Andrew V. Turnbull

Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat.

Interleukin-6 (IL-6) is a neurotrophic cytokine expressed in both neurons and glia. The present study shows that cerebral ischemia produced by permanent occlusion of the middle cerebral artery (MCAO) produces a dramatic increase in IL-6 bioactivity in the ischemic hemisphere within 2 hours of MCAO (167 ± 55 IU versus sham: 50 ± 35 IU), with further increases at 8 hours (3,456 ± 1,162 IU) and 24 hours (6,088 ± 1,772 IU). In a separate series of experiments, intracerebroventricular injection of recombinant IL-6 (3,100 or 31,000 IU) significantly reduced ischemic brain damage after MCAO (to 52% and 65% of controls, respectively). The large increase in endogenous IL-6 bioactivity in response to ischemia, together with the marked neuroprotection produced by exogenous IL-6 suggest that this cytokine is an important endogenous inhibitor of neuronal death during cerebral ischemia.

Corticotropin-Releasing Factor (CRF) and Endocrine Responses to Stress: CRF Receptors, Binding Protein, and Related Peptides

Abstract Corticotropin-releasing factor (CRF) is a 41 -amino acid neuropeptide, which is recognized as a critical mediator of complimentary, stress-related endocrine, autonomic, and behavioral responses in mammalian species. CRF belongs to a family of structurally related peptides including frogskin sauvagine and fish urotensin I. The effects of CRF and related peptides are mediated by two distinct receptors, which differ in their anatomical distribution, as well as in their pharmacological characteristics. In addition, CRF is bound with high affinity by a CRF binding protein (CRF-BP), which is a putative inhibitor of CRF action. CRF is probably not the sole endogenous ligand for CRF receptors or the CRF-BP, since a second mammalian member of the CRF family, urocortin, has recently been identified. This article describes recent findings with respect to CRF, its receptors, binding protein, and CRF-related peptides, which provide further insights into the role and mechanisms of CRF action in stress responses.

Mechanisms of Hypothalamic-Pituitary-Adrenal Axis Stimulation by Immune Signals in the Adult Rata

Abstract: Immune stimulation increases the activity of the HPA axis, a phenomenon directly or indirectly mediated through cytokines. We have used two models, the peripheral administration of endotoxin (LPS) or turpentine‐induced tissue injury to show that corticotropin‐releasing factor (CRF) and vasopressin (VP), hypothalamic peptides released by cytokines, play a dominant role in the increased ACTH measured in these two paradigms. In turn, CRF and VP synthesis and/or release is modulated by catecholamines, prostaglandins (PGs), and nitric oxide (NO). These secretagogues are produced in the periphery and/or the central nervous system (CNS) in response to increased cytokine levels and act on CRF/VP neurons and nerve terminals. Finally, endotoxemia and local tissue inflammation may upregulate brain levels of tumor necrosis factor α, interleukin‐1β, and/or interleukin‐6, providing yet another mechanism through which the occurrence of systemic inflammation is conveyed to the brain. The relative importance of brain or peripheral intermediates appears to depend on the site at which cytokine levels are increased. We have shown, for example, that peripheral, but not brain, PGs are important in mediating the neuroendocrine influence of blood‐borne cytokines, while PGs in the CNS play a role in situations characterized by elevated brain immune proteins. NO, on the other hand, restrains the response of the HPA axis to circulating, but not brain cytokines. These results illustrate the complexity of the mechanisms involved in the stimulation of the HPA axis and suggest that their specific involvement depends on the type, intensity, and duration of immune stimulation.

Urocortin, a corticotropin-releasing factor-related mammalian peptide, inhibits edema due to thermal injury in rats

Urocortin is a recently characterized mammalian peptide which appears to be an endogenous ligand for corticotropin-releasing factor (CRF) receptors, in particular CRF receptor type 2. The effect of rat urocortin on protein extravasation and edema, produced by immersion of the paws of anesthetized rats in 58 degrees C water for 5 min, was compared to that of rat/human CRF. Urocortin administered i.v. dose-dependently inhibited the leakage of Evans blue dye into the skin of the footpad and the increase in paw weight, with a potency 6.6-6.7 times greater than CRF. alpha-Helical CRF-(9-41), a more potent antagonist of type 2 than type 1 CRF receptors, completely reversed the inhibition of edema produced by either CRF or urocortin, at a dose (200 nmol/kg) that did not affect adrenocorticotropin secretion induced by either peptide. These data indicate that urocortin is a potent inhibitor of heat-induced edema, and that this action is mediated by CRF receptors, most likely CRF receptor type 2.

