Chapter 11 Neurohypophyseal hormones in the integration of physiological responses to immune challenges

D. Poulain, S. Oliet and D. Theodosis (Eds.) Progressin Brain Research,Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 11

Neurohypophyseal hormones in the integration of physiological responses to immune challenges K r i s z t i n a J. K o v 4 c s * Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Budapest, Hungary

Keywords: Paraventricular nucleus; Vasopressin; Corticotropin-releasing hormone; Cytokine; Allergy; Anaphylaxis; Brattleboro rat

The neuro-immuno-endocrine web There is a multidirectional communication between the immune, neuroendocrine and central nervous systems. Mediators released from activated immune cells may reach directly or indirectly the central nervous system and activate cell groups that are involved in autonomic, neuroendocrine and behavioral regulation. Lymphoid organs are densely innervated and immune competent cells are equipped with neurotransmitter and hormone receptors. Some hormones and neuropeptides, secreted upon immune activation, display cytokinelike effects, while cytokines may directly stimulate hormone release. Corticosterone, the end-product of the hypothalamo-pituitary-adrenocortical regulatory axis, possesses negative feedback influence not only on the central nervous system targets, but also has profound immunosuppressive effects. Dysfunction of the neuroendocrine-immune communication has been implicated as an etiologically important factor in certain forms of autoimmunity, immunosuppression and hypersensitivity. * Correspondence to: K.J. Kov4cs, Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Szigony u. 43. H-1083. Budapest, Hungary. Tel.: +36-1-210-9952; Fax: -t-36-1-210-9429; E-mail: kovacs @koki.hu

The paraventricular nucleus of the hypothalamus as the key integrator of physiological responses to immune stimuli The hypothalamus contains neurosecretory cells that make up the final pathway by which the brain governs several autonomic and endocrine functions. These neurons serve as cellular transducers to integrate stimulatory and inhibitory drives, which may be neural or humoral (hormonal) in nature, and provide relevant output to modulate hypophyseal hormone secretion. Much is known on the location, neurochemical phenotype and secretory capabilities of neurosecretory effector neurons, and of the organization and chemical coding of their major afterents. However, how these come to be linked to form operational circuits is still not well characterized.

Functional architecture of the PVN The hypothalamic paraventricular nucleus contains three functionally distinct neuron populations that are clustered in distinct subdivisions of the nucleus (Swanson and Sawchenko, 1980). Magnocellular neurosecretory neurons release vasopressin (AVP) and oxytocin (OXY) at the posterior pituitary to serve homeostatic functions of water balance and reproduction. Parvocellular corticotropin-releasing hormone (CRH) containing neurons project to hy-

128 pophyseal portal vasculature at the external zone of the median eminence to provide the central control of the hypothalamo-pituitary-adrenocortical (HPA) axis and initiate the neuroendocrine stress cascade (Antoni et al., 1983; Swanson et al., 1983). Cells in the dorsal, ventral and lateral parvocellular subnuclei give rise long descending projections that target the periaqueductal gray, parabrachial nucleus, nucleus of the solitary tract, rostral ventrolateral medulla, as well as sympathetic and parasympathetic preganglionic cells in the medulla and spinal cord (Swanson and Sawchenko, 1980; Sawchenko and Swanson, 1982a; Saper, 1995). These neurons are involved in the coordination of autonomic functions to endocrine and behavioral responses. The neuropeptide phenotype of hypothalamic visceromotor neurons is complicated by several factors and depends on the sensitivity of the detection. An updated list of co-expressing peptides that are contained in parvo- and magnocellular neurosecretory neurons is found in a recent review of Burbach et al., 2001. In contrast to the neurosecretory neurons, autonomic projection neurons do not contain a universal identifying neuropeptide marker, subsets of these cells express OXY, AVP or CRH (Sawchenko and Swanson, 1982b). The phenotypic variation of neuropeptide gene expression has functional significance for instance co-expression, co-packing and co-secretion of synergistically acting neuropeptides under high-demand conditions. In one well studied model, withdrawal of the negative feedback signal from the HPA axis by adrenalectomy, results in upregulation of AVP expression in the parvocellular neurosecretory system (Kiss et al., 1984; Sawchenko et al., 1984; Sawchenko, 1987) where its transcription is restrained under basal conditions (Kovacs et al., 2000). Acute and chronic exposure to stressors also result in vasopressinergic phenotype in CRH-secreting parvocellular neurons (Bartanusz et al., 1993, 1994; Makino et al., 1995; Kovacs and Sawchenko, 1996a,b; Ma et al., 1997a,b). Oxytocinergic neurons became CRH immunoreactive in response to chronic salt loading (Young, 1986; Dohanics et al., 1990; Watts, 1992, 2001; Kovacs and Sawchenko, 1993), while lesion of the paraventricular nucleus provoke CRH mRNA expression in the oxytocin-containing neurons of the supraoptic nuclei (Palkovits et al., 1997). The expression of AVP and

