BRAIN,BEHAVIOR, AND IMMUNITY 4, 129-138 (1990)

Immune Function in Lines of Mice Selected for High or Low Degrees of Behavioral Asymmetry

ESTERFRIDE,'ROBERT L. COLLINS,* PHIL SKOLNICK, AND PRINCE K. ARORA

Laboratory of Neuroscience, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and *Jackson Laboratory, Bar Harbor, Maine 04609

Cerebral lateralization has been suggested to play a regulatory role in immune func- tion. In this study, several measures of immune function were evaluated in mice selec- tively bred for either a strong (HI) or weak (LO) degree of behavioral asymmetry (paw preference) and compared to an unselected control population (HET). Both HI and LO animals had fewer spleen cells but higher degrees of rH]thymidine incorporation into DNA (on a per cell basis) than HET mice. However, only HI mice had lower immune functions compared to HET controls manifest as reduced mixed leukocyte reaction (MLR), cytotoxic T lymphocyte (CTL) activity, and natural killer (NK) cell activity. These findings indicate that although both extremes in the degree of paw preference may be associated with deviations from the norm, a high degree of behavioral lateralization is associated with decreased immune responsiveness in this animal model. 0 1990 Academic

Press, Inc.

INTRODUCTION

Cerebral asymmetry and lateralization of behavioral functions have been de- scribed in many species, including man (for review, see Glick, 1985; Geschwind & Galaburda, 1987). While a relationship between an abnormal pattern of cerebral lateralization and autoimmune disease has been proposed (Geschwind & Behan, 1982, 1984), several studies in man failed to find evidence for a relationship be- tween left or right handedness and immune function (Burke, Yeo, Vranes, Garry, & Goodwin, 1988; Pennington, Smith, Kimberling, Green, & Haith, 1987; Van Strien, Bouma, & Bakker, 1987). However it is unclear whether a deviation in the directionality of asymmetry from the predominant right-handedness, rather than a decrease in the strength of hemispheric specialization, underlies anomalous pat- terns of cerebral Iateralization in man (Bryden, 1982). Hence it is possible that the degree of handedness, rather than the direction (left or right), is associated with immune function.

Cortical ablation studies in rodents indicate that the left and right cerebral hemisphere each exert a differential control on immune function (Renoux, Bi- ziere, Renoux, Guillaumin, & Degenne, 1983; Neveu, Taghzouti, Dantzer, Simon, & LeMoal, 1986; Bameoud, Neveu, Vitiello, & LeMoal, 1988; Neveu, 1988). In order to examine whether there is a relationship between the degree and/or di- rection of behavioral lateralization and immune function, we employed lines of mice which were selectively bred for high (HI) and low (LO) degrees of behavioral

’ To whom all correspondence should be addressed. Present address: The Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, P.O. Box 1172, Jerusalem 91010, Israel

129 0889-1591/90 $3.00 Copyri&t 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

130 FRIDE ET AL.

asymmetry based on their performance in a paw preference test (Collins, 1985). HI line animals express a strong preference for using one paw over the other, while the LO mice express only weak paw preference. These mice were compared with the heterogeneous (HET) parent population.

MATERIALS AND METHODS

Animals

Male mice of the HI, LO, and HET lines were obtained from Jackson Labo- ratory (Bar Harbor, ME) at l&l 1 weeks of age. The common ancestral stock was produced by means of a diallel cross from six inbred mouse strains (BALB/cJ, C57BL/6J, DBA/2J, LP/J, RF/J, and SIWJ) and two partially inbred stocks of M. castaneus and M. molossinus. Shipments of generations 33-36 of HI and LO lines and generations 20-23 of HET control mice were supplied. Upon arrival (in groups of 3-5) at the NIH vivarium, animals were kept with their original cage- mates in order to minimize stress and fighting. They were housed in plastic cages (15 x 25 x 12.5 cm) on a 12-h light cycle (lights on at 0700 h) with free access to food and water and were acclimated to the NIH conditions for 2 weeks before use. Female NFR/N mice (Veterinary Resources Branch DRS, NIH) were used as a source of allogeneic stimulator spleen cells. C57BL/6, DBA/2, and BALB/c mice were obtained from the National Cancer Institute (Frederick, MD).

