Neuroimmunoendo Crine Actions During Longterm Elevation Of Brain Corticotropinreleasing Hormone

CRH, a neuropeptide widely distributed in the brain, is the key mediator of the overall responses of the organism to stress. CRH not only functions as the principal neuropeptide regulating ACTH release, but is also responsible for autonomic and behavioural responses associated with stress (for references see Vale et al., 1997). Moreover, central administration of CRH reduces natural killer cell activity, pointing to a role of CRH in the regulation of the activity of the immune system (Irwin et al., 1987).

Disturbances of the brain CRH system are thought to be implicated in several stress-related disorders such as major depression and anorexia/bulimia, but also in Alzheimer's disease. There is evidence that major depression is associated with an increased central CRH drive. Patients suffering from major depression have elevated levels of CRH in the cerebrospinal fluid (Nemeroff, Widerlov, Bissette, Walleus, Karls-son, Eklund, Kilts, Loosen, & Vale, 1984), increased numbers of CRH expressing neurons and CRH mRNA levels in their PVN (Raadsheer, Hoogendijk, Stam,Tilders, & Swaab, 1994; Raadsheer, Van Heerikhuize, Lucassen, Hoogendijk, Tilders, & Swaab, 1995) and reduced CRH receptor density in the frontal cortex (Owens & Nemeroff, 1993). In addition, these patients present many symptoms reminiscent of the effects of centrally administered CRH in experimental animals, such as an activated HPA axis, increased sympathetic outflow, anxiety, increased emotionality, and loss of appetite and sexual interest (Owens & Nemeroff, 1991; Holsboer & Barden, 1996). In contrast, a role for a hypoactive CRH system is suggested in the pathophysiology of Alzheimer's disease, as indicated by the decreased CRH content and the upregulation of CRH receptors in postmortem cerebral cortex of patients (De Souza, Whitehouse, Kuhar, Price, & Vale, 1986). Moreover, cognitive impairment in patients with Alzheimer's disease seems to be associated with a reduced amount of CRH in the cerebrospinal fluid (Pomara, Singh, Deptula, LeWitt, Bissette, Stanley, & Nemeroff, 1989).

From the above-described functions of CRH and the putative involvement of disturbed CRH functioning in brain diseases, it is clear that a well-balanced CRH

Figure 5, Schematic drawing of llie long-term i.c.v, C.RH-intused rat. CRH is pumped into the ventricular system during 7 days via an i.c.v. cannula connected to a minios-motic pump (Al/a Corporation, Palo Alto, U.S.A.). In addition, some endocrine and physical data are depicted.

The long-term i.c.v. CRH-infused rat

The long-term i.c.v. CRH-infused rat

In Vivo Treatment * Long-term central CRH-Infusion (0.1 -1 ng/h, 7 days)

Neuroendocrine and Physical Effects

» Plasma ACTH and Corticosterone

• Anterior pituitary POMC mRNA

• Adrenal weight

* Spleen and Thymus weight

* Body weight gain system is essentia! for health. Although CRH system disturbances have been suggested in major depression and Alzheimer's disease, until now, studies in humans could not identify which physiological and behavioural abnormalities, associated with these illnesses, are caused by hyper- or hyposecretion of CRH. Experimental paradigms for the investigation of central CRH dysfunction in humans are rather limited. Despite the extensive molecular and neuroanatomical characterization studies on the CRH system and the study of physiological and behavioural effects of acutely administered CRH, hardly any investigations have focussed on the neurobiological consequences of long-term central CRH dysfunction. The latter should be of prime concern given that chronic disturbances in this system are most likely to occur during and to be involved in the development of stress-related disorders. We have, therefore, developed an animal model of central CRH hyperactivity, i.e. the long-term i.c.v. CRH-infused rat (Figure 5). Rats are i.c.v, infused with CRH for seven days via a miniosmotic pump, positioned subcutaneously in the dorsal region, connected to an i.e.v. cannula via polyethylene tubing (Alza Corporation, Palo Alto, U.S.A.). This animal model enables the study of the consequences of a disturbed CRH system Sot physiological, immune, neurochemical, and behavioural processes under baseline conditions and after stressful challenges. Because 1) CRH plays an important role in neuroimmimoendocrine interactions (Irwin et al, 1987; Rivier & Rivest, 1993), and 2) immune system alterations have been described in major depression (Maes, 1995; Sluzewska et al., 1996), we decided to assess the effects of peripheral inflammation in the long-term i.c.v. CRH treated rat.

