Biomedical subjects

P E Gottschall

Publications and source records attributed to P E Gottschall.

At least 19 recordsLinked to original sources

PubMed2002

Association between protease-specific proteolytic cleavage of brevican and synaptic loss in the dentate gyrus of kainate-treated rats.

Proteolytic fragments generated by ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs)-mediated cleavage of the aggregating chondroitin sulfate proteoglycan, brevican, have been identified, but not localized in the CNS. The purpose of this study, using kainate-induced CNS lesion, was to examine the spatial and quantitative relationship between ADAMTS1 and 4 mRNA expression and ADAMTS-mediated cleavage of brevican (as determined by the abundance of the neo-epitope QEAVESE at the C-terminal of the cleaved brevican G1 domain). In untreated rats, in situ hybridization and reverse transcriptase polymerase chain reaction indicated that ADAMTS4 expression was higher than ADAMTS1 and was localized to hippocampus, temporal lobe and other areas of cortex, striatum and hypothalamus. ADAMTS4 mRNA expression in these regions correlated with the presence of the QEAVESE neo-epitope, which was concentrated in perineuronal nets and in neuropil. In rats that seized after kainate, there was a dramatic elevation in ADAMTS1 and ADAMTS4 transcript that correlated and co-localized with a robust elevation in an extractable, 55-kDa fragment of brevican in temporal lobe and hippocampus. This fragment consisted, at least in part, of the ADAMTS-cleaved epitope G1-QEAVESE. The kainate-induced elevation in this ADAMTS-cleaved fragment was localized to amygdaloid and thalamic nuclei, hippocampus, caudate-putamen, cingulate cortex, and the outer molecular layer of the dentate gyrus where it was accompanied by a robust elevation in ADAMTS1 and 4 mRNA and a 28% decline in synaptic density 5 days after kainate.Thus, complexes of extracellular matrix proteins that exist in perineuronal nets and in the neuropil are cleaved by specific matrix-degrading proteases at early time points during excitotoxic neurodegeneration. The observed ADAMTS-induced cleavage of brevican in the dentate outer molecular layer is closely associated with diminished synaptic density, and may, therefore, contribute to synaptic loss and/or reorganization in this region.

ADAM Proteins↗

PubMed2001

Number of Abeta inoculations in APP+PS1 transgenic mice influences antibody titers, microglial activation, and congophilic plaque levels.

There have been several reports on the use of beta-amyloid (Abeta ) vaccination in different mouse models of Alzheimer's disease (AD) and its effects on pathology and cognitive function. In this report, the histopathologic findings in the APP+PS1 doubly transgenic mouse were compared after three, five, or nine Abeta inoculations. The number of inoculations influenced the effects of vaccination on Congo red levels, microglia activation, and anti-Abeta antibody titers. After three inoculations, the antibody titer of transgenic mice was substantially lower than that found in nontransgenic animals. However, after nine inoculations, the levels were considerably higher in both genotypes and no longer distinguishable statistically. The number of inoculations influenced CD45 expression, an indicator of microglial activation. There was an initial upregulation, which was significant after five inoculations, but by nine inoculations, the extent of microglial activation was equivalent to that in mice given control vaccinations. Along with this increased CD45 expression, there was a correlative reduction in staining by Congo red, which stains compact plaques. When data from the mice from all groups were combined, there was a significant correlation between activation of microglia and Congo red levels, suggesting that microglia play a role in the clearance of compact plaque.

Alzheimer Disease↗

PubMed2000

Regional and age-related expression of gelatinases in the brains of young and old rats after treatment with kainic acid.

Due to the possible detrimental impact of local inflammatory responses in neurodegenerative disease, it was of interest to measure the expression of extracellular matrix-degrading enzymes, a group of proteases that are induced during an inflammatory response, in the brains of old and young animals in a model of neuronal death. Doses of kainic acid were administered that resulted in comparable hippocampal pyramidal neuron loss in young and old F344/BN hybrid rats, even though each age group received widely differing doses. Two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were differentially induced with respect to time after kainic acid in sensitive brain regions in both young and old rats. However, the elevation of MMP-9 in the temporal lobe 12 h after injection in old rats was significantly greater than that observed in young animals. These results suggest that early and late induction of MMPs may play a role in neuronal death and repair mechanisms, respectively, and that inflammatory mechanisms in the central nervous system (CNS) of old rats are exaggerated compared to young rats.