OXY in the magnocellular neurosecretory cells is also not mutually exclusive, i.e. during lactation, coexistence of AVP and oxytocin was revealed in the SON (Mezey and Kiss, 1991; Glasgow et al., 1999). Afferent control of the hypothalamic neurosecretory neurons Activity of the hypothalamic paraventricular neurons is governed by neural and/or hormonal influences. Generally, magnocellular neurons receive neural inputs from restricted sources, while the afferent control of parvocellular neurons is much more diverse and includes direct and indirect pathways that convey relevant information from all major sensory modalities of the brain. In addition to sensory inputs, cognitive and emotionally related influences also have a great impact on the stress-related parvocellular neurons, although the anatomical pathways that may mediate these effects are not well characterized (Sawchenko et al., 1996). CRH-secreting parvocellular neurons receive significant monoaminergic innervation: adrenergic axons arise from the ventrolateral medulla (C1) and nucleus of the solitary tract (C2); noradrenergic inputs originate from A2 and A6 regions and from local hypothalamic sources (A12AI4) (Sawchenko and Swanson, 1982b; Liposits et al., 1986a,b; Liposits, 1990). 5-HT-positive fibers that make synaptic contacts with CRH neurons in the parvocellular subdivision (Liposits et al., 1987) appear to arise from three distinct serotoninergic cell groups (B7, B8 and B9) in the midbrain (Sawchenko et al., 1983). Parvocellular neurosecretory neurons receive GABAergic inputs from local interneurons (Roland and Sawchenko, 1993). GABA-containing profiles make symmetric, inhibitory synaptic contacts with CRH neurons and it is estimated that one-third of all synapses that innervate these cells is GABAergic (Mikl6s and Kovfics, submitted). Magnocellular neurons are themselves sensitive to changes of osmotic concentration; however, both AVP and OXY neurons receive inputs from osmosensitive structures of the lamina terminalis that lies outside of the blood-brain barrier and may also confer hormonal and immune signals (Miselis, 1981, 1982; Leng et al., 1982). Descending pathways from the subfornical organ (SFO) and the vascular organ of the lamina terminalis (OVLT) provide glu-

129 tamatergic stimulatory and GABAergic inhibitory projections to the hypothalamic magnocellular neurons. Some of the inputs that originate in lamina terminalis relayed in the median preoptic nucleus, which has strongly been implicated in the inhibitory control of hypothalamic neurosecretory cells (Saper and Levisohn, 1983). It has been recently shown (Leng et al., 2001) that osmotic stimuli coactivate excitatory and inhibitory inputs to the magnocellular neurons and results in increased neuronal firing linear to the osmotic challenge. These findings are in good agreement with those of Theodosis (Theodosis and Poulain, 1993) revealing ultrastructural signs of increased GABAergic inputs under high demand physiological conditions such as lactation. The osmosensitive pathways highlighted above have also been implicated in coordination of CRH expression in parvo- and magnocellular neurons in response to chronic salt loading (Kovacs and Sawchenko, 1993). A drop in blood pressure produces a reflex release of AVP from the posterior pituitary, and it is thought that the pressor effect requires doses of vasopressin considerably higher than those needed for maximal antidiuresis (Koob et al., 1985). Baroreceptors, under basal conditions, inhibit vasopressinergic magnocellular neurons indirectly, via the A6 catecholaminergic cell group. Stimulatory influences are mediated through the nucleus of the solitary tract (NTS) and relayed in the A1 noradrenergic cell group of the caudal ventrolateral medulla that preferentially target AVP neurons (Sawchenko and Swanson, 1982b; Day and Sibbald, 1988, 1990; Day et al., 1992). NTS provides direct innervation to oxytocinergic magnocellular neurons that includes ascending peptidergic pathways and noradrenergic input from the A2 cell group (Sawchenko and Swanson, 1982a,b; Cunningham and Sawchenko, 1988; Plotsky et al., 1989). Neurosecretory cells in the PVN and SON receive histaminergic inputs from discrete cell clusters in the posterior hypothalamus/tuberomamillary region (Panula et al., 1984, 1990). Histamine has been shown to be involved in physiological functions relevant in immune-neuroendocrine crosstalk, including food-intake, thermoregulation, water balance and regulation of the HPA axis (Ookuma et al., 1993; Bealer and Abell, 1995; Kang et al., 1995; Bugajski et al., 1996; Hatton and Yang, 1996; Sakata et al., 1997).

Afferent inputs to the hypothalamic motoneurons together with their potential to express an array of functionally distinct effector molecules and supply them to autonomic, endocrine and behavioral centers constitute the hardware of the regulatory circuit. The aim of the present chapter is to reveal the organization of those operational circuits that are recruited in a situation dependent manner to mediate immune signals to the brain and integrate physiological responses to immune challenges.

Immune regulation in brief (from a neuroendocrine perspective) Immune responses are regulated by antigen presenting cells and by Th (helper) lymphocyte subclasses Thl and Th2, which secrete different sets of cytokines and promote cellular and humoral immunity, respectively (Fearon and Locksley, 1996). Any immune challenge that threatens the homeostasis should be regarded as stressor. Indeed, activation of either form of immunity, activate the HPA axis and results in a complex series of centrally mediated autonomic reaction that serve to cope with the challenge. Thl and Th2 responses are mutually inhibitory, and hormones play a definitive role in the regulation of Thl/Th2 balance (Elenkov and Chrousos, 1999; Elenkov et al., 2000). For instance, stress has been regarded as immunosuppressive (Besedovsky et al., 1986). Indeed, corticosterone inhibits proinflammatory cytokines and suppress cellular immunity, but activation of the HPA axis boosts humoral immunity (Elenkov and Chrousos, 1999). Recent evidence indicates a CRHmast cell-histamine regulatory loop (Theoharides, 1996; Theoharides et al., 1998), and catecholamines released during sympathoadrenal activation also selectively inhibit cellular immunity and drive towards a Th2-mediated humoral immunity (Elenkov et al., 2OO0).