Paw Preference

Animals were scored at the Jackson Laboratory for paw preference in an “un- biased world” as described previously (Collins, 1985). A total of 50 paw entries was observed. The degree of paw preference was assessed by [PPE = abs(RPE - 25) + 251, where PPE is the number of preferred paw entries, RPE is the number of right paw entries, and abs stands for the absolute value (of RPE - 25). The direction of paw preference was defined as right when an animal had made more than 25 right paw entries, and left, when it had made more than 25 left entries (Collins, 1985).

Preparation of Spleen Cells

Animals were killed by cervical dislocation (0900-1008 h). Spleens were re- moved immediately and placed in Hanks’ balanced salt solution (HBSS, GIBCO, Grand Island, NY) containing 10% heat inactivated fetal bovine serum (HFBS, Hazleton Research Products, Lenexa, KS). Single cell suspensions were prepared as described by Arora and Shearer (1981). In brief, spleens were minced, passed through a sterile nylon screen, and washed twice in HBSS + 10% HFBS. The cells were counted by trypan blue exclusion.

Mixed Leukocyte Reaction (MLR)

Mixed leukocyte reaction (MLR) assay was performed in 96-well flat bottom microtiter plates (Costar, Carbundy, MA) using 1 x lo6 responder spleen cells in

IMMUNE FUNCTION AND BEHAVIORAL ASYMMETRY 131

100 $/well of tissue culture medium (TCM) consisting of RPMI-1640 medium (GIBCO) supplemented with 2 mM r.-glutamine, 1% nonessential amino acids, 1% sodium pyruvate (Bioproducts Walkersville, MD), 5 x lop5 M 2-mercapto- ethanol, penicillin/streptomycin, gentamycin (GIBCO), and 10% HFBS (Hazle- ton). Cells were cultured in the presence or absence of irradiated (2000 rad) allogeneic female NFR/N stimulator spleen cells (4 X lo5 in 100 ~1) which had been freed of erythrocytes by treatment with ammonium chloride lysing solution (ACK, NIH Media Unit, Bethesda, MD). The culture plates were incubated at 37°C in 95% air, 5% CO2 for 4 days. Cultures were pulsed with 1 l&i of [3H]thymidine (sp act 6.7 Ci/mmol; DuPont-New England Nuclear, Boston, MA) for the final 20-24 h. The cells were harvested with a PHD cell harvester (Cam- bridge Technology, Cambridge, MA) and L3H]thymidine uptake measured in a liquid scintillation counter (Beckman LS 5801). MLR ratio was defined as the quotient of [3H]thymidine incorporated in the presence and absence (basal up- take) of allogeneic stimulator cells.

In Vitro Generation of Cytotoxic T Lymphocytes (CTL)

Cytotoxic effector cells were generated in 24-well flat bottom tissue culture plates (Costar) as described previously (Arora 8c Shearer, 1982). In brief, respond- ing spleen cells (5 x 106) were co-cultured with 2000 rad-irradiated (‘37Cs source, Gammator Isomedix, Parsippany, NJ) female allogeneic NFRN stimulator spleen cells (2 x 106, ACK lysed) in TCM. The culture plates were incubated for 5 days under the same conditions as the MLR cultures.

Cytotoxicity Assays

CTL and natural killer (NK) cell activities were assessed by a “Cr release assay (Arora & Shearer, 1981). Briefly, effector cells in lOO+ aliquots were added in triplicate (CTL) or quadruplicate (NK) to microtiter U-bottom wells (Costar). Target cells for CTL were 48-h concanavalin A (Sigma Chemical Co., St. Louis, MO) stimulated blast spleen cells. For NK, YAC-1 tumor cells were used as targets. Target cells were labeled with 200 l&i of Na,‘lCrO, (DuPont-New En- gland Nuclear, Boston, MA), washed twice in HBSS containing 10% HFBS and 3 ml Hepes buffer (GIBCO). After counting, target cells were added (100 t.~l) to the microtiter wells containing effector spleen cells, such that different effector:target cell (E:T) ratios were evaluated. The plates were centrifuged for 3 min at 400 rpm and incubated at 37°C for 4 h in a 95% air-5% CO, atmosphere. After incubation, the plates were centrifuged for 3 min at 800 rpm and the supematant was collected with a Titertek Supematant Collection System (Skatron, Inc., Sterling, VA) and counted in a Beckman Auto Gamma scintillation spectrometer. The percentage of lysis was determined as described (Arora & Shearer, 1981) using the equation:

EXP. CPM - SPONT. CPM % Lysis = MAX. CPM - SPONT. CPM x loo’

132 FRIDE ET AL.

where EXP. CPM was defined as the counts released into the medium by target cells when assayed with effector spleen cells; MAX. CPM defined as the counts released by target cells in the presence of 2.5% Triton X-100 (no effecters), and SPONT. CPM as the counts released by target cells in the presence of medium alone.