Age Factors↗

PubMed1999

Activated isoforms of MMP-2 are induced in U87 human glioma cells in response to beta-amyloid peptide.

Prior studies using rat primary hippocampal cultures indicated induction of matrix metalloproteinases (MMPs) in response to beta-amyloid (A beta). Hence, it was of interest to determine whether MMP activity in a human cell line is influenced by A beta. A beta, but not interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS), stimulated an active form of MMP-2 in human U87 glioblastoma cells, as well as increased the expression of the well-known activator of MMP-2, membrane-type (MT)-MMP. Activation experiments carried out with amino phenyl mercuric acetate (APMA), immunoprecipitation, as well as immunoblotting, suggest that the lower molecular weight, gelatin-degrading activity was an activated form of MMP-2. Furthermore, it was demonstrated that a synthetic furin convertase inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, decreased the production of A beta-induced active MMP-2 in U87 cells. The induction of MMP-3 by cytokines, but not by A beta, suggests that the effect of A beta on MMP-2 is selective. Although A beta stimulated tissue inhibitor of metalloproteinase-1 (TIMP-1), there was no obvious effect of A beta on TIMP-2 production in U87 cells. These results demonstrate that A beta induces an active form of MMP-2 likely by increasing the expression of MT-MMP in a human glioblastoma cell line. Active MMP-2 may degrade A beta or act on ECM components critical in neuronal survival mechanisms and possibly play a role in Alzheimer's disease (AD) neuropathology.

Amyloid beta-Peptides↗

PubMed1998

Regional and differential expression of gelatinases in rat brain after systemic kainic acid or bicuculline administration.

Indirect evidence from in vitro studies implicates a functional role for matrix metalloproteinases (MMPs) in the central nervous system (CNS), including induction of neuronal migration during development and enhancement of neurite extension. Few reports have documented the expression of these enzymes in the brain, especially after injury in vivo. The objective of this study was to determine whether MMPs are expressed in various regional areas of rat brain after administration of the neurotoxin, kainic acid. Limbic motor seizures and neuronal degeneration were induced in Sprague-Dawley rats by systemic administration of kainate (10 mg/kg). Rats were subsequently divided into convulsive and non-convulsive groups, after observing their behaviour in response to the drug. Animals were killed 6, 12, 24, 72 and 168 h (7 days) after injection of kainate. Gelatinases were extracted from various brain regions and assayed by gelatin-substrate zymography. Levels of glial fibrillary acidic protein (GFAP) in corresponding regions were measured by ELISA. In the absence of treatment, MMP-2 and MMP-9 activities were expressed differentially in various brain regions with the highest levels in the hippocampus and the lowest in the cerebellum. In areas from convulsive rats, MMP-9 activity was markedly elevated at 6 h, and reached a maximum at 12 h after injection of kainate (8.1-fold hippocampus, 7.7-fold diencephalon, 7.2-fold striatum, 5.7-fold frontal cortex, 5.5-fold cerebellum, 2.6-fold midbrain). MMP-2 activity was induced more than two-fold in the hippocampus, diencephalon and striatum, to a lesser extent in the frontal cortex and midbrain, and was unchanged in the cerebellum, 72 h after injection. Neither MMP activity was altered in any brain region derived from non-convulsive rats. Treatment with the GABAA antagonist, bicuculline, resulted in increased levels of MMP-9, 12 h after drug administration, but no change in levels of MMP-2 up to 3 days following treatment. GFAP levels were induced 3 days after kainic acid injection in brain regions where MMP-2 was elevated. Nissl staining displayed the classical, regional neurodegeneration in kainate-treated animals that exhibited seizures. No obvious degeneration was detected in kainate-treated, non-convulsive rats or bicuculline-treated animals. These data demonstrate that MMP-9 and MMP-2 are differentially expressed with respect to time after kainic acid injection, and suggest that they are regulated by convulsion and/or neurodegenerative-associated mechanisms, respectively. Although similar in catalytic activity, MMP-9 and MMP-2 may play different roles in response to kainic acid-induced seizure and neuronal degeneration.

Animals↗

PubMed1997

Zymographic measurement of gelatinase activity in brain tissue after detergent extraction and affinity-support purification.

