Correlation of aggression with serum IgM level in autoimmune-prone NZB mice

Developmental Brain Research 159 (2005) 145 – 148 www.elsevier.com/locate/devbrainres

Short Communication

Correlation of aggression with serum IgM level in autoimmune-prone NZB mice Kazuhiro Nakamura a,*, Hiroyuki Nishimura b, Sachiko Hirose a b

a Department of Pathology, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Department of Biomedical Engineering, Toin Human Science and Technology Center, Toin University of Yokohama, Yokohama 225-8502, Japan

Accepted 23 July 2005 Available online 6 September 2005

Abstract Neurological symptoms are often found in patients with systemic lupus erythematosus, an autoimmune disease. We found an enhanced aggression in young autoimmune-prone NZB mice before expression of autoimmune hemolytic anemia, which was accompanied by an increase in neural activity in the accessory olfactory bulb. The performance of aggressive behavior was correlated with serum IgM level. These results indicate that IgM class autoantibodies could be implicated in brain dysfunction without apparent pathological changes of autoimmune disease. D 2005 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Neural-immune interactions Keywords: Aggressive behavior; SLE; NZB mouse; IgM

Systemic lupus erythematosus (SLE), a chronic inflammatory disease showing lupus-like symptoms, often accompanies symptoms of neurological disorders [19]. Like human SLE, spontaneous SLE-prone MRL/lpr, BXSB, New Zealand Black (NZB) and NZB  New Zealand White (NZW) F1 (B/W F1) strains of mice show alterations in behavior such as increased anxiety, learning disturbances, impaired motor coordination, altered sensitivity to pain stimuli and reduced exploration [11,14 –16]. It is hypothesized that multiple neuroimmunological pathways are involved in the pathogenesis of the neural symptoms. Genetic factors can also contribute to the symptoms in autoimmune model mice [11]. There are two major path-

Abbreviations: AOB, accessory olfactory bulb; B/W F1, NZB/NZW F1; GRL, granule cell layer; HPA, hypothalamic – pituitary – adrenal; MTL, mitral/tufted cell layer; NZB, New Zealand Black; NZW, New Zealand White; SLE, systemic lupus erythematosus * Corresponding author. Fax: +81 3 3813 3164. E-mail address: [email protected] (K. Nakamura). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.07.008

ways by which autoimmune processes may affect behavior. First, autoimmunity can cause pathology in specific organs and/or contribute to abnormal function of the endocrine glands, which can affect brain functions. Second, several immune factors access the brain. Direct binding of autoantibodies to the brain cells [13] or indirect effect of immune factors such as cytokines, immune complexes and infiltration of lymphoid cells also potentially affect the behavior. NZB mice spontaneously develop autoimmune hemolytic anemia, in association with anti-erythrocyte autoantibodies [3]. Proliferation of B1 cells and IgM hypergammaglobulinemia including autoantibodies to thymocytes and nuclear components occurs early in life before the onset of autoimmune hemolytic anemia in NZB mice. However, IgG hypergammaglobulinemia is not seen in young NZB strain. By using young NZB mice, therefore, we can study the effect of IgM class autoantibodies on the behavioral changes without possible effects of autoimmune hemolytic anemia. In addition, we can exclude the contribution of IgG hypergammaglobulinemia.

146

K. Nakamura et al. / Developmental Brain Research 159 (2005) 145 – 148

The progress of autoimmune disease might affect aggression in animals because the latency of the animal to attack an intruder correlated significantly with the experimental autoimmune encephalomyelitis disease score [4]. In the present investigation, we found an enhanced aggression in NZB mice compared to non-autoimmune C57BL/6 and NZW mice, and the relation between performance of an aggressive behavior and serum IgM level was studied. Male C57BL/6, NZB, NZW and B/W F1 mice at 8 weeks old were used for the behavioral analysis. Mice obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan) were maintained in our laboratory according to guidelines of Juntendo University. To evaluate aggressive behavior, we carried out resident – intruder test. The animal housed individually for 1 month prior to the behavioral test was confronted with a group-housed C57BL/6 male mouse in its home cage, which induces strong territorial behavior in the residential animal [21]. Compared to C57BL/6 and NZW mice, higher proportion of NZB and B/W F1 resident mice bit the intruder within 5 min, whereas no NZW mice bit within 5 min (Fig. 1). Likewise, the latency to the first attack was shorter in the NZB and B/W F1 mice. The numbers of biting were also larger in NZB and B/W F1 mice. We then asked whether the behavioral change accompanies the functional alteration of the brain. Pheromones are involved in the regulation of various aspects of behavior including aggressive behavior in mammals [5,9]. The vomeronasal system is known to play an important role in pheromone recognition [5,9], consisting of the vomeronasal organ, the accessory olfactory bulb (AOB) and the higher vomeronasal centers receiving afferents from the AOB. It was reported that an increased protein product of the immediate – early gene fos in the mitral and granular layers of AOB after exposure to an intruder was observed in lactating mice displaying fierce aggression towards novel, male mice but not in virgin mice [1]. To evaluate neuronal activities in the AOB during the aggressive behavior, we