Immune challenges activate parvoceilular neurosecretory neurons Increased production of pro-inflammatory cytokines released by activated macrophages or lymphocytes, that are effector elements of the cellular immunity, represent an essential feature of the early events

130 of immune activation called acute phase response. Interleukin-l{3 (IL-I~3), interleukin-6 (IL-6), tumor necrosis factor (TNFc~) play a pivotal role in mediating central and peripheral host responses in that most are mimicked by exogenous administration of these cytokines or by bacterial endotoxin lipopolysaccharide (LPS) that triggers their sequential release (Chensue et al., 1991). Immune challenges commonly activate the HPA axis. Although there are reports in the literature on the direct action of cytokines and LPS on the adrenal gland and pituitary hormone secretion, since the inseminating reports from 1987 (Berkenbosch et al., 1987; Sapolsky et al., 1987) the consensus has been shifted towards acknowledgement the paraventricular nucleus as the major hypothalamic target to govern HPA axis activity in response to immune challenges. In addition to LPS, IL-1, IL-6 and TNF stimulate ACTH secretion and induce immediate-early gene markers of neural activity in the PVN (Elenkov et al., 1992; Grinevich et al., 2001). Lines of evidence supporting the CRH as the major target of IL-1 are the following: first, immunoneutralization with CRH antibody completely prevent ACTH release provoked by acute local inflammation following turpentine injections (Turnbull et al., 1998), and by IL-1 (Sapolsky et al., 1987) or IL-6 (Kageyama et al., 1995); second, IL-1 selectively depletes CRH containing terminals (Berkenbosch et al., 1987), and third, IL-1 injections induce CRH transcription in the parvocellular neurons (Suda et al., 1990; Harbuz et al., 1992; Kakucska et al., 1993; Ericsson et al., 1994; Rivest and Rivier, 1994). It is noteworthy that a single administration of IL-6 does not induce c-fos and CRF expression in the PVN (Vallieres et al., 1997), although lesioning the PVN abolishes the ACTH response to systemic IL-6 (Kovacs and Elenkov, 1995), suggesting involvement of other ACTH releasing moieties of paraventricular origin such as AVP. In line with this hypothesis, IL-6-induced ACTH response was significantly suppressed by central injection of AVP antibody (Kageyama et al., 1995). It is also possible that single exposure to IL-6 is insufficient in activating hypothalamic neurons. Recently, it has been shown, that pretreatment with LPS upregulates IL-6 receptors and amplifies the effect of IL-6 on the CRH expression in the PVN (Vallieres and Rivest, 1999).

Circulating TNFc~ also triggers ACTH (Kovacs and Elenkov, 1995) and corticosterone secretion (van der Meer et al., 1996), induces c-fos mRNA in the stress-related circuitry including the paraventricular nucleus, increases CRH transcription in the parvocellular neurons (Tolchard et al., 1996; Nadeau and Rivest, 1999) and stimulates CRH release in vitro (Spinedi et al., 1992). Inducible expression of vasopressin gene in the parvocellular neurosecretory neurons of the paraventricular nucleus has a pivotal regulatory role in activation of HPA axis responses to different stressors, including immune challenges (De Goeij et al., 1992a,b; Kovacs and Sawchenko, 1996a,b; Ma et al., 1997a,b; Aubry et al., 1999). Moreover, evidence has recently been accumulated supporting the view that AVP is the primary regulated variable governing HPA function during stress, which maintains the axis activity, particularly under conditions of prolonged or repeated stimulation (De Goeij et al., 1992a,b; Chowdrey et al., 1995; Makino et al., 1995; Ma et al., 1997a,b; Aubry et al., 1999; Aguilera and Rabadan-Diehl, 2000). Vasopressin, released to the hypophyseal portal circulation acts on the VPI~ receptors on the pituitary corticotropes (Antoni et al., 1984) and potentiates the effect of CRH and other secretagogues on ACTH secretion (Lutz-Bucher et al., 1980; Giguere and Labrie, 1982; Gillies et al., 1982; Vale et al., 1983). Acute systemic administration of interleukin1 or LPS has been shown to deplete both AVPcontaining and AVP-deficient subtypes of CRH neurosecretory axons (Whitnall et al., 1992; Whitnall, 1993). This effect seems to be different from other systemic stressors such as immobilization, insulininduced hypoglycemia or colchicine treatment that specifically target the VP-containing CRH terminals (Whitnall, 1989). Moreover, a single cytokine injection may result in delayed and long-lasting increase of AVP stores in the external zone of the median eminence (Schmidt et al., 1995, 1996, 2001). Although IL-1 induced ACTH responses have been thought to primarily be driven by CRH, single IL-1 challenge results not only a phenotypic shift to AVP co-storing terminals, but also result in a 'functional shift' of the response: rats that have been pre-exposed to IL- 1 show marked IL-1-induced depletion of AVP stores upon repeated exposure. These changes might be

131 due to hyperreactivity of noradrenergic nerve terminals in the PVN seen in response to IL-1 injection (Schmidt et al., 1995, 1996, 2001).

