Cerebral vessels express interleukin 1beta after focal cerebral ischemia

Rapid and marked increased levels of expression of interleukin 1beta (IL-1beta) mRNA have been detected in animal models of cerebral ischemia. However, the protein production of IL-1beta and the cellular sources of IL-1beta are largely undefined after cerebral ischemia. In the present study, we have measured the cellular localization of IL-1beta protein in brain tissue from non-ischemic and ischemic mice using immunohistochemistry. Male C57B/6J (n=45) mice were subjected to middle cerebral artery (MCA) occlusion by a clot or a suture. The mice were sacrificed at time points spanning the period from 15 min to 24 h after onset of the MCA occlusion. Non-operated and sham-operated mice were used as control groups. A monoclonal anti-IL-1beta antibody was used to detect IL-1beta. In the non-operated and sham-operated mice, a few IL-1beta immunoreactive cells were detected scattered throughout both hemispheres. IL-1beta immunoreactive cells increased in the ischemic lesion as early as 15 min and peaked at 1 h to 2 h after MCA occlusion. IL-1beta immunoreactivity was detected in the cortex of the contralateral hemisphere 1 h after ischemia. By 24 h after onset of ischemia, IL-1beta immunoreactivity was mainly present adjacent to the ischemic lesion and in the non-ischemic cortex. IL-1beta immunoreactivity was found on endothelial cells and microglia. This study demonstrates an early bilateral expression of IL-1beta on endothelium after MCA occlusion in mice.     

D1 and D2 dopamine receptor function in the striatum: coactivation of D1- and D2-dopamine receptors on separate populations of neurons results in potentiated immediate early gene response in D1-containing neurons

D1- and D2-dopamine receptor-mediated regulation of immediate early gene levels in identified populations of neurons in the striatum was examined with quantitative in situ hybridization histochemical techniques. Levels of messenger RNA (mRNA) encoding the immediate early genes zif268 and c-fos were examined in two experiments in rats with unilateral lesions of the nigrostriatal dopamine pathway. In a dose-response study, animals were treated with doses of 0.5, 1.0, and 1.5 mg/kg of the D1 agonist SKF-38393 either alone or in combination with the D2 agonist quinpirole (1 mg/kg). Levels of immediate early gene mRNAs 60 min following drug treatments showed a dose-related increase to the D1 agonist alone and a potentiation to combined D1 and D2 against treatment. In a second experiment, in animals receiving 1 mg/kg SKF-38393 either alone or in combination with 1 mg/kg quinpirole, the level of zif268 mRNA was measured with a double-labeling method in striatal neurons containing enkephalin mRNA, a marker of D2-containing neurons, and in neurons not containing enkephalin, putative D1-containing neurons. In the dopamine-depleted striatum, D1 agonist treatment alone did not affect enkephalin-positive neurons but significantly elevated zif268 mRNA levels in nearly all enkephalin-negative neurons. Combined D1 and D2 agonist treatment further increased zif268 mRNA levels in this population of enkephalin-negative neurons and decreased zif-268 mRNA levels in enkephalin-positive neurons. These data indicate that the synergistic response to combined D1- and D2-receptor stimulation is mediated by interneuronal interactions involving the activation of D1 and D2 receptors on separate populations of striatal neurons.     

HIV-1 gp120 modulates hypothalamic cytokine mRNAs in vivo: implications to cytokine feedback systems

HIV-1-derived envelope glycoprotein 120 (gp120) may play an important role in HIV-1 neuropathology. Gp120 may act through mediators including proinflammatory cytokines. Here, we investigated the regulation of the IL-1 beta system [IL-1 beta, IL-1 receptor type I (IL-1RI), IL-1 receptor antagonist (IL-1Ra), IL-1 receptor accessory proteins (IL-1R AcP I and II)], TNF-alpha, TGF-alpha, and TGF-beta 1 mRNAs in the hypothalamus of Wistar rats in response to the chronic intracerebroventricular (ICV) microinfusion (via osmotic minipumps) of HIV-1 gp120 (100, 500, and 1000 ng/24 h for 72 h). Gp120 increased IL-1 beta, IL-1Ra, TNF-alpha, and TGF-beta 1 mRNAs. Gp120-induced cytokine mRNA profiles were highly intercorrelated in the same samples. Levels of IL-1RI, IL-1R AcP I and II, and TGF-alpha did not change significantly, and levels of GAPDH mRNA were constant. The data suggest potential cytokine-cytokine interactions with positive (IL-1 beta<-->TNF-alpha) and negative (IL-1Ra-->IL-1 beta; TGF-beta 1-->IL-1 beta/TNF-alpha) feedback in gp120 action. A dysregulation of the balance between stimulatory and inhibitory cytokine mechanisms may participate in the initiation, propagation, and/or aggravation of HIV-1 neuropathology.     

Systemic liberation of interleukin-1 beta and interleukin-1 receptor antagonist in the perioperative phase of liver transplantation

We measured systemic serum levels of interleukin-1 receptor antagonist (IL-1ra), interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) during the preoperative, anhepatic, and postreperfusional phases up to the 7th postoperative day in 60 patients undergoing orthotopic liver transