Noxious stimulation enhances release of cytokine-induced neutrophil chemoattractant from hypothalamic neurosecretory cells.

Neuroscience Research 27 (1997) 181 – 184

Rapid communication

Noxious stimulation enhances release of cytokine-induced neutrophil chemoattractant from hypothalamic neurosecretory cells. Keiko Matsumotoa,b, Koji Koikeb, Akira Miyakeb, Kazuyoshi Watanabed, Kiyoshi Konishic, Hiroshi Kiyamaa,e,* a

Department of Neuroanatomy, Biomedical Research Center, Osaka Uni6ersity Medical School, 2 -2 Yamadaoka, Suita, Osaka 565, Japan b Department of Obstetrics and Gynecology, Osaka Uni6ersity Medical School, 2 -2 Yamadaoka, Suita, Osaka 565, Japan c Department of Biochemistry, Toyama Medical and Pharmaceutical Uni6ersity Faculty of Medicine, Sugitani, Toyama 930 -01, Japan d Institute of Cytosignal Research, Hiromachi, Shinagawa-ku, Tokyo 140, Japan e Department of Anatomy, Asahikawa Medical College, 4 -5 -3 -11 Nishikagura, Asahikawa 078, Japan Received 26 August 1996; accepted 27 November 1996

Abstract Cytokine-induced neutrophil chemoattractant (CINC), a member of IL-8 family in rat and a counter part of human growth-related oncogene product (GRO), was demonstrated to be synthesized in hypothalamic neurons and released in peripheral blood in response to noxious stimulation. CINC immunoreactive neurons were found mainly in supraoptic nucleus, while positive fibers were found in the median eminence and the posterior lobe of pituitary gland. Colchicine injection into the third ventricle demonstrated additional CINC positive neurons in both parvo- and magno-cellular parts of paraventricular nucleus, and an increase of positive fibers in the external layer of the median eminence. After formalin injection into the foot pad, CINC immunoreactivity substantially increased in the posterior lobe of the pituitary gland and in the external layers of the median eminence. In addition, a parallel increase of CINC level was noticed in the peripheral blood. It is suggested from the present results that, in response to a noxious stimulation, CINC is released from hypothalamic neurosecretory cells via both the anterior and posterior pituitary system. This cytokine mediated noxious response from CNS to peripheral tissue, would be a novel type of cross-talk between nervous and immune system. © 1997 Elsevier Science Ireland Ltd. Keywords: Cytokine-induced neutrophil chemoattractant; Growth-related oncogene product; Noxious stimulation; Pain; IL-8

Cytokine-induced neutrophil chemoattractant (CINC) is a member of the IL-8 family in rat and a counterpart of human growth-related oncogene product (GRO) (Watanabe et al., 1989a,b). Human IL-8 is a chemoattractant for neutrophils and T-lymphocytes, while GRO has a chemotactic activity for neutrophils comparable to that of IL-8 (Watanabe et al., 1991). On the other hand, CINC is a chemotactic cytokine for rat neutrophils which is induced in response to IL-1, tumor necrosis factor (TNF), and lipopolysaccharide (LPS) * Corresponding author: Fax: +81 166 66 1172.

(Iida et al., 1992). CINC induces transient neutrophil accumulation without lymphocyte and monocyte migration and is considered as a neutrophil-specific chemoattractant. Many studies have been done on CINC in relation to the local inflammation. Recently, a few CINC-like immunoreactive cells were observed in the rat anterior pituitary gland (1–3% of all cells in the anterior pituitary) (Koike et al., 1994). It was also shown that CINC stimulated the secretion of prolactin (PRL), growth hormone (GH) and adrenocorticotropin (ACTH), but suppressed the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH)

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from rat anterior pituitary cells (Sawada et al., 1994). These findings suggest that CINC may play a role as a modulator of anterior pituitary function. On the other hand, in our previous study, we found that CINC-like immunoreactivity existed in the supraoptic nucleus (SON), the median eminence (ME) and the posterior lobe of pituitary gland, and that CINC synthesis was activated in the paraventricular hypothalamic nucleus and released into peripheral blood in response to immobilization stress (Sakamoto et al., 1996). Thus, CINC is also considered to play an important role in hypothalamic psychological stress response. To reveal another range of CINC reaction, here we examined if noxious stimulation could also elicit a similar CINC response. All experiments were performed on adult male Sprague-Dawly (S-D) rats, specific pathogen free and weighing 150 g. They were divided into three groups. In the first experimental group (n =4), colchicine (0.05 mg in 0.01 ml saline) was injected into the third ventricle, and animals were allowed to survive for 48 h. Animals were, then, deeply anesthetized with excessive dose of sodium pentobarbital and perfused through the aorta with 80 ml of saline followed by 400 ml of Zamboni’s fixative. The brain was removed and postfixed in the same fixative, and immersed in 0.1 M phosphate buffer containing 30% sucrose for 1 day. Then, frozen sections of 20 mm of thickness were cut on a cryostat. In the second experimental group (n =20), animals were exposed to noxious chemical stress. These rats were bilaterally subcutaneously injected with 0.1 ml of 5% formalin into each hind foot pad. This noxious stimulation has been used as the model of the painful stress (Dubuisson and Dennis, 1977; Bullit, 1990; Senba et al., 1993). At 0, 2,4, and 8 h after the injection of formalin, five animals at each time were anesthetized with sodium pentobarbital, and blood samples were drawn intracardially before they were perfused through the aorta in the same way as the first experimental group. The third group (n=2) were just perfused with the same fixative without any treatment for the control study. In addition to the hypothalamus, the pituitary gland were removed, post fixed in the same fixative and immersed in 0.1 M phosphate buffer containing 30% sucrose for 1 day. Then, frozen sections of 10 mm of thickness were cut on a cryostat. The sections were incubated with antibody against CINC at the concentration of 2 mg/ml diluted in 0.1 M phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 1% bovine serum albumin (BSA) for 1 day at 4°C. The subsequent procedure for the immunohistochemistry was followed by Vector’s protocol (Vectastain ABC kit, Vector). The optical density of CINC-like immunoreactivity was measured using a microcomputer imaging device (Imaging Research Inc.). Twenty pituitary sections were used at each time. The optical density of CINC-like immunoreactivity in the anterior pituitary was used as control at