Activation of magnocellular neurosecretory system by immune stimuli In addition to the parvocellular neurons, immune challenges also affect neurohypophyseal hormones secreted by the magnocellular neurons of the PVN and in the supraoptic nucleus. The situation is complicated by the fact that axons from the magnocellular neurosecretory cells 'in passage' through the median eminence are able to release AVP or OXY into the hypophyseal portal blood and contribute to the regulation of hormone secretion by the anterior lobe of pituitary (Holmes et al., 1986; Antoni et al., 1988). The stimulating effect of IL-1 on plasma AVP and OXY concentration seems to depend on glucocorticoid hormones. Intact animals do not display increases in circulating neurohypophyseal hormones, however in adrenalectomized rats a substantial increase was detected following IL-1 injection (Chover-Gonzalez et al., 1994; (Harbuz et al., 1996). Systemic application of IL-1 or LPS result in expression of activation markers c-fos and NGFI-B in the magnocellular neurons. Co-localization studies revealed that immunological insults seem to target oxytocinergic profiles rather than vasopressinergic neurons in the PVN and SON (Ericsson et al., 1994; Rivest and Laflamme, 1995). However, OXY and AVP heteronuclear RNA (hnRNA) levels were unchanged in the magnocellular neurons of the hypothalamus of LPS treated rats (Rivest and Laflamme, 1995). Recently, type I interleukin-1 receptor (IL-1R1) protein and IL-1 receptor antagonist were detected in the magnocellular compartment of the PVN and cells in the SON as well as in accessory magnocellular neurons. IL-1R1 was localized in vasopressin-immunoreactive neurons that provide a means by which IL-1 directly affect VP neurosecretion (Diana et al., 1999). In humans, IL-6 administration activates magnocellular AVP secreting neurons and results in a significant elevation of plasma AVP levels (Mastorakos et al., 1994). In line with these observations, highly elevated plasma AVP (and corticosterone) lev-

els were found in response to restraint stress in transgenic mice that constitutively express IL-6 under the control of GFAP (glial fibrillary acidic protein) promoter targeted to CNS astrocytes (Raber et al., 1997). Elevated plasma AVP levels in these mice are paralelled by hyperplasia of the adrenal cortex and medulla, while their ACTH response is blunted. Magnocellular neurons may also be activated secondarily by viscerosensory stimuli posed by cardiovascular responses seen in endotoxemia or after cytokine treatment (Weinberg et al., 1988; Rogausch et al., 2000; Xia and Krukoff, 2001). A unique feature of vasopressin is that this neuropeptide has a significant modulatory effect on the immune system, therefore it represent an additional means by which neuroendocrine and immune systems communicate. AVP modulates cellular immunity via its enhancement of the autologous mixed lymphocyte response (Bell et al., 1992). The response may be mediated through V1 vasopressin receptors (Bell et al., 1993). AVP and OXY are capable of replacing interleukin 2 (IL-2) requirement for T cell mitogen induction of y-interferon in mouse spleen cultures (Johnson et al., 1982; Johnson and Torres, 1985); vasopressin potentiates primary antibody responses (Croiset et al., 1990). Increased AVP secretion may participate in susceptibility to autoimmune and inflammatory diseases. Lewis rats display blunted HPA responses to stress and immune challenges, but have augmented systemic AVP secretion that collectively potentiate Thl-mediated cellular immunity and renders this strain to be sensitive to inflammatory (adjuvant-induced arthritis, AA) and autoimmune (experimental allergic encephalomyelitis, EAE) diseases (Patchev et al., 1992; Chowdrey et al., 1995; Harbuz et al., 1997; Chikanza et al., 2000; Huitinga et al., 2000; Sternberg, 2001). A proinflammatory role of AVP in Lewis rats is supported by the fact that immunoneutralization with AVP antibodies attenuates the inflammatory responses (Patchev et al., 1993). AVP have also been implicated in human pathophysiologies, elevated circulating vasopressin levels were measured in patients with rheumatoid arthritis (Chikanza et al., 2000). Manipulation of the neuroendocrine-immune communication at this level would offer strategies to treat chronic inflammatory and autoimmune diseases.

