrapolated directly to man is questionable, may nonspecifically stimulate some of the cells to since the frequency of radiation we used (2450-MHz) mature before they are activated by antigen. When sig- allows for almost complete penetration of microwaves nals produced by injection of an antigen reach them, at through the body of the mouse but would allow for only least some of them will be already in a late, unresponsive superficial penetration of the tissues in man [Baranski stage. The discrepancy between our data and observa- and Czerski, 1976]. Studies on the effects of micro- tions previously reported by Czerski [1975], who found an increase in the IgM response to SRBC immunization after long-term exposure to microwaves, may be explained by differences both in the exposure-immunization schedule and in the exposure conditions of the two studies. Czerski first exposed animals, then immunized them and assayed for response, while in our study the animals were first immunized and then exposed before the response was evaluated. Nevertheless,

waves on the immune system are in their infancy; until experiments are performed in different laboratories, under different exposure conditions, and with different species of animals, firm conclusions cannot be drawn.

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Immune response of mice to 2450MHz microwave radiation: Overview of immunology and empirical studies...

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