4.1. Characteristics of Long-Term i.c.v. CRH-infused Rats under Basal

Conditions and during a Peripheral Inflammatory Challenge

4.1.1. The Immune System. In a first series of experiments the effects of long-term i.c.v. CRH infusion on the immune system were investigated (Labeur, Arzt, Wiegers, Holsboer, & Reul, 1995). CRH-infused rats showed a marked involution of the spleen and the thymus. In vitro T-cell proliferation was tested in primary spleno-cytes collected at day seven of CRH treatment. As expected, splenocytes of long-term CRH-treated animals had reduced proliferative responses to the mitogen concanavalin A. Surprisingly, however, it was found that IL-2 levels were elevated in the supernatant of splenocyte cultures of CRH-infused rats, despite the high level of circulating glucocorticoid in this animal model (see below). This is most likely caused by a reduced uptake/internalization of IL-2 resulting from a diminished gene expression of the IL-2 receptor a chain as observed in splenic T cells of CRH-treated animals. The effects of long-term elevation of brain CRH on T cell proliferation and IL-2 receptors may be evoked by substances released from the adrenal gland, because adrenalectomy prior to CRH infusion prevents these changes in immune function (Labeur et al., 1995).

Next, effects of the CRH treatment on the level of the cytokines were investigated. It was observed that seven days of i.e.v. CRH treatment elevated the basal levels of IL-lp mRNA expression in splenocytes. Moreover, in vitro stimulation of the splenocytes with LPS induced a higher increase in IL-ip mRNA in splenocytes of long-term i.c.v. CRH-infused rats than in those of control animals (Labeur et al., 1995). These results point to a possible hyperresponsiveness of IL-1 in the immune system of long-term i.c.v. CRH-infused animals, which is underscored by experiments on the in vivo effects of LPS on cytokines. On day seven of the CRH treatment, rats were i.p. injected with LPS or saline. After administration of saline, plasma levels of bioactive IL-1, TNF, and IL-6 were below the detection limit of their respective assays. However, administration of LPS caused a much more pronounced elevation in circulating levels of IL-1 and IL-6, but not of TNF, at three hours after the injection in long-term CRH-infused rats than in control rats (Linthorst, Flachskamm, Hopkins, Hoadley, Labeur, Holsboer, & Reul, 1997). Hence, it may be concluded that the immune system of long-term CRH-infused rats responds with a much higher release of IL-1 and IL-6 into the circulation during inflammatory challenges. This may represent a compensatory mechanism for the elevated levels of glucocorticoids which are well known to negatively regulate cytokines and cytokine actions (Munck & Guyre, 1991). On the other hand, this situation may be potentially dangerous, given the involvement of cytokines in septic shock and tissue degeneration (Dinarello, 1991).

4.1.2. The HPA Axis. Long-term infusion of CRH enhanced plasma levels of ACTH and corticosterone (Labeur et al., 1995). In addition, microdialysis experiments on day seven of treatment showed that free corticosterone levels were elevated in CRH-infused rats, resulting in a disappearance of the normal diurnal rhythm. Such a condition is also observed in chronic stress and major depression (Owens & Nemeroff, 1991; Holsboer & Barden, 1996). Other observations pointing to a sustained hyperactive HPA axis are hyperplasia of the adrenals, involution of the thymus and elevated expression of proopiomelanocortin (POMC) mRNA in the anterior pituitary of CRH-treated rats (Labeur et al., 1995; Linthorst et al., 1997).