132 Activation of autonomic-related projection neurons in the PVN in response to immune stimuli The third major visceromotor cell cluster in the paraventricular nucleus, the one that give rise to long descending projections to brainstem and spinal cord plays a crucial role in governing autonomic responses to immune challenges. Infectious and inflammatory diseases are often accompanied with changes in thermoregulation (fever), sleepiness, loss of appetite, hypotension etc. Some components of these responses may be mediated by CRH, AVP and OXY-containing neurons in the dorsal, ventral and lateral parvocellular subdivision of the PVN. Rapid, but persisting activation of these neurons was revealed by using c-los and/or NGFI-B immediateearly gene markers following LPS (Elmquist et al., 1993, 1996; Rivest and Laflamme, 1995), IL-1 (Ericsson et al., 1994; Rivest and Rivier, 1994) and TNF-c~ (Nadeau and Rivest, 1999). Increased peripheral sympathetic activity that has been reported following IL-1 or LPS injections may directly be related to the activation of the this parvocellular cell group (Berkenbosch et al., 1989). Combination of retrograde tracing from different levels of the intermediolateral cell column of the spinal cord and LPS-induced c-Fos immunocytochemistry revealed the dorsal parvocellular subdivision of the PVN as the hypothalamic center mediating fever-inducing effects of endotoxin (Zhang et al., 2000). It is striking that a relatively small number of PVN neurons (100/side) may orchestrate pattern of sympathetic responses to endotoxemia such as stimulating brown adipose tissue, heart, the adrenal medulla and vascular tone in the tail artery. These responses then collectively serve to elevate body temperature, i.e. induce fever. The neurochemical phenotype of PVN neurons that mediate these effects has not been characterized. However, the distribution of PVNderived, oxytocin-containing terminals in the sympathetic preganglionic cell column of the spinal cord shows overlapping pattern with those that are activated by LPS and are involved in fever generation (Swanson and Sawchenko, 1980).

Afferent pathways that mediate immune signals to hypothalamic effector neurons Much of our recent knowledge on the activated cells and extended pathways that are responsive to a given stimulus came from application of immediate-early gene induction based functional anatomical mapping (Sagar et al., 1988; Morgan and Curran, 1989, 1991; Kovacs, 1998). Identification of these activation markers, in combination with anatomical tracing methods and in situ techniques that reveal the phenotype and transcriptional capacity of the activated profiles is a powerful tool to identify functional units in the rodent brain (Ceccatelli et al., 1989; Chan et al., 1993; Zhang et al., 2000). Interleukin-1

Because of the existence of the blood-brain barrier to circulating macromolecules such as IL-1, the manner in which blood-borne cytokines signal to the hypothalamic effector neurons remained problematic. Transduction through the circumventricular organs that lack blood-brain barrier, active transport through the barrier and local signaling from the brain vasculature have all been proposed as possible mechanism for immune-to-brain signaling (Katsuura et al., 1990; Elmquist et al., 1997). To identify central targets of circulating IL-1, distribution of IL-1 receptors (IL-1 R1) has been compared to those cell groups that display c-fos induction in response to IL-1 injections. Expression of the IL-1R1 in rats is restricted to the major barrier structures including meninges, choroid plexus, ependymal lining of the ventricles and endothelial cells of the brain capillaries (Ericsson et al., 1995). However, IL-1 responsive neurons are found in the paraventricular nucleus and in cell groups recognized as engaged with interoceprive information processing. These structures include the NTS, ventrolateral medulla, lateral parabrachial nucleus, central nucleus of amygdala and bed nucleus of stria terminalis (BNST) (Ericsson et al., 1994; Rivest and Rivier, 1994). Lesioning experiments clearly suggested that activation of CRHsecreting neurons in the PVN is dependent on the ascending catecholaminergic pathways (Ericsson et al., 1994). These findings together with localization of IL-1 receptors in the medulla are compatible with

133 the hypothesis that paracrine effects of prostaglandin PGE2, released from the perivascular cells in response to IL-1 challenge, acting through prostanoid receptor-expressing local catecholaminergic neurons that project to the PVN, contribute to the HPA axis activation by IL-1 (Ericsson et al., 1997). Involvement of the abdominal vagus nerve in mediation of cytokine signals to the central stressrelated circuitry was also proposed (Gaykema et al., 1995). Indeed, immune-related cells, that express ILl in response to LPS challenge were found in the close proximity to the abdominal vagus (Goehler et al., 1999), and transection of the abdominal vagus has been shown to be capable of eliminating LPS-induced neural activation in the brainstem and hypothalamus (Wan et al., 1993), but exclusively after intraperitoneal, but not after intravenous administration of the endotoxin. Signaling through the circumventricular organs is supported by several reports (Katsuura et al., 1990; Xia and Krukoff, 2001). However, the concentration of IL-1 required to induce activation markers in the vascular organ of the lamina terminalis is about a magnitude higher than those for c-fos induction in the medullary catecholaminergic neurons. In line with these findings, transection of descending inputs from the circumventricular organs to the hypothalamic stress-related neurons did not prevent transcriptional activation of CRH-secreting neurons by systemic IL-1 (Ericsson et al., 1994). Tumor necrosis factor There are two cell surface TNF receptors in the rat brain. P55-TNF receptor mRNA is constitutively expressed in the circumventricular organs, choroid plexus, leptomeninges ventricular ependyma and along the brain microvasculature. However, the other form, p75 is undetectable under basal conditions. Both TNF receptors are robustly upregulated in barrier-associated structures in the brain, including capillary endothelium, in response to immune challenges. (Nadeau and Rivest, 1999). In contrast to the localization of TNF receptors to the barrier structures, cells that respond to a systemic TNF challenge are revealed throughout the brain notably along the stress-related circuitry that stereotypically induced following other challenges (Nadeau and

Rivest, 1999). These results suggest that circulating TNF activates directly the capillary endothelial cells which in turn may produce soluble molecules such as prostaglandins that act on a paracrine manner stimulating autonomic and neuroendocrine centers. Together, immune challenges in general, and proinflammatory cytokines in particular, activate the hypothalamic stress-related neurons through pathways that show similarities to those that are commonly recruited by other stressors. However, the primary sensory information originates from widespread sources including cytokine-responsive cells at the barrier structures and transduced by secondary signaling molecules.