performed c-fos immunohistochemistry. Two hours after the aggressive behavior, the mice were deeply anesthetized by injection of pentobarbital Na. Then, they were perfused with 4% paraformaldehyde in 0.1 M PB, and the olfactory bulbs of the mice were dissected out from the skull and postfixed overnight at 4 -C in the same fixative solution. The olfactory bulbs were immersed in 0.1 M PB containing 30% sucrose until they sank. Serial sagittal sections of the olfactory bulb (50 Am in thickness) were cut on a freezing microtome. Eight sagittal sections in the middle region of the AOB were selected for the immunohistochemical analysis for c-fos expression. Immunohistochemistry and data analysis were done essentially as described [20]. Briefly, after elimination of endogenous peroxidase activity by incubation with 3% hydrogen peroxide in absolute methanol, the sections were rinsed in 0.3% Triton X-100 in 50 mM PBS (PBST, pH 7.4) for 45 min with three changes of buffer and then blocked with a solution of 1% bovine serum albumin (BSA) in PBST (BSA-PBST) for 1 h at room temperature. The sections were subsequently incubated with the c-fos antibody (Ab-5, Oncogene Science, Cambridge, MA; diluted to 1:40,000 with BSA-PBST) for approximately 60 h at 4 -C. They were incubated further with biotinylated anti-rabbit IgG (7.5 Ag/ml; Vector Labs, Burlingame, CA) in BSA-PBST for 1 h at room temperature, and then with avidin – biotin complex (1:100; Amersham, Buckingham, UK) in BSA-PBST for 1 h at room temperature. Each step was followed by four washes for 15 min each with PBST. After the last wash, the sections were incubated with the chromogen AEC (3-amino-9-ethylcarbazole) and 0.0025% hydrogen peroxide in the same buffer for 5 min. The reaction was stopped by transferring the sections into PBST. Then, the sections were mounted onto glass slides. Quantitative analysis of the number of c-fos-positive cells in the mitral/tufted cell layer (MTL) and the granule cell layer (GRL) of the AOB was done. Each layer was

Fig. 1. Resident – intruder test of C57BL/6 (n = 7), NZB (n = 12), NZW (n = 12) and B/W F1 (n = 8) mice. The male mouse was evaluated by the percentage of mouse showing biting (A), latency to the first attack (B) and number of biting (C) during 5 min. **P < 0.01.

K. Nakamura et al. / Developmental Brain Research 159 (2005) 145 – 148

divided into two parts (rostral and caudal areas) bisected by the anterior – posterior axis between the extreme anterior and extreme posterior points of the AOB. The numerical density of the Fos-positive cells in each area of each layer was calculated in individual sections. Fos-positive nuclei were observed in the glomerular layer, MTL and GRL in the male mice after the behavior. The densities of Fos-positive mitral/tufted and granule cells after aggressive behavior were larger in rostral regions in MTL and GRL of NZB than those of NZW and C57BL/6 mice (Fig. 2), indicating that neural activity in rostral AOB was enhanced in NZB mice. Apparent differences were not observed in caudal regions of AOB. In a previous study, exposure of ICR male mice to BALB/c males resulted in an increase in c-fos immunoreactivity in a cluster of cells located almost exclusively in the caudal AOB in ICR mice [7]. This caudal cluster of activated cells did not appear to require the overt display of aggressive behavior. The dominant activation in the caudal region was not observed in NZB mice. Spatially distinct activation pattern in AOB might depend on the degree of aggression. As a control, we evaluated the densities of Fos-positive cells using female intruder. The densities in MTL and GRL were larger in NZW mice than NZB mice [12]. Thus, the enhanced Fos expression in NZB mice seems to be specific for aggressive behavior. We then used backcrossed mice produced by crossing female B/W F1 mice with male NZW mice. The perform-

