Frank Berkenbosch

Appearance of interleukin-1 in macrophages and in ramified microglia in the brain of endotoxin-treated rats: a pathway for the induction of non-specific symptoms of sickness?

The presence and cellular localization of interleukin-1 beta immunoreactivity (irIL-1) in and around the brain was investigated using immunocytochemistry on Bouins fixed vibratome brain sections of control and endotoxin-treated rats. Peripheral administration of endotoxin resulted in the appearance of irIL-1 in cells in the meninges, choroid plexus, brain blood vessels and in non-neuronal cells in the brain parenchyma. Using monoclonal and polyclonal antibodies to macrophage and astrocyte antigens, the endotoxin-induced irIL-1 positive cells could be identified as macrophages in the meninges and choroid plexus (ED2), perivascular cells (ED2) and ramified microglial cells (GSA-I-B4 isolectin). Our data demonstrate a pathway for the induction of non-specific sickness symptoms in response to endotoxin.

Repeated stress-induced activation of corticotropin-releasing factor neurons enhances vasopressin stores and colocalization with corticotropin-releasing factor in the median eminence of rats.

Stress-induced release of corticotropin-releasing factor (CRF) and vasopressin (AVP) was studied in rats by measuring the decline of CRF and AVP stores in the median eminence after blockade of fast axonal transport with colchicine (5 micrograms per rat intracisternally). Quantitative immunocytochemistry was used to detect changes in CRFi and AVPi in the external zone of the median eminence (ZEME) selectively. Immobilization stress induced a fast ACTH response to 1,000-2,000 pg/ml which was associated with a fall in both CRFi and AVPi of 34% during the first 30 min. This is followed by different time courses of further AVPi and CRFi depletion. In addition, we investigated the effect of repeated daily stress exposure on CRFi and AVPi in the ZEM 1 day after stress exposure. Repeated daily immobilization for 9 or 16 subsequent days did not affect the CRFi stores in the ZEME, but increased the AVPi stores to 161 +/- 13% and 218 +/- 11% respectively. Quantitative analysis of electron microphotographs of repeatedly handled rats showed a mean density of CRF positive profiles in the ZEME of 45.5 +/- 2.5 per 500 microns 2 of which 25% also stained for pro-AVP-derived peptides. After 9 subsequent days of immobilization the total density of CRF-positive profiles remained unchanged, but the fraction of CRF swellings that also stained for pro-AVP-derived peptides increased approximately 2-fold. We conclude that (1) the secretion of AVPi and CRFi from the ZEME are independently controlled, indicating differential activation of AVP containing and AVP deficient CRF neurons during acute immobilization, and (2) repeated stress leads to plastic changes in hypothalamic CRF neurons resulting in increased AVP stores and colocalization in CRF nerve terminals.

Neuroendocrine, sympathetic and metabolic responses induced by interleukin-1

Effects on turnover of vasopressin (AVP) in the hypothalamus and on secretion of pituitary hormones, catecholamines and insulin after intraperitoneal injection of recombinant interleukin-1 (beta) (IL-1) were investigated in male wistar rats. Intraperitoneal administration of IL-1 in a dose (1 microgram) that maximally activated pituitary-adrenal activity failed to alter plasma concentrations of prolactin, luteinizing hormone and melanocyte-stimulating hormone. Rats chronically cannulated in the right jugular veins showed a time-related increase in plasma corticosterone concentrations in response to intraperitoneal administration of IL-1 that lasted up to 4 h. In the same rats, plasma epinephrine (E) and norepinephrine (NE) concentrations were only slightly elevated (2-fold increase) at 30 min and at 1 h after IL-1 administration. Unlike in endotoxin-resistant C3H/HeJ mice, where IL-1 induces hypoglycemia, IL-1 did not affect plasma concentrations of glucose and insulin in Wistar rats. In the zona externa of the median eminence, IL-1 stimulated corticotropin-releasing factor (CRF) turnover at an approximate rate of 15%/h, but did not cause a concomitant change in AVP turnover as can be observed after insulin-induced hypoglycemia. Since half of the hypothalamic CRF neurons have been shown to costore AVP, the data favor the view of a selective effect of IL-1 on a subtype of CRF neurons. We conclude that pituitary-adrenal activation in response to Il-1 is caused by CRF secretion from a subtype of CRF neurons (not storing AVP) in the rat hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of interleukin-1β in Lewis rat brain during experimental allergic encephalomyelitis by immunocytochemistry at the light and ultrastructural level

Interleukin-1 beta (IL-1) is a cytokine which exerts many biological effects during inflammation. In the present study, experimental allergic encephalomyelitis (EAE) was induced in Lewis rats. During the various stages of EAE, the presence of IL-1 in the brain was investigated using immunocytochemistry at both the light and ultrastructural level. Ten days after immunization, IL-1 immunoreactivity was found in brains of animals which at this time showed mild clinical signs. Outside the blood-brain barrier, IL-1 was localized in the cytoplasm of meningeal macrophages and perivascular cells. Within the brain parenchyma, IL-1 immunoreactivity was distributed in perivascular lesions in the cytoplasm of infiltrated macrophages and activated microglia. On day 13, animals had developed a full blown EAE. At this stage the number of lesions with IL-1-positive cells had increased. In the remission phase (day 25), lesions with IL-1-positive cells could still be detected but were less pronounced as compared to day 13. Other presumptive IL-1-producing cell types like endothelial cells or astrocytes were, at none of the various stages, found to stain for IL-1.