Transcriptional changes in the parvoceilular neurosecretory cells in response to immune stimuli Under basal conditions, ongoing transcription from both CRH and AVP genes is undetectable in the parvocellular neurons, only a few neurons show nuclear signal when hybridized with CRH intronic probes (Herman et al., 1991, 1992; Kovacs and Sawchenko, 1996a,b). We have previously shown that ether stress-induced activation of CRH and AVP genes in the parvocellular compartment follows different time courses with peak hnRNA responses occurring at 5 min and 2 h, respectively (Kovacs and Sawchenko, 1996a,b). Based on the timing of certain coexpressing transcription factors in the parvocellular neurons, it seems likely that transcriptional activation of AVP and CRH in the same neurons involve distinct mechanisms. Rapid inducibility of CRH gene by stress is compatible with rapid phosphorylation of CREB; while delayed activation of AVP expression in the CRH cells might involve de novo synthesized transcription factors, such as AP1. It is also important to emphasize that the AVP, and not the CRF, gene is the principal target of glucocorticoid-mediated transcriptional suppression during ether stress (Kovacs et al., 2000). Upregulation of CRH transcription in the parvocellular neurosecretory neurons occurs in response to LPS, IL-1 and TNF (Kakucska et al., 1993; Ericsson et al., 1994; Rivest and Rivier, 1994; Nadeau and Rivest, 1999). Using intron-specific probes for in situ hybridization, transient induction of AVP hn-

134 RNA signal has been revealed in the parvocellular neurons following LPS treatment, while other reports did not detect significant changes in AVP mRNA levels (Juaneda et al., 1999). Particular effects of proinflammatory cytokine injections on parvocellular AVP gene expression remain to be analyzed. Notably, LPS-induced co-expression of neurotensin and CCK mRNA has been recently reported in CRH neurons, that may also contribute to the functional plasticity of the parvocellular neurosecretory system responses to inflammation (Juaneda et al., 1999, 2001). The stimulatory effect of immune challenges on CRH transcription seems to be dependent on the ascending catecholaminergic pathways originating in the nucleus of the solitary tract and ventrolateral medulla (Ericsson et al., 1994; Sawchenko et al., 2000). Although this pathway is commonly activated by physical stressors and immune challenges it is not clear if stimulus-transcription coupling recruits similar transcription factors. Inducible expression of immediate early genes, such as c-fos, and NGFIB has been detected in the parvocellular neurons following LPS, IL-1 etc. (Chan et al., 1993; Chang et al., 1993; Ericsson et al., 1994; Rivest and Rivier, 1994). It remains to be determined whether these particular transcription factors are directly involved in regulation of CRH gene expression responses during immune challenges. Signal transduction pathways and transcription factors, specific for immune challenges, such as NFKB, STAT etc. and their involvement in induction of neuropeptide gene expression in the hypothalamus have not been extensively studied (Kovalovsky et al., 2000). NFKB is a nuclear factor bound to enhancer region of the gene that encodes K light chain of antibody molecules in B cells. NF•B is ubiquitously expressed in many cells and forms a transcriptionally inactive complex with an inhibitory protein referred to as IKB. Upon phosphorylation in response to wide range of extracellular signals, boB dissociates from the complex and NFKB translocates to the nucleus and regulates expression of many genes, most of which play essential role in immunity and inflammation (O'Neill and Kaltschmidt, 1997). Expression of NFKB and IKB has been detected in the CNS. NF~cB in astrocytes, microglia and endothelial cells of the brain capillaries may signal

inflammation, injury and viral infections, whereas neuronal NF•B has been implicated in synaptic plasticity, neuronal development and neurodegenerative diseases (van der Burg and van der Saag, 1996; O'Neill and Kaltschmidt, 1997). Systemic LPS, IL-1 and TNF has been described to induce NFKB activity at the barrier-associated structures in the brain, that is consistent with the hypothesis that immune-tobrain signaling is not mediated directly by immune mediators, such as cytokines, it rather involve reception of immune signals at the barrier structures and induction of secondary messenger molecules and activation of classic neural networks to activate effector neurons (Rivest et al., 2000). It is noteworthy that NFKB activity in the brain and in the periphery is restrained by glucocorticoids, pointing to another level of immune-endocrine interactions (van der Burg and van der Saag, 1996).