147

Fig. 3. Scatterplot of latency to the first attack during aggressive behavior on the x-axis versus serum total IgM level on the y-axis. Note the inverse correlation of the latency and serum total IgM level.

ances of the aggressive behavior in 61 B/W F1  NZW backcrossed progeny were assessed, and immunological indexes were also examined in these mice. The immunological indexes examined were B1 cell ratio, CD4/CD8 ratio and total IgM in peripheral blood. These were evaluated using flow cytometry and ELISA as described [2]. As shown in Fig. 3, total IgM was inversely correlated with the latency to the first attack (Pearson, r = 0.42; P = 0.001). This result implies that the animals having high IgM tend to attack the intruder. In the present study, we presented correlation between IgM hypergammaglobulinemia and aggression in young NZB mice. B/W F1 mice are generally considered to be the

Fig. 2. Neural activities in rostral and caudal regions of AOB during aggressive behavior of C57BL/6, NZB and NZW mice. Representative examples (upper panels) and summary data (lower panels) for the number of c-fos-positive cells in mitral/tufted and granule cell layers of AOB from C57BL/6 (n = 14, number of mouse), NZB (n = 13) or NZW (n = 13) mice. **P < 0.01. MTL, mitral/tufted cell layer; GRL, granule cell layer. Scale bar, 200 Am.

148

K. Nakamura et al. / Developmental Brain Research 159 (2005) 145 – 148

best model available for human SLE because of the high occurrence in females and the severe fatal lupus nephritis [17]. Although autoimmune disease of B/W F1 mice is assumed to be led by class switch of IgM to high affinity IgG class autoantibodies, IgM hypergammaglobulinemia is also observed in young B/W F1 mice. When we assessed aggressive behavior in B/W F1 male mice, their performances were similar to that of NZB mice. This observation supports the association between IgM and aggression. The number of sniffing was not different between resident NZB and NZW mice during aggressive behavior (data not shown), suggesting that the enhanced neural activity in AOB of NZB mice seemed not to simply reflect an increased exposure to pheromone of the intruder. Rather, activities in AOB could be modulated via efferent input from other brain regions. AOB receives efferent projections from amygdala, and neurons in hypothalamus project to amygdala [6,10]. Hypothalamus and amygdala have close relationship with aggression. Autoimmune-prone mice including NZB, NZW, B/W F1 and MRL/lpr show changes in the activity of hypothalamic –pituitary –adrenal (HPA) axis [8], and antigen-induced immune stimulation induces elevation of IgM class antibodies and changes in the HPA axis [18]. In the Nissl staining, we found no gross anatomical abnormalities either in the AOB, amygdala or hypothalamus in NZB mice (data not shown). Together, IgM class autoantibodies might trigger the functional changes in HPA axis in NZB mice, thereby modulating reactivity to the intruder. The heightened sensitivity to environmental variables has to be also considered. Further careful examinations, particularly neural activities in the amygdala and hypothalamus, have to be done to clarify the relationship among IgM, HPA axis and aggression.

Acknowledgments We thank Dr. Yokosuka (St. Marianna University) for technical advice. This work was supported in part by research grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan.

References [1] N.S. Hasen, S.C. Gammie, Differential fos activation in virgin and lactating mice in response to an intruder, Physiol. Behav. 84 (2005) 681 – 695. [2] S. Hirose, H. Tsurui, H. Nishimura, Y. Jiang, T. Shirai, Mapping of a gene for hypergammaglobulinemia to the distal region on chromosome 4 in NZB mice and its contribution to systemic lupus erythematosus in (NZB  NZW)F1 mice, Int. Immunol. 6 (1994) 1857 – 1864.