Several methods have been developed for the measurement of gelatinase activity from various tissues using detergent extraction. Gelatin-affinity chromatography has been employed for the large-scale purification of gelatinases from conditioned medium obtained from cultured cells. The objective of this paper was to develop a rapid method whereby gelatinase activity could be extracted from regional brain tissues without tedious, intervening purification steps. After Triton X-100 extraction and gelatin-Sepharose 4B purification of rat brain tissue extracts, two major activities were observed on gelatin zymograms. These were identified as gelatinase A and B using co-migration with astrocyte-derived enzymes and inhibition of activity by tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). The non-ionic detergents, Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) were equally effective in extracting activities from brain tissue. Little difference in recovery was observed among 0.1, 1 and 10% concentrations of Triton X-100. The method developed here was capable of recovering gelatinase activities from rat brain tissue over a 4-10-fold range using gelatin zymography for the measurement of activity. It is possible that this method may be modified for the measurement of gelatinases in tissues such as biopsy samples of gliomas or astrocytomas or other cancers where gelatinases are thought to play a role in tumor invasion and/or metastasis.

Animals↗

PubMed1996

beta-Amyloid induction of gelatinase B secretion in cultured microglia: inhibition by dexamethasone and indomethacin.

Since administration of anti-inflammatory drugs has been reported to possibly slow the progression of Alzheimer's disease, an effort was made to define effector functions induced by beta-amyloid (A beta) which may be inhibited by these drugs. Rat microglia in culture were treated with various A beta fragments and gelatinase (GLase) activity was determined using gelatin-substrate zymography; the ability of indomethacin (INDO) and dexamethasone (DEX) to inhibit A beta-stimulated GLase, specifically matrix metalloproteinase-9 (MMP-9), activity was also evaluated. A beta (1-38) and A beta (1-40) were effective in stimulating MMP-9 activity in a dose-dependent fashion. Co-treatment with INDO (50 microM) or DEX (100 nM) resulted in a 54% and 66% inhibition, respectively, of A beta (1-40)-stimulated MMP-9 production in microglia.

Amyloid beta-Peptides↗

PubMed1996

Effects of phenobarbital and interleukin-6 on cytochrome P4502B1 and 2B2 in cultured rat hepatocytes.

The objectives of this study were to characterize further the effects of phenobarbital (PB) on cytochrome P4502B1 and 2B2 (P4502B1/2) enzyme activity and immunoreactivity in rat hepatocytes and to investigate the mechanism(s) mediating the ability of interleukin-6 (IL-6) to inhibit this induction. PB caused a concentration-dependent increase in benzyloxyresorufin O-deethylase (BROD) activity with maximal effects (a 25-fold increase) at concentrations of 0.3 to 1 mM. The induction of BROD activity was linear over 24 hr of exposure. Immunoblot profiles of P4502B1/2 agreed with measurements of enzyme activity. In addition to inducing P4502B1/2, PB (0.75 mM) also increased the levels of P450 reductase by approximately 2-fold following a 24-hr exposure to PB. When IL-6 was added concomitantly with or up to 12 hr after the addition of PB, the PB induction of BROD activity and immunoreactivity was inhibited significantly. When 18 hr elapsed between the time of addition of PB and IL-6, the inhibitory effects of IL-6 were no longer apparent, suggesting that the actions of IL-6 were mediated by early events in the induction process. IL-6 did not affect the PB induction of P450 reductase. To determine whether IL-6 altered the degradation of P4502B1/2, hepatocytes were exposed to PB for 24 hr, then washed, and the loss of BROD activity and immunoreactivity following incubation with a protein synthesis inhibitor was measured. IL-6 did not alter the rate of loss of either enzyme activity or immunoreactivity, indicating that the effects of IL-6 could not be attributed to the enhanced degradation of P4502B1/2. Results suggest that the inhibition of PB-induced BROD activity by IL-6 is due to an action on early cellular and molecular events in the induction process.

Animals↗

PubMed1996

Increased production of matrix metalloproteinases in enriched astrocyte and mixed hippocampal cultures treated with beta-amyloid peptides.

Growing evidence supports the notion of a functional relationship between the presence of the beta-amyloid (A beta) peptide and the production of inflammatory mediators in and around neuritic plaques of Alzheimer's disease. Tissue remodeling enzymes that are critical in peripheral inflammatory responses are the matrix metalloproteinases (MMPs), enzymes produced by neurons and glia. Thus, it was of interest to determine whether A beta may alter the expression of MMPs in glial and neuronal cultures. It was demonstrated that A beta (1-40) is a potent stimulator of MMP-9 and MMP-2 activity in addition to inducing the expression of a lower molecular weight, unidentified gelatinase activity in mixed hippocampal and astrocyte cultures. Shorter fragments of A beta were less effective in stimulating the production of these enzymes. The lower molecular weight activity was observed only in response to A beta, and not after treatment with various cytokines. In addition, both cultures express MMP-3 (stromelysin-1) in response to A beta peptides. These results suggest that MMPs may play a role in the development or progression of neuritic plaques, i.e., abnormal neurite outgrowth.

Amyloid beta-Peptides↗

PubMed1995

Increased production of gelatinase B (matrix metalloproteinase-9) and interleukin-6 by activated rat microglia in culture.

Activated macrophages produce several matrix metalloproteinases (MMPs), a family of extracellular matrix (ECM)-degrading enzymes, during wound healing and in other inflammatory states. In response to brain injury, brain microglia become "activated," in a way similar to peripheral tissue macrophages, a process which includes differentiation and probably invasion and proliferation. Little is known about the ECM-degrading MMPs that are secreted by microglia upon activation. Thus, it was of interest to determine whether activated microglia secrete MMPs. Conditioned media samples obtained from cultured microglia that were stimulated with various activating agents were subjected to gelatin-substrate zymography. Microglia constitutively express low levels of a 94-kDa gelatinase (GLase) activity. Treatment with LPS, zymosan, and fixed Staphylococcus aureus for 24 hr stimulated the activity of the 94-kDa GLase, 4-20-fold, in a dose-dependent manner. Addition of INF gamma inhibited the LPS-stimulated activity of MMP-9. LPS, zymosan, and fixed Staphylococcus aureus also stimulated the secretion of IL-6 from microglia in a dose-dependent manner. The 94-kDa GLase activity was Ca++ dependent, it was inhibited by 1,10-phenanthroline, and it was activated by organomercurial compounds. When immunoblots were performed using specific antisera against the 94-kDa gelatinase B (MMP-9) with untreated and LPS-stimulated conditioned medium samples, a 94-kDa immunopositive band was observed. Thus, it appears that the 94-kDa GLase is gelatinase B (MMP-9). These results indicate that activators of peripheral macrophages are potent secretagogues for the MMPs in cultured microglia. The ability of activated microglia to secrete MMPs suggests that these enzymes may play an important function in the brain parenchyma during inflammatory states.

Animals↗

PubMed1995

Differential effect of cytokines on the phenobarbital or 3-methylcholanthrene induction of P450 mediated monooxygenase activity in cultured rat hepatocytes.

Cultured rat hepatocytes have been used to compare the relative activities of cytokines to inhibit the phenobarbital (PB) or 3-methylcholanthrene (MC) induction of cytochrome P4502B1 and 2B2 (P4502B1/2) or P4501A1 and 1A2 (P4501A1/2), respectively. Recombinant cytokines tested were human interleukin-6 (IL-6), interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta, respectively), and rat gamma-interferon (INF gamma). Hepatocytes were cultured in the presence of 2 mM PB or 1 microgram MC/mL culture medium for 24 hr with or without the cytokines. Benzyloxyresorufin and ethoxyresorufin O-dealkylase (BROD and EROD, respectively) activities were determined as indices of P4502B1/2 and P4501A1/2, respectively. All cytokines produced a concentration-dependent inhibition of the PB induction of BROD activity. IL-1 beta and IL-6 were approximately equipotent with IC50 values of 1-2 U/mL, causing greater than 90% inhibition of PB induction of BROD activity at a concentration of 50 U/mL culture medium. IL-1 alpha tended to be less active. PB induction of BROD activity was also inhibited by INF gamma, but higher concentrations (62.5 to 500 U/mL culture medium) were required. All cytokines were less effective in inhibiting the MC induction of EROD activity than the PB induction of BROD activity. IL-1 beta and IL-6, at 50 U/mL culture medium, inhibited EROD induction by only 35% compared with the greater than 90% inhibitory effect on the PB induction of BROD activity. INF gamma was ineffective in inhibiting EROD activity at the concentrations studied. Western immunoblot analysis indicated that the cytokines prevented the ability of the inducers to increase the expression of P4502B1/2 and P4501A1/2 immunoreactive proteins, and this effect correlated with their inhibitory effect on induction of enzyme activity. The results suggest that inducible isoforms of cytochrome P450 differ in their susceptibility to regulation by the cytokines, and that cytokines possess differential activity to inhibit the induction of P450 isoforms, with IL-1 beta and IL-6 being the most effective.