Allergic reactions: another example of immune-neuroendocrine integration In contrast to infectious and inflammatory responses outlined above, hypersensitivity is predominantly mediated and governed by T helper 2 (Th2) cytokines. Allergic reactions occur immediately following the contact with innocuous allergens and clinically manifested as local reactions as hay fever, eczema, asthma, urticaria and food allergy, while anaphylaxis is a systemic allergic reaction (Ewan, 1998). Initial contact with an allergen results in IgE production by B cells, which then bound to the IgE receptors (FceRI) on the surface of mast cells and basophils (sensitization) and IgE cross-linking results in the release of vasoactive, chemotactic mediators, such as histamine and serotonin, proteoglycans and enzymes (Wasserman, 1990). Mast cell mediators cause capillary leakage, edema, and smooth muscle contraction which collectively may lead to severe symptoms of anaphylactic shock, including hypotension, asphyxia, and respiratory arrest (Ewan, 1998). In rats, a specific syndrome can be provoked by injecting foreign proteins, which shows all symptoms of general allergic reactions and referred to as anaphylactoid reaction as it occurs after the primary contact with the allergen (Fig. 1). Experimentally, anaphylactoid reactions can be induced by i.p. or i.v. injection of egg white, ovalbumin, compound 48/80 etc.

135

Fig. 1. Anaphylactoid reaction in rats. Rats were injected intravenously with egg white (EW) or saline (CONTR) 1 h before the photographs were taken. First signs of the edema are detected around 30 min and persist up to 6-8 h post-injection. In addition to the edema in paws and legs, anaphylactoid reaction results in scratching and rubbing around the nose, head and genitalia, labored respiration, drop in blood pressure, polydipsia, and reduced exploration.

Anaphylactoid reaction results in activation of the HPA axis Egg white and 4 8 / 8 0 challenge results in elevation of ACTH and corticosterone plasma levels (Foldes et al., 2000); the kinetics of the response is similar to those seen in case of other systemic stress models. To define the circuitry underlying these effects, c-Fos was used as a marker to identify central neurons that are responsive to allergic challenges. Fos protein was detected throughout the PVN, including the CRH-expressing parvocellular neurons, the OXY and AVP-containing magnocellular elements as well as the autonomic projection neurons with a peak of 9 0 - 1 2 0 min after challenge (Figs. 2 and 3). Extrahy-

pothalamic sites of neural activation show striking similarities with the gross pattern of immediate-early gene induction seen in response to systemic LPS and IL-1, and included structures involved in central interoceptive information processing and autonomic regulation (Foldes et al., 2000). In addition, circumventricular structures including the vascular organ of the lamina terminalis, subfornical organ also became c-Fos-positive following egg white injection.

Possible sites of immune-to-brain communication during anaphylactoid reactions Immune mediators, such as histamine and serotonin (5-HT) released from activated mast cells and ba-

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Fig. 2. Neuronal activation in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) in response to immune challenges. Bright field photomicrographs showing c-Fos immunoreactive cell nuclei 2 h following intravenous injection of egg white (EW) or bacterial lipopolysaccharide,LPS. These challenges can be regarded as experimental models of Th-2 mediated hypersensitivity and Th- 1mediated inflammatory reactions, respectively. Abbreviations: 3V, third ventricle; 0% optic tract; mpd, dorsal medial parvocellular subdivision; mpv, ventral aspect of medial parvocellular subdivision; pm, posterior magnocellular. Scale bar: 100 gm. From Foldes et al., 2000.

sophil leukocytes, are very likely to play an essential role in immune-brain communication (Foldes et al., 2000; Matsumoto et al., 2001), since pretreatment with chromolyn (a mast cell stabilizer) attenuated the symptoms and hormonal responses seen during anaphylactoid reactions. Because neither of these mediators can passively cross the b l o o d brain barrier, the manner in which they reach the brain parenchyma and affect hypothalamic neurosecretory neurons remains to be established. Potential sites through which blood-borne signals transduced

into central responses include: (1) circumventricular organs, that lack blood-brain barrier (Katsuura et al., 1990); (2) stimulation of peripheral sensory nerves that may be associated with immune cells, including mast cells (Johnson and Krenger, 1992; Gaykema et al., 1995; Goehler et al., 1999); (3) interaction with the brain microvasculature, that involves release of secondary signaling molecules (Elmquist et al., 1997); and (4) involvement of central mast cells (Theoharides et al., 1995; Matsurnoto et al., 2001). Whatever the signaling mechanism is, it is

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Transcriptional activation of CRH and AVP genes in the neurosecretory neurons in response to allergic insults To determine if the activation of hypothalamic neurosecretory neurons is reflected in the stimulation of target gene expression, in situ hybridization techniques with intron-specific probes were used to follow the timing of CRH and vasopressin gene expression in the hypothalamus. We found a reliable upregulation of CRH transcription in the parvocellular compartment very rapidly (15 min) following anaphylactoid reaction (Figs. 4 and 5). This timing of CRH gene activation is similar to that found in response to ether stress (Kovacs and Sawchenko, 1996a,b) or to blockade of corticosteroid synthesis (Herman et al., 1992), but occurs earlier than following restraint (Ma et al., 1997a,b) or systemic LPS treatment (Rivest and Laflamme, 1995). Rapid stimulation of CRH transcription is more likely compatible with activation of preformed transcription factors (such as CREB phosphorylation) than induction of de novo synthesized transcription factors such as AP-1 (Guardiola-Diaz et al., 1994; Kovacs and