Inhibition of Gonadotropin-Induced Testosterone Secretion by the Intracerebroventricular Injection of Interleukin-1β in the Male Rat1

The intracerebroventricular (icv) injection of the proinflammatory cytokine interleukin (IL)-1β is known to significantly decrease plasma LH levels in the male rat, thereby lowering testosterone (T) secretion. We show here that central administration of this cytokine (20–80 ng) also inhibits T secretion in response to human CG (hCG), an effect that is apparent already when IL-1β is injected 15 min before hCG. This phenomenon is independent of LH secretion because lowering LH levels with the potent GnRH antagonist Azaline B neither mimics nor affects the suppressive influence of icv IL-1β on the hCG-induced T secretory response. Elevations in plasma corticosterone levels do not seem to play a role either, because icv IL-1β is able to blunt hCG-induced T secretion in animals whose corticosterone has been removed by adrenalectomy or reduced by the administration of antibodies to CRF. Furthermore, the observation that icv IL-1β inhibits the T response to hCG before elevations in plasma IL-6 concentrations are...

Inhibition of Tumor Necrosis Factor-α Action within the CNS Markedly Reduces the Plasma Adrenocorticotropin Response to Peripheral Local Inflammation in Rats

The present study tested the hypothesis that the cytokine tumor necrosis factor-α (TNF-α) is an important CNS mediator of the hypothalamo-pituitary-adrenal (HPA) axis response to local inflammation in the rat. Recombinant murine TNF-α administered directly into the cerebroventricles of normal rats produced a dose-dependent increase in plasma adrenocorticotropin (ACTH) concentration. Local inflammation induced by the intramuscular injection of turpentine (50 μl/100 gm body weight) also produced an increase in plasma ACTH, peaking at 160–200 pg/ml at 7.5 hr after injection (compared with 10–30 pg/ml in controls). Intracerebroventricular pretreatment with either 5 μl of anti-TNF-α antiserum or 1–50 μg of soluble TNF receptor construct (rhTNFR:Fc) reduced the peak of the ACTH response to local inflammation by 62–72%. In contrast, intravenous treatment with the same doses of anti-TNF-α or rhTNFR:Fc had no significant effect on the ACTH response to local inflammation. Although these data indicated an action of TNF-α specifically within the brain, no increase in brain TNF-α protein (measured by bioassay) or mRNA (assessed using either in situ hybridization histochemical or semi-quantitative RT-PCR procedures) was demonstrable during the onset or peak of HPA activation produced by local inflammation. Furthermore, increased passage of TNF-α from blood to brain seems unlikely, because inflammation did not affect plasma TNF-α biological activity. Collectively these data demonstrate that TNF-α action within the brain is critical to the elaboration of the HPA axis response to local inflammation in the rat, but they indicate that increases in cerebral TNF-α synthesis are not a necessary accompaniment.

Sprague-Dawley Rats Obtained from Different Vendors Exhibit Distinct Adrenocorticotropin Responses to Inflammatory Stimuli

The purpose of this work was to compare the plasma adrenocorticotropin (ACTH), corticosterone and interleukin-6 (IL-6) responses that rats of the outbred Sprague-Dawley strain obtained from two different vendors: Charles River (CR) and Harlan (HSD). Basal plasma ACTH and IL-6 concentrations were similar in rats from either vendor (HSD or CR), while CR animals exhibited slightly elevated corticosterone levels in late afternoon. Inflammatory stimuli such as lipopolysaccharide (LPS) (1 µg/kg, i.v.) or turpentine (50 µl/100 g, i.m.) which induce the production of endogenous cytokines, produced a significantly larger ACTH response in CR, compared to HSD rats, while the overall corticosterone responses were comparable in both rat groups. This could probably not be accounted for by a greater ACTH responsiveness in CR rats per se because CR and HSD rats showed similar peak ACTH responses to electrofootshock. Furthermore, in contrast to when the stimulus was one that induced endogenous cytokine production, the administration of exogenous interleukin-1β (IL-1β, 200 ng/kg, i.v.) produced a 2-fold greater rise in plasma ACTH concentrations in HSD rats compared to CR rats. The plasma IL-6 responses to the inflammatory stimuli showed a similar pattern to ACTH, with LPS and turpentine tending to pruduce greater IL-6 responses in CR rats, though these differences were not statistically significant. In contrast HSD rats had a significantly greater IL-6 response to IL-1β than did CR rats. Collectively, these results show that Sprague-Dawley rats obtained from different commercial sources can differ in immune-neuroendocrine responses to inflammatory stimuli.