Effect of axonal transport blockade on corticotropin-releasing factor immunoreactivity in the median eminence of intact and adrenalectomized rats: relationship between depletion rate and secretory activity

Male Wistar rats were anaesthetized, injected intracisternally (i.c.) with saline or colchicine and were decapitated at various time intervals. Trunk blood was collected for the determination of immunoreactive adrenocorticotropic hormone (ACTHi) by radioimmunoassay (RIA) and of corticosterone by a fluorometric assay. Changes in corticotropin-releasing factor (CRF) content of the median eminence (ME) were assessed by quantitative immunocytochemistry (QICC) on cryostat sections of ME preparations or by RIA of CRF in ME-extracts. Administration of colchicine resulted in a long-lasting and dose-dependent stimulation of ACTHi secretion. At a dose of 25 micrograms, high plasma ACTHi levels were found for up to 24 h. A dose of 5 micrograms per rat, that has been reported to effectively block vasopressin transport in paraventricular-neurohypophyseal neurons, resulted in a small elevation of plasma ACTHi. In intact rats, i.c. administration of saline of saline containing 5 micrograms of colchicine had no effect on the CRF content in the ME. In contrast, colchicine caused a depletion of the CRF stores in the ME of 1-week adrenalectomized (ADX) rats. The disappearance rate was 9.2%/h as measured by RIA and 11.2%/h as measured by QICC. When plasma corticosterone and ACTHi were normalized by giving ADX rats corticosterone in drinking water, the colchicine-induced depletion of the CRF stores was fully prevented. We conclude that the rate of decline of CRF in the ME of rats treated with a non-toxic dose of colchicine to block axonal transport is positively correlated to the secretory activity of CRF neurons of the paraventricular-infundibular system.

Distribution of interleukin 1β immunoreactivity within the porcine hypothalamus

Abstract The presence and localization of interleukin 1β immunoreactivity (IL 1β i.r.) was studied in the hypothalamus of four healthy, male pigs at 7 months of age, using immunocytochemical techniques on 100 μ vibratome and 10 μ paraffine sections. IL 1β i.r. was found in neuronal cell bodies and their processes within nuclei and fiber of the fiber tracts of the hyphotalamus as well as in varicose fibers, terminals and deposits within the median eminence. In addition, IL 1β i.r. was found in the walls of several, but not all, blood vessels and in very few glial cells.

Sympathoadrenal Activity Facilitates Beta-Endorphin and Alpha-MSH Secretion but Does Not Potentiate ACTH Secretion during Immobilization Stress

The potential involvement of the sympathoadrenal system in stress-induced secretion of peptides from the intermediate lobe of the pituitary gland and the activation of the pituitary-adrenal axis was studied. Male Wistar rats were subjected to control procedures, to sympathectomy by chronic administration (8 weeks) of guanethidine and/or to medullectomy by adrenal enucleation 9 weeks prior to exposure to forced immobilization stress for various periods of time. In intact or sham-operated rats, immobilization caused a prompt increase of circulating norepinephrine, epinephrine (EPI), corticosterone and of immunoreactive adrenocorticotropic hormone (ACTHi), alpha-melanocyte-stimulating hormone (alpha-MSHi) and beta-endorphin (beta-ENDi). Peak levels of pituitary hormones were found after 10 min of stress exposure, but fell to less than 30% of these levels after 2.5 h of immobilization. Adrenal medullectomy, which abolished the stress-induced release of EPI, reduced the acute increase of plasma alpha-MSHi and beta-ENDi, but did not influence the acute increase of plasma ACTHi during immobilization stress. Also in medullectomized plus sympathectomized rats, the initial stress response of circulating ACTHi was not different from that of controls. Adrenal medullectomy with or without additional sympathectomy caused a marked increase in plasma ACTHi concentrations after prolonged stress exposure. We conclude that: catecholamines originating from the adrenalmedulla facilitate the stress-induced secretion of intermediate lobe peptides (alpha-MSHi, beta-ENDi); catecholamines from the sympathoadrenomedullary system do not contribute to the acute release of ACTH during immobilization stress; the sympathoadrenomedullary system is involved in the secondary reduction of circulating ACTHi levels seen during prolonged stress.