Intraperitoneal injection of LPS elevated free corticosterone levels in both i.c.v. CRH-infused and control rats (Figure 6A). Whereas the maximum levels reached in the two treatment groups were similar, the rise in free corticosterone was significantly delayed in long-term CRH-infused animals (Linthorst et al., 1997). Hence, these data indicate that the HPA axis in long-term CRH-infused rats is still able to respond to an inflammatory challenge, despite the higher level of circulating glucocorticoids and the possible desensitization of CRH receptors (see 4.1.3.). However, a pathophysiological consequence of such delayed response in corticosterone may be that glucocorticoid-

facilitated and glucocorticoid-regulated processes are executed (too) late (Wiegers & Reul, 1998; Wiegers et al., 1995).

4.1.3. Body Temperature and Locomotion. We have used a biotelemetry method to assess the effects of long-term i.c.v. infusion of CRH on body temperature and locomotor activity. Continuous i.c.v. infusion of CRH increased body temperature during the light as well as the dark period of the diurnal cycle, resulting in a flattened circa-dian rhythm especially during the first 3-4 days of treatment. Moreover, the i.c.v. treatment enhanced locomotor activity during the light and the dark period of the first 2-3 days (Linthorst et al., 1997). The fading-out of the CRH-induced increases in body temperature and locomotion over the treatment period points to a desensitization of central CRH effector mechanisms. Such a desensitization may take place at the level of the CRH receptors, because various situations which elevate CRH levels, including chronic stress and adrenalectomy, decrease CRH receptor density and CRH mRNA concentrations in the anterior pituitary, the hypothalamus and/or the frontal cortex (Anderson, Kant, & De Souza, 1993; De Souza, Insel, Perrin, Rivier, Vale, & Kuhar, 1985; Hauger, Millan, Lorang, Harwood, & Aguilera, 1988; Luo, Kiss, Rabadandiehl, & Aguilera, 1995; Makino, Schulkin, Smith, Pacak, Palkovits, & Gold, 1995; Tizabi & Aguilera, 1992).

Intraperitoneal administration of LPS (100|ig/kg body weight) caused a marked increase in body temperature in control animals, whereas this fever response was highly attenuated in long-term i.c.v. CRH treated rats (Figure 6B). Long-term peripherally CRH-infused rats displayed normal LPS-induced fever responses. Peripherally and i.c.v. CRH-infused animals showed similar elevations in free corticosterone levels under baseline conditions, Thus, the unchanged fever response in peripherally CRH-treated rats suggests that elevated levels of corticosterone are not responsible for the attenuated fever that was observed in long-term i.c.v. CRH-infused subjects (Linthorst et al., 1997).

4.1.4. Hippocampal Serotonergic Neurotransmission and Behavioural Activity. Long-term i.c.v. infusion of CRH had no consequences on basal extracellular levels of 5-HT and 5-HIAA in the hippocampus as assessed on day 7 of treatment using in vivo microdialysis (Linthorst et al., 1997). This is a striking observation, because we have shown that acute i.c.v. injection of CRH caused a profound increase in both 5-HT and 5-HIAA in the hippocampus (Reul & Linthorst, 1997). Hence, it may be hypothesized that long-term i.c.v. CRH treatment results in pertinent regulatory changes in 5-HT neurotransmission; an effect possibly related to desensitization of CRH receptors (see 4.1.3.).

Intraperitoneal injection of LPS is known to increase hippocampal extracellular levels of 5-HT and 5-HIAA (see The LPS-induced rise in 5-HT was, however, significantly reduced in long-term i.c.v. CRH-infused rats (Figure 6C). Moreover, a delayed elevation of 5-HIAA after i.p. administration of LPS was found in i.c.v. CRH-treated animals (Linthorst et al., 1997). These observations clearly show that long-term elevation of brain CRH resulted in an attenuated responsiveness of the hippocampal 5-HT system to an acute stressful challenge. Whether CRH receptor desensitization or putative alterations in brain cytokines (IL-1?) are underlying the reduced responsiveness in 5-HT needs to be further elucidated.

LPS (100pg/kg body weight i.p.) induced sickness behaviour, as indicated by a reduced behavioural activity of the animals, in both i.c.v. treatment groups (CRH and vehicle). The induction of sickness behaviour was, however, delayed by about 1.5-2 hr in long-term i.c.v. CRH-treated rats (Figure 6D) (Linthorst et al., 1997). Because sick-

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