Influence of Carbon Monoxide, and its Interaction with Nitric Oxide, on the Adrenocorticotropin Hormone Response of the Normal Rat to a Physico-Emotional Stress

We determined whether the gas carbon monoxide (CO) altered the adrenocorticotropin hormone (ACTH) response to mild inescapable electrofootshocks, and whether it interacted with nitric oxide (NO). Peripheral injection of the NO synthase (NOS) inhibitor Nwnitro‐l‐arginine‐methylester (l‐NAME), a compound which readily crosses the blood–brain barrier, produced the expected blunting of the ACTH response to the shocks. This effect was mimicked by other arginine analogues such as l‐nitroarginine (l‐NNA) and NG‐methyl‐l‐arginine (NMMA). The subcutaneous (s.c.) administration of the heme oxygenase (HO) blockers tin mesoporphyrin (SnMP) or tin protoporphyrin (SnPP) significantly decreased brain HO levels, indicating that both compounds had penetrated the brain. Blood pressure showed a modest increase in response to SnMP, and no change after SnPP. SnMP and SnPP both decreased shock‐induced ACTH release, though the magnitude of this effect was slightly less than that of l‐NAME. The influence of SnPP was further augmented in rats with concomitant blockade of NO formation, which suggests that both NO and CO are necessary for the full response of this axis to electrofootshocks. Finally, the ability of SnPP to significantly blunt the expression of the mRNA for the immediate early gene NGFI‐B in the paraventricular nucleus (PVN) of rats exposed to shocks, indicates that the influence of CO was exerted on hypothalamic neuronal activity. Collectively, our results show that NO and CO exert a stimulatory effect on the HPA axis response to mild electrofootshocks, and that at least part of this influence takes place on hypothalamic neurons and/or their afferents.

Intracerebroventricular Passive Immunization. I. The Effect of Intracerebroventricular Administration of an Antiserum to Tumor Necrosis Factor-α on the Plasma Adrenocorticotropin Response to Lipopolysaccharide in Rats1

The present study tested the hypothesis that the cytokine tumor necrosis factor-α (TNF-α) is an important central nervous system mediator of the rat hypothalamo-pituitary-adrenal (HPA) axis response to the iv administration of lipopolysaccharide (LPS; 5 μg/kg). LPS produced a rapid (within 30 min) rise in plasma TNF-α levels, which preceded elevations in plasma ACTH (commencing at 45 min). Despite a lack of detectable TNF-α biological activity in the brain 30 min to 2 h after LPS administration, intracerebroventricular (icv) pretreatment (−20 h) with 5 μl anti-TNF-α antiserum significantly delayed the onset of the plasma ACTH response to LPS, suggesting that TNF-α acts within the brain. However, we also noted that the icv infusion of anti-TNF-α 20 h earlier produced experimentally significant concentrations of the same anti-TNF-α antibodies in systemic blood. This suggested the possibility that the effect of this antiserum was due to its leakage to the periphery. Indeed, 5 μl anti-TNF-α administered iv at...

Interleukin-1β Regulates Pituitary Follistatin and Inhibin/Activin βB mRNA Levels and Attenuates FSH Secretion in Response to Activin-A

Activins and follistatins regulate all levels of the reproductive axis, including the pituitary where they stimulate and inhibit FSH production, respectively. Gonadotropes are known to express inhibin/activin βB and activin-B (βBβB) functions as an autocrine modulator of FSH production. By contrast, the mRNA for the activin-binding protein, follistatin, is present in most pituitary cells and folliculo-stellate cells may be the major source of the protein secreted by the anterior pituitary. Interleukin-1β (IL-1β) is one of several cytokines known to also influence the reproductive axis. IL-1β inhibits the hypothalamo-pituitary-gonadol (HPG) axis by suppressing GnRH and gonadal steroid production. Because several pituitary cell types, including follistatin-producing folliculo-stellate cells, are targets of IL-1β, cytokine effects on gonadotrope function were evaluated using cultured rat anterior pituitary cells. Activin-A (0.01 to 1 nM; 24 h) increased basal FSH secretion ∼2-fold. IL-1β (0.005 to 0.5 mM) by...