Corticotropin-releasing factor immunostaining in the rat spinal cord and medulla oblongata: An unexpected form of cross-reactivity with substance P

By use of two antisera (alpha-CRFA, alpha-CRFB) raised against conjugates of o-CRF and bovine thyroglobulin, cryostat sections of formaldehyde-fixed gelatin models containing o-CRF can be stained. The staining intensity was quantitated by use of an automated microfluorimeter and was shown to be dependent on the concentration of o-CRF (1-300 microM) added to the gel. Determination of the CRF staining intensity after incorporation of o-CRF-related peptides and fragments indicated that both antisera reacted with the C-terminal region of o-CRF. They showed poor cross reactivity with r-CRF fixed in the gel. In the same models, r-CRF could be immunostained efficiently by use of an antiserum (alpha-CRFC) raised to a conjugate of r-CRF and thyroglobulin. This antiserum reacted with the N-terminal and midportion parts but not with the C-terminal fragment of o-CRF fixed in the gels. By use of both o-CRF antisera nerve fibers can be stained in the rat hypothalamus (median eminence) and in the medulla oblongata (spinal trigeminal tract and nucleus) and spinal cord (dorsal horn). Immunoinhibition experiments showed that o-CRF caused a concentration-dependent quenching (0.001-1 microM) of the immunostaining of o-CRF-containing models, rat median eminence and medulla oblongata preparations. alpha-CRFC also stained CRF immunoreactive (CRFi) fibers in the rat hypothalamus with an equal distribution to that found with the o-CRF antisera. However, no immunostaining was found in the spinal trigeminal nucleus and tract and in the dorsal horn, indicating that these fibers store different CRF-related products from those found in the hypothalamus. The CRFi in the medulla oblongata and spinal cord induced by alpha-CRFA was completely abolished 1 week after treatment of adult rats with capsaicin, a substance known to deplete Substance P (SP) from those areas. Gels incorporated with SP showed a concentration-dependent increase (range 10-1000 microM) in immunostaining with both o-CRF sera but not with the r-CRF antiserum. In addition, incubation of o-CRF sera with SP caused a concentration-dependent quenching (range 10-100 microM) of immunostaining in SP-containing models. SP at a concentration of 100 microM was also effective in quenching the CRFi in the dorsal horn and spinal trigeminal area. Quenching was also obtained with the C-terminal part of o-CRF (range 0.002-0.1 microM), which indicates that both CRF antisera contain an immunoglobulin which recognizes determinants on CRF as well as on SP.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroendocrinology of Interleukin-1

Inflammatory processes and infectious diseases induce a constellation of host responses referred to as acute phase response (1,2). These responses include changes in immunologic, metabolic, neurologic, and endocrinologic functions. Although many of its components are far from being understood, it is generally believed that the acute phase response serves to regain normal homeostasis.

Is Vasopressin Preferentially Released from Corticotropin‐Releasing Factor and Vasopressin Containing Nerve Terminals in the Median Eminence of Adrenalectomized Rats?

The effects of adrenalectomy (ADX) on the amounts of immunoreactive corticotropin‐releasing factor (CRFj) and arginine vasopressin (AVPi) that are stored in the zona externa of the median eminence (ZEME) were investigated by means of quantitative immunocytochemistry. Although ADX of male Wistar rats for 1 week or 4 weeks did not affect CRFi in the ZEME as compared to sham‐operated or intact controls, AVPi showed a progressive accumulation. The ratio of AVPi over CRFi in the ZEME had already increased 1 day after ADX. However, it should be noted that the exact changes in CRFi and AVPj as measured by radioimmunoassay and/or quantitative immunocytochemistry were dependent on the substrain of rats used. The secretion rate of CRFi and AVPj was estimated in 1 week and 4 week ADX rats, by measuring the disappearance rate of CRFi and AVPi from the ZEME after blockade of fast axonal transport, by a low non‐toxic dose of colchicine (5 μg per rat). In contrast to intact rats, where this dose of colchicine did not affect CRFi or AVPi in the ZEME, ADX rats showed a progressive depletion of the CRFi and AVPi stores as measured 2.5 and 5 h later. In 1 week ADX rats, CRFi and AVPi both disappeared at a rate of 7% to 8% of their stores per hour. In contrast, after 4 weeks of ADX the fractional disappearance rates of CRFi and AVPi were different and were 3% and 8% of the content per hour, respectively. This indicates that in long‐term ADX rats the chance of AVP being released from the ZEME is more than twice that of CRF. Since most, if not all, of the CRF; containing nerve terminals in the ZEME of ADX rats costore AVP, we hypothesize that in long‐term ADX rats AVP may be preferentially secreted from AVP and CRF costoring nerve terminals.