Statistics

AlI data were analyzed using two-way analyses of variance (ANOVA). CTL and NK activities data were analyzed by ANOVA for repeated measures (E:T ratio). Individual comparisons were made by Student-Newman-Keuls tests (SNK). Correlations were calculated with Pearson correlation coeffkients.

Paw Preference

RESULTS

In the sample used in this study, the line distribution of the degree of paw preference reflected that of the larger population (Collins, 1985). Preferred paw entry (PPE) scores were 35.5 + 1.9 (N = 15), 38.9 k 3.0 (N = ll), and 41.7 + 2.2 (N = 13) for LO, HET, and HI mice, respectively. These differences were not statistically significant, but line differences were highly significant for a larger population (N = 293) from which the present sample was drawn (data not shown).

Natural Killer (NK) Cell Activiry

NK activity in HI asymmetry mice was significantly lower than in HET mice. Differences in NK activity ranged between 45 and 49% at E:T ratios of 25:l to 1OO:l (F(2,13) = 3.6, p < .06; see Fig. 1).

25.

20 z2 2 15,

= 10

5

0' 25 50 75 100 EFFECTOR : TARGET CELL RATIO

FIG. 1. NK cell activity in HI, LO, and HET mice. l , HET; W, LO; A, HI. *Different from HET mice C.p < .05). +Different from LO mice (p < .OS). Six mice from each line were used; data from two HI mice were discarded due to technical errors.

IMMUNE FUNCTION AND BEHAVIORAL ASYMMETRY

Spleen Cell Number, Basal i3HjThymidine Uptake, and MLR

133

A significantly higher number (44-76%) of spleen cells was found in HET mice compared to both the HI and LO lines [F(2,43) = 5.9, p < .Ol]. Unstimulated (basal) [3H]thymidine uptake differed between lines [F(2,25) = 3.6, p < .05], with a 67-70% higher uptake found in LO and HI mice compared to HET controls (Table 1). Line differences among cpm values of [3H]thymidine uptake by allo- geneically stimulated spleen cells were not significant [F(2,25) = 2.9, p < . 10; see Table 1,3rd data column], but the decrease in MLR ratio in HI mice, as compared to either LO or HET animals, was highly significant [F = (2,25) = 8.3, p < .002, Table 1, last column]. Similar data were obtained in a replication of this experi- ment (data not shown).

Cytotoxic T Lymphocyte (CTL) Response

The three lines differed in CTL response [F(2,36) = 3.8, p < .04; interaction effect between line and E:T ratio: Hotelling’s F(4,68) = 5.4, p < .Ol]. This effect was due to the lower CTL activity in HI mice as compared to both LO and HET animals (Fig. 2). CTL activity was also measured against the same allogeneic stimulator cells (NFR/N) in the three inbred ancestral stocks with known H-2 haplotypes [BALB/c (H-2d), DBA/2 (H-2d), and C57BL/6, @YZ-~~)]. Table 2 shows that the CTL response was similar for all three strains.

Association between Left or Right Paw Preference and Immune Function

No ambidextrous mice were present in the sample studied. No differences were found between animals with a left as compared to those with a right paw prefer- ence in any of the parameters evaluated (Table 3). Moreover, no significant cor- relations were found between immune parameters and paw preference for indi- vidual mice (data not shown).

TABLE 1 [3H]Thymidine Uptake and Mixed Leukocyte Reaction (MLR) in Spleen Cells of High, Low

Asymmetric, and Heterogeneous Mice

09 Cell no.

(10Yspleen)”

Uptake (cpm)

Basal Stimulated MLR ratid

LO (10) 65 + 5.5’ 41222 -+ 7799’ 101080 2 18695 2.2 -+ .17 HET (11) 115 + 15.5 27729 f 1904 62173 + 7676 2.2 + .17 HI (7) 80 + 12.0’ 46299 f 7801’ 61737 2 10452 1.3 + mc

o Since not all spleens yielded sufficient cells to perform each assay, the sample sizes from which cell numbers are derived are larger than those applicable to MLR. Sample sizes were 18, 14, and 14 for LO, HET, and HI, respectively.

b Stimulated/basal [3H]thymidine uptake. c Significantly diierent from HET mice (p < .05).