Animals↗

PubMed1995

Cytokines regulate gelatinase A and B (matrix metalloproteinase 2 and 9) activity in cultured rat astrocytes.

Under a tightly regulated expression mechanism, matrix metalloproteinases degrade extracellular matrix proteins and are thought to play a role in injury repair and tumor metastasis in peripheral tissues. Little is known about the function of matrix metalloproteinases or agents that regulate their production in adult brain; however, it has been shown that the activity of a calcium-dependent metalloproteinase is elevated in Alzheimer's hippocampus. The goals of this study were to determine whether cultured rat astrocytes produce matrix metalloproteinases and to identify agents that regulate protease activity. Enriched astrocyte cultures were prepared from brains of 1-day-old rat pups, and experiments were performed 13 days later. Gelatinase activity in astrocyte conditioned medium was determined using zymography with gelatin copolymerized with acrylamide in the gel. Under basal conditions after a 24-h incubation, rat astrocytes produce gelatinases of 58 and 66 kDa. On stimulation of astrocytes with lipopolysaccharide, interleukin-1 alpha or -beta, or tumor necrosis factor-alpha for 24 h, a dose-dependent increase in the activity of the 58- and 66-kDa gelatinases and the induction of a 94-kDa gelatinase occurred. All three astrocyte-derived proteases showed maximal activity in the presence of millimolar levels of Ca2+, their activity was inhibited in the presence of 1,10-phenanthroline, and their proenzymes were cleaved and activated after incubation with p-aminophenylmercuric acetate. Using immunoblotting, immunopositive bands at the respective molecular sizes indicated that the 58-kDa gelatinase was gelatinase A (matrix metalloproteinase 2) and the 94-kDa activity was gelatinase B (matrix metalloproteinase 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Regulation of interleukin-6 (IL-6) secretion in primary cultured rat astrocytes: synergism of interleukin-1 (IL-1) and pituitary adenylate cyclase activating polypeptide (PACAP).

Interleukin-6 (IL-6) is a pleiotropic cytokine that is produced by astrocytes and microglia and may act as a trophic factor in the nervous system. These experiments were intended to identify neuroactive agents that regulate IL-6 production in primary cultured rat astrocytes. Addition of either lipopolysaccharide (LPS) or human recombinant interleukin-1 beta (IL-1 beta) to rat astrocytes in culture stimulated IL-6 secretion. However, LPS was significantly more efficacious in eliciting IL-6 production compared to IL-1 beta. Co-addition of the specific IL-1 receptor antagonist (IL-1ra) completely inhibited IL-1 beta-induced IL-6 secretion but did not affect LPS-stimulated IL-6 production during a 6 h incubation period. Two neuroactive peptides, pituitary adenylate cyclase activating polypeptide (PACAP38) and vasoactive intestinal peptide (VIP), stimulated IL-6 production either alone or in combination with IL-1 beta. PACAP38 was significantly more potent in stimulating IL-6 compared to VIP. Results from these experiments indicate that LPS is an effective inducer of IL-6 production in rat astrocytes. This effect of LPS is independent of astrocyte IL-1 production since the IL-1ra was unable to inhibit LPS-stimulated IL-6 secretion. Also, the neuropeptides PACAP38 and VIP are potential secretagogues for IL-6 secretion, and both peptides synergize with IL-1 to stimulate IL-6 secretion in rat astrocytes.

Adenylyl Cyclases↗

PubMed1993

Infectious disease, interleukin-1 and central nervous system.

Host mediators are responsible for inducing many of the nonspecific, common symptoms of illness. Several of these responses, including the induction of fever, increased levels of glucocorticoid, and increased sleep are mediated by the central nervous system (CNS). Interleukin-1 appears to be an important, but not the only, cytokine involved in activating these processes. Numerous unresolved questions remain regarding cytokine activation of the CNS, such as the neuroanatomical and neurochemical pathways involved in these responses; why these responses vary in magnitude and duration; what are the roles of individual cytokines in inducing these responses during infectious disease.

Acute-Phase Reaction↗

PubMed1992

Increased circulating interleukin-1 and interleukin-6 after intracerebroventricular injection of lipopolysaccharide.