each section. For the CINC assay in the peripheral blood, samples were drawn before the rats were perfused, and separated into sera and clots. The sera were assayed for CINC by the enzyme-linked immunosorbent assay (ELISA) as reported by DeForge and Remick (DeForge and Remick, 1991). The rabbit polyclonal antibody against CINC was raised as described previously (Koike et al., 1994), and IgG was isolated from the antisera using a protein A-agarose column (MASS 1 mg Protein A, Dainihon, Osaka). All other chemicals were commercial materials of the highest purity available and were used without further purification. Strong CINC immunoreactive fibers were observed in the external layer and the external part of internal

Fig. 1. Photomicrographs of immunohistochemical staining for CINC-like immunoreactivity after the colchicine treatment in the ME (A), the SON (B) and the PVN (C). Bar = 0.1 mm. Abbreviations 3V: 3rd ventricle, ox: optic chiasma.

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Fig. 2. Photomicrographs of immunohistochemical staining for CINC-like immunoreactivity in the posterior pituitary at 0 h (A), 2 h (B), 4 h(C) and 8 h (D) after the injections of formalin as pain stimulation. Bar=0.03 mm.

layer of the median eminence (ME) (Fig. 1A) and the posterior lobe of pituitary gland (Fig. 2A). In addition, CINC immuno-stained large neurons were also found in the supraoptic nucleus (SON) (Fig. 1B). The treatment by axonal transport blocker colchicine markedly enhanced CINC-like immunoreactivity in ME and SON. Furthermore, this treatment also revealed CINC immunoreactive neurons mainly in the parvocellular and partly in magnocellular part of paraventricular hypothalamic nucleus (PVN) (Fig. 1C) where its immunoreactivity was almost negative without the colchicine treatment. Two, four and eight hours after formalin injection, CINC immunoreactivity in the posterior lobe of the pituitary gland was markedly increased in density (Fig. 2). The optical density unit of CINC immunoreactivity

increased about 3-fold at 2 and 4 h and thereafter, gradually decreased to basal level (Fig. 3). In addition, we measured the change of serum CINC level induced by noxious stimulation (Fig. 4) and found a significant increase of more than 3-fold of the CINC level in the peripheral blood at 2 and 4 h. Subsequently, CINC gradually decreased to basal level. This alteration profile of serum CINC level was consistent with that of its optical density imunoreactivity observed in the posterior pituitary. The present study firstly demonstrated that CINC was enhanced in its expression and released from the hypothalamic neurons in response to peripheral noxious stimulation. The increase of CINC immunoreactivity in the posterior lobe of pituitary gland was presumably due to the enhanced expression of CINC

Fig. 3. The change of optical density unit of CINC-like immunoreactivity after formalin stimulation. *PB 0.001 vs. at 0 h, **PB0.01 vs. at 0 h by unpaired Student’s t-test.

Fig. 4. The change of the concentration of serum CINC after formalin stimulation. The unit of the concentration (y axis) is ng/ml. *PB 0.005 vs. at 0 h, **PB0.01 vs. at 0 h by unpaired Student’s t-test.

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mainly in the SON and partly in the magnocellular sub-nucleus of PVN. In addition, it could be concluded from the dramatic increase of CINC in the external layer of the median eminence that its induction originated in the parvocellular part of PVN. Colchicine treatment is known to cause damage or stress to neurons, leading to various gene expressions. Thus, this treatment does not exactly demonstrate CINC synthesizing cells under normal condition, but shows, at least, those which are able to produce CINC. In a previous work (Sakamoto et al., 1996), we have already demonstrated that CINC is released from pituitary gland in response to immobilized stress. Accordingly, in response to noxious stimulation, CINC appears to be released in the peripheral circulation via two distinct pathways. These include a release from magnocellular neurohypophyseal neurons in PVN and the SON via the posterior lobe. As the increase of CINC immunoreactivity observed in the posterior lobe did parallel the increase of CINC level in the pheripheral blood, this coincidence would suggest that CINC is released from the posterior lobe. Another CINC release would be originated from the parvocellular neurons in the PVN, the axons of which were found to terminate in the external layer of ME. The CINC released from the ME neurons might circulate via portal vein, although there remains some possibilities that CINC could affect some population of the anterior pituitary cells. In fact, CINC has been found to stimulate the secretion of PRH, GH and ACTH in vitro (Sawada et al., 1994). In particular, ACTH secretion may be significant as a noxious stress response. In any way, the noxious stimulation seems to affect the two distinct hypothalamic CINC releasing pathways, namely the anterior and posterior pituitary system. Although the exact functional significance of CINC response to noxious stimulation is still obscure, the acute increase of CINC in peripheral blood which is regulated by the CNS may be the trigger to the initial activation and the migration of neutrophils in peripheral blood to painfull or inflammatory regions. The present finding is the first demonstration of the release of a cytokine member to peripheral targets in

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response to noxious stimulation, and this cytokine mediated signal transduction from neurons to immune cells could be identified as a novel type of cross-talk between the neural and the immune systems.

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