134 FRIDE ET AL.

0 * ' 10 20 30 40

Effector : Target Cell Ratio FIG. 2. CTL response in HI, LO, and HET mice. 0, HET; W, LO; A, HI line. *Different from

HET, p < .05. +Different from LO, p < .05. Fifteen LO, 11 HET, and 13 HI mice were used.

DISCUSSION

The present findings demonstrate significant differences in immune function in mice selectively bred for a low (LO) or high (HI) degree of behavioral asymmetry compared with a heterogeneous (HET) population. Thus, several measures of immune function were different in both lines with extremes of paw preference compared to the unselected population. Whether these differences reflect changes in a specific population of lymphocytes may be of importance in understanding the functional differences between the lines. Such studies are currently in progress. Several recent studies have found that aspects of immune function are under asymmetric control using extensive surgical ablations of the cerebral hemispheres in female rodents (Bameoud et al., 1988; Neveu, 1988; Neveu et al., 1986; Renoux et al., 1983). In a model for autoimmune disease, Neveu, Betancur, Bameoud, Preud’homme, Aucouturier, LeMoal, and Vitiello (1989) recently showed an ear- lier onset of auto-antibody synthesis in left-pawed as compared to right-pawed female but not male mice. The present findings in male mice suggest the existence of a relationship between immune function and the degree of behavioral

TABLE 2 CTL Activity in DBA/Z, BALBk, and C57BW6 Mice

CTL response (% lysis)

E:T ratio 40: 1 20: 1 1O:l 5:l WI

DBz#2 (5) 69.2 + 1.5 57.6 f 2.6 42.1 + 2.6 32.5 f 2.9 BALBic (5) 70.8 + 0.7 59.5 2 1.9 43.2 k 1.0 33.1 + 0.7 C57BW6 (5) 73.0 + 1.9 59.9 f 2.1 49.0 2 2.6 37.3 + 2.2

Note, CTL activity was assayed as described in Methods using the same target cells (NFR/N) as in Fig. 1. This experiment was performed in order to determine whether differences in H-2 could explain the lower CTL response in HI compared to LO mice.

IMMUNE FUNCTION AND BEHAVIORAL ASYMMETRY 135

TABLE 3 Immune Function in Mice with Left or Right Paw Preference

Preference

Left (M Right 09

CTL (40: 1) CTL (20: 1) CTL (10: 1) NK (100: 1) NK (50: 1) NK (25: 1) Cell no.

(x lO%pIeen)

MLR (cpm) Basal uptake Stimulated uptake

MLR ratio

69 k 4.6 45 + 9.3 30 f 4.9 23 f 2.1 14 f 1.5 9* 1.2

108 ?I 12.5

27044 2 859 52636 2 3217

2.0 + .I3

(4) (4) (4) (8) (8) 03)

(4)

(4) (4) (4)

60 2 9.4 41 2 7.9 30 2 7.2

18 +- 4.3 11 f 3.0 I f 1.5

120 2 24.0

28111 f 3035 67623 + 11731

2.3 f .24

(7) (7) (7) (6) (6) (6)

(7)

(7) (7) (7)

Note. Only data from HET mice are shown. Values from the other two lines or the pooled data were similar. NK cell activity was measured in animals from a different shipment from those used for MLR and CTL assays resulting in different sample sizes.

asymmetry rather than the direction (left-right) of cerebral lateralization. Hence, further studies are required to investigate whether the mechanism which deter- mines the relationship between directionality of behavioral asymmetry and im- mune function (Bameoud et al., 1988; Neveu et al., 1986, 1989; Renoux et al., 1983) is similar to that influencing the association between the degree of asym- metry and immune function as observed in our study.

In toto, the present data suggest that a high degree of behavioral asymmetry is associated with lower immune function. One possible explanation for the differ- ences observed among the lines is the differential distribution of the major histo- compatibility complex (MHC, H-2 in mice) in HI, LO, and HET mice (Collins, Carlson, & Nadeau, 1985). According to the hypothesis proposed by Geschwind and Behan (1982), genes controlling function of the immune system may also influence development of cerebral lateralization through the mediation of testos- terone. Alternatively, the H-2 complex on Chromosome 17 (Gorer, Lymen, & Snell, 1948) may be localized near the gene(s) affecting cerebral lateralization. The HI and LO lines differ mainly in the distribution of the ZZ-2b (found in LO, but not in HI mice) and H-2d (found in HI and only marginally in LO mice) haplotypes (Collins et al., 1985). Whereas the progenitor stock C57BL/6 and possibly, LPIJ would have contributed the H-2b haplotype in LO mice, the H-2d haplotype could have been derived from either BALB/c or DBA/2 strains. Since CTL activity in response to the same allogeneic stimulator cells (NFR/N) was similar in the three strains tested (Table 2), it is unlikely that the differences in CTL response could be explained by the distribution of genes within the H-2 complex (mapped at the K and D subregions) in the HI, LO, and HET strains. The other two aspects of immune function (MLR and NK activity) which were studied are each regulated