[3] S. Hirose, Y. Jiang, Y. Hamano, T. Shirai, Genetic aspects of inherent B-cell abnormalities associated with SLE and B-cell malignancy: lessons from New Zealand mouse models, Int. Rev. Immunol. 19 (2000) 389 – 421. [4] A. Kavelaars, C.J. Heijnen, R. Tennekes, J.E. Bruggink, J.M. Koolhaas, Individual behavioral characteristics of wild-type rats predict susceptibility to experimental autoimmune encephalomyelitis, Brain Behav. Immun. 13 (1999) 279 – 286. [5] E.B. Keverne, Mammalian pheromones: from genes to behaviour, Curr. Biol. 12 (2002) R807 – R809. [6] G.A. Kevetter, S.S. Winans, Connections of the corticomedial amygdala in the golden hamster: I. Efferents of the ‘‘vomeronasal amygdala,’’ J. Comp. Neurol. 197 (1981) 81 – 98. [7] A. Kumar, C.A. Dudley, R.L. Moss, Functional dichotomy within the vomeronasal system: distinct zones of neuronal activity in the accessory olfactory bulb correlate with sex-specific behaviors, J. Neurosci. 19 (1999) RC32. [8] O. Lechner, Y. Hu, M. Jafarian-Tehrani, H. Dietrich, S. Schwarz, M. Herold, F. Haour, G. Wick, Disturbed immunoendocrine communication via the hypothalamo – pituitary – adrenal axis in murine lupus, Brain Behav. Immun. 10 (1996) 337 – 350. [9] M. Luo M, L.C. Katz, Encoding pheromonal signals in the mammalian vomeronasal system, Curr. Opin. Neurobiol. 14 (2004) 428 – 434. [10] N. Moreno, A. Gonzalez, Hodological characterization of the medial amygdala in anuran amphibians, J. Comp. Neurol. 466 (2003) 389 – 408. [11] K. Nakamura, Y. Xiu, M. Ohtsuji, G. Sugita, M. Abe, N. Ohtsuji, Y. Hamano, Y. Jiang, N. Takahashi, T. Shirai, H. Nishimura, S. Hirose, Genetic dissection of anxiety in autoimmune disease, Hum. Mol. Genet. 12 (2003) 1079 – 1086. [12] K. Nakamura, H. Nishimura, S. Hirose, Unpublished Data. [13] M. Ohgaki, G. Ueda, J. Shiota, H. Nishimura, S. Hirose, H. Sato, T. Shirai, Two distinct monoclonal natural thymocytotoxic autoantibodies from New Zealand black mouse, Clin. Immunol. Immunopathol. 53 (1989) 475 – 487. [14] B. Sakic, H. Szechtman, J.A. Denburg, Neurobehavioral alterations in autoimmune mice, Neurosci. Biobehav. Rev. 21 (1997) 327 – 340. [15] L.M. Schrott, L.S. Crnic, Anxiety behavior, exploratory behavior, and activity in NZB  NZW F1 hybrid mice: role of genotype and autoimmune disease progression, Brain Behav. Immun. 10 (1996) 260 – 274. [16] L.M. Schrott, L.S. Crnic, Increased anxiety behaviors in autoimmune mice, Behav. Neurosci. 110 (1996) 492 – 502. [17] T. Shirai, S. Hirose, Preface and overview: genetics of SLE; a sine qua non for identification, Int. Rev. Immunol. 19 (2000) 289 – 295. [18] M. Stenzel-Poore, W.W. Vale, C. Rivier, Relationship between antigen-induced immune stimulation and activation of the hypothalamic – pituitary – adrenal axis in the rat, Endocrinology 132 (1993) 1313 – 1318. [19] J.J. Sweet, N.A. Doninger, P.C. Zee, L.I. Wagner, Factors influencing cognitive function, sleep, and quality of life in individuals with systemic lupus erythematosus: a review of the literature, Clin. Neuropsychol. 18 (2004) 132 – 147. [20] M. Yokosuka, M. Matsuoka, R. Ohtani-Kaneko, M. Iigo, M. Hara, K. Hirata, M. Ichikawa, Female-soiled bedding induced fos immunoreactivity in the ventral part of the premammillary nucleus (PMv) of the male mouse, Physiol. Behav. 68 (1999) 257 – 261. [21] H. Yoshimura, K. Watanabe, N. Ogawa, Psychotropic effects of ginseng saponins on agonistic behavior between resident and intruder mice, Eur. J. Pharmacol. 146 (1988) 291 – 297.