Sawchenko, 1996a,b). In agreement with previous reports, AVP hnRNA was detected in the acknowledged hypothalamic seats of vasopressin synthesis, including the supraoptic and suprachiasmatic nuclei and the magnocellular subdivision of PVN (Herman, 1995; Kovacs and Sawchenko, 1996a,b). This basal level of transcription was clearly upregulated in the magnocellular neurosecretory neurons in rats that were exposed to anaphylactoid challenge (Fig. 4). In addition, allergic insults provoke an increase of AVP expression in the parvocellular subdivision of the PVN that peaks parallel with CRH transcription (Figs. 4 and 5) It is worthy of mention, however, that this pattern of timing of parvocellular AVP induction is different from that have been detected following ether stress. Previous studies described distinct timing for transcriptional activation with peak of CRH and AVP hnRNA responses to ether at 5 and 120 min after stress, respectively. We have interpreted the delayed AVP expression as a result of stress-induced rise in circulating corticosterone levels that may restrain AVP, but not CRH transcription. Indeed, adrenalectomized rats with or without low level of corticosterone supplementation display advanced peaks of AVP hnRNA responses to ether stress (Kovacs et al., 2000). The time course of AVP hnRNA seen in response to anaphylactoid challenge shows

138

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Fig. 4. Time course of CRH and AVP hnRNA responses to anaphylactoid challenge. Darkfield autoradiograms from similar rostrocaudal levels of the PVN showing nuclear hybridization signals obtained using an intron-specific cRNA probes at key time points. From low resting levels, rats show a marked increase of CRF hnRNA signal in the dorsal aspect of medial parvocellular subdivision that peaks at 15 min after egg white injection. CRH hybridization signal 2 h after challenge is not different from uninjected controls. Substantial basal levels of AVP hnRNA expression are apparent in the magnocellular subdivision which were significantly further increased up to 2 h post-challenge. Egg white induced a rapid and transient increase of AVP expression in the parvocellular subdivision that was maximal 15 min after injection.

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time (min) Fig. 5. Effects of anaphylactoid reactions. (A) Hypotension, induced by intravenous injection of egg white. (B) Time course of transcriptional induction of CRF and AVP genes in the medial parvocellular subdivision of the paraventricularnucleus. Mean ± SEM number of hnRNA expressing cells. Note the rapid and transient activation of both genes. similarities to that described following hypotensive hemorrhage (Chan et al., 2002), and might involve different regulatory mechanisms governing AVP expression in response to ether stress and immune challenge. It is noteworthy that aspects of steroid feedback that may delay AVP transcription are not detectable in the hemorrhage model (Thrivikraman and Plotsky, 1993). Whether this is also the case in response to anaphylactic reactions remains to be established. The regulation of AVP gene expression in the parvocellular versus magnocellular paraven-

tricular subdivisions might be different. Although AVP hnRNA levels are rapidly increase after egg white injection, the upregulation of AVP expression in the magnocellular neuron population seem to be prolonged. Anaphylactic responses are accompanied with transient hypotension that may initially trigger vasopressin expression in the magnocellular neurons. Edema in the paws and legs is another symptom seen during anaphylactoid reactions, which is maintained long after the injection of the provoking agent and might contribute to the increasing demand for pos-

140 terior pituitary hormone secretion. Accordingly, we find blunted edema formation, and more interestingly blunted ACTH and c-fos responses to anaphylactoid challenges in vasopressin-deficient Brattleboro rats. Conclusion All three functional domains of the hypothalamic paraventricular nucleus are involved in the regulation of physiological responses to immune challenges and neurohypophyseal hormones, expressed in all visceromotor cell types play a pivotal role in these effector functions (Fig. 6). Neural pathways, hormonal and transcriptional changes, which are recruited in response to activation either Thl or Th2 immune responses, show similarities to each other and to those that are stimulated by other stressors. Dyscommunication in the neuro-immuno-endocrine web results in certain neuro- and immunopathologies, while manipulation of this regulatory system should open new therapeutic avenues in treatments of chronic inflammatory, autoimmune and allergic diseases.

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Fig. 6. Schematic summary of immune-challenge-induced physiological responses governed by distinct visceromotor parts of the paraventricular nucleus. Increased expression, synthesis and release of CRH and AVP at parvocellular compartment results in stimulation of hypothalamo-pituitary-adrenocortical axis. Corticosterone, the end-product of this regulatory system, suppress inflammatory responses, but stimulate Th-2 mediated cellular immune responses. Neurohypophyseal hormones released at the posterior pituitary are involved in the regulation of osmo- and cardiovascular responses to immune challenges. Autonomic projection neurons in the PVN orchestrate a pattern of sympathetic responses to immune challenges.

Abbreviations ACTH AVP CNS CRH EAE GABA hnRNA HPA IL- 1, -6 LPS NFKB NGFI-B OVLT OXY PVN SFO SON STAT Th TNF VP 5-HT

adrenocorticotropin arginine vasopressin central nervous system corticotropin-releasing hormone experimental allergic encephalomyelitis y-aminobutyric acid heteronuclear RNA hypothalamo--pituitary-adrenocortical interleukin(- 1, -6) lipopolysaccharide nuclear factor kappa B nerve growth factor-induced protein B vascular organ of the lamina terminalis oxytocin paraventricular nucleus of the hypothalamus subfornical organ supraoptic nucleus signal transducers activators of transcription T-helper tumor necrosis factor vasopressin serotonin

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