136 FRIDE ET AL.

by different MHC antigens. MLR is determined by the H-21 region and may receive additional regulatory input from a non-MHC locus in mice (Mls; Paul 1989; Roitt, Brostoff, & Male, 1985). Further, it is unlikely that the lower NK activity in HI mice is explained by the line differences in H-2 distribution, since previous studies (Stuttman & Cuttito, 1980; Kiesling & Wigzel, 1979) have shown that the three ancestral inbred strains tested (BALB/c, DBA/2, and C57BL/6) are all in the intermediate category of NK response. Thus, the consistently lower immune activity observed in the HI line for the parameters studied here (MLR, CTL, and NK activity) suggests that the association between behavioral asym- metry and immune function cannot be explained by the differential distribution of the H-2 complex among the lines.

Although no significant correlations were observed between paw preference and immune parameters for individual mice, the sample size used in the present study may not be sufficiently large to evince significant associations between these diverse variables. No differences in immune function among the HET, HI, and LO lines were found between animals with left/right paw preferences (Table 3). In contrast, Neveu, Barneaud, Vitiello, Betancur, and LeMoal(1988) reported that C3H mice with left paw preferences had higher mitogen-induced lymphocyte proliferation than animals with right preferences. However, recent data from our laboratory indicate that differences in immune function such as mitogen-induced cell proliferation between mice exhibiting a left/right paw preference appear to be a strain-dependent phenomena (Fride, Collins, Skolnick, & Arora, in press). This strain dependence would be consistent with the lack of correlation between hand- edness and immune function reported in several human studies (Pennington et al., 1987; Van Strien et al., 1987; Burke et al., 1988). Nonetheless, the degree of behavioral lateralization was clearly associated with immune function, with HI mice exhibiting reduced MLR, CTL, and NK responses compared to HET con- trols.

The reduced immune function observed in mice with a high degree of behavioral lateralization appears inconsistent with the hypothesis that a higher degree of cerebral asymmetry is of greater adaptive value than a more symmetric brain in man (Bradshaw, 1978; Blakeslee, 1980; Geschwind & Galaburda, 1987) as well as in rodents (Glick, Zimmerberg, & Jerussi, 1977; Zimmerberg, Strumpf, & Glick, 1978; Denenberg, Sherman, Rosen, & Galaburda, 1988). However, Glick et al. (1977) observed that rats and mice with either an extremely high or low degree of behavioral asymmetry displayed decreased learning ability as compared to ani- mals with moderate degrees of lateralized behavior. Thus, it is possible that both extremes of asymmetry may affect behaviors/neuroendocrine parameters which ultimately impact on physiological functions. This is consistent with the sugges- tion (Glick et al., 1977; Bradshaw, 1978; Geschwind & Galaburda, 1987) that both extremely strong or weak degrees of cerebral lateralization may be maladaptive traits. Thus, the lower immune response in HI mice together with the observation that both HI and LO mice also had a lower number of spleen cells (44-760/o) and a higher basal [3H]thymidine incorporation into DNA (67-70%) than HET mice (Table 1) are consistent with that suggestion.

The neuroanatomical substrate(s) resulting in a lowered immune function in HI

IMMUNE FUNCTION AND BEHAVIORAL ASYMMETRY 137

mice are unknown. However, Cassells, Wahlsten, and Collins (in press) have recently observed that the size of the corpus callosum of HI mice was significantly smaller than that of LO mice. This finding leads us to suggest that changes in interhemispheric communication due to alterations in callosal development may underlie some of the changes in immune function in HI mice as reported here. This hypothesis is currently under investigation.

ACKNOWLEDGMENTS

We thank Ms. Lisa Burton for expert behavioral testing. Portions of this work were supported in part by grant #NO001489-J-1534 from the Office of Naval Research to PKA. RLC was supported in part by NIH grant GM 23618. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.

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Received September 12, 1989