Recently, it was demonstrated that intracerebroventricular (icv) injection of interleukin-1 (IL-1) stimulated circulating interleukin-6 (IL-6) to a greater degree than intravenous (i.v.) injection of IL-1. The goal of this study was to compare the efficacy of lipopolysaccharide (LPS), injected both icv and i.v., on circulating concentrations of IL-1 and IL-6. Both i.v. and icv injection of LPS stimulated plasma levels of IL-1 to a similar degree. However, i.v. injection of LPS was significantly more efficacious than icv injection of LPS in elevating circulating IL-6. These results demonstrate that like i.v. injection of LPS, icv injection of LPS stimulates plasma levels of IL-1 and IL-6. Increases in circulating cytokines during infectious diseases which are limited to the central nervous system may serve to activate peripheral functions of an acute phase response.

Animals↗

PubMed1991

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification.

The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.

Adenylyl Cyclases↗

PubMed1991

Glucocorticoid upregulation of interleukin 1 receptor expression in a glioblastoma cell line.

Interleukin 1 (IL-1) is a multifunctional cytokine produced by numerous cell types, including cells of the central nervous system (CNS). In the CNS, IL-1 produced by glia is thought to support trophic functions after brain injury. However, little is known about whether the IL-1 receptor (IL-1R) is expressed in brain cells and how these receptors might be regulated. Analysis of IL-1R expression in the human glioblastoma cell line U-87 MG indicated the presence of a specific, saturable, and high-affinity (dissociation constant = 104 +/- 14 pM) binding site, which was of moderately high density (1,228 +/- 156 sites/cell). Incubation of U-87 MG cells with cortisol or dexamethasone for as little as 6 h resulted in an upregulation of IL-1R expression, which could be blocked by coincubation with cycloheximide or actinomycin D. IL-1 beta downregulated the expression of its own binding site. Upregulation of the IL-1R by glucocorticoids (GCs) appeared to be coupled to the release of interleukin 6 (IL-6), since IL-1 was significantly more potent in inducing IL-6 release in U-87 cells that were preincubated in the presence of GCs compared with cells incubated in the absence of GCs. These results suggest that IL-1 acts on glial cells via a high-affinity receptor and indicate that GCs may amplify or prolong the actions of IL-1 in the CNS.

Cell Line↗

PubMed1991

Inhibition of mitogen-stimulated proliferation of murine splenocytes by a novel neuropeptide, pituitary adenylate cyclase activating polypeptide: a comparative study with vasoactive intestinal peptide.

Two novel polypeptides known as pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and a shorter form of the peptide corresponding to the N-terminal 27 residues (PACAP27) were isolated from ovine hypothalamus. The N-terminal 28 residues of PACAP show 68% homology with vasoactive intestinal peptide (VIP). VIP has been reported to have specific binding sites in lymphocytes and inhibit mitogen-stimulated lymphocyte proliferation through a receptor-mediated stimulation of cAMP-dependent protein kinase. Using concanavalin A-induced proliferation of murine splenocytes as a model system, we now report that both PACAP38 and PACAP 27 can inhibit the proliferation of these cells in the same dose-dependent manner as VIP. The minimal effective concentration of the PACAPs was 10(-10)-10(-9) M. However, neither PACAP affected lipopolysaccharide-induced proliferation of murine splenocytes. The binding of [125I]PACAP27 to these splenocytes was rapid, time dependent, reversible, and proportional to the numbers of murine splenocytes. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites. The dissociation constant (Kd) was 0.86 +/- 0.24 nM and the maximal binding capacity (Bmax) was 1.13 +/- 0.39 fmol/10(6) cells for the high affinity binding site. The low affinity binding site had a Kd of 0.13 +/- 0.03 microM with a Bmax of 73.5 +/- 9.5 fmol/10(6) cells. PACAP38 and VIP displaced the binding of [125I]PACAP27 in the same manner as PACAP27 and Scatchard analyses indicated the presence of two classes of binding sites with Kd and Bmax similar to those for PACAP27. Furthermore, when [125I]VIP was used as a radiolabeled ligand, PACAP27 and PACAP38 displaced the [125I]VIP binding to the same degree as unlabeled VIP. Scatchard analysis indicated that there was no significant difference of the Kd or Bmax between PACAP and VIP. Taken together, these data suggest that PACAPs bind to a site similar or identical to that used by VIP which inhibit the proliferation of murine splenocytes induced by concanavalin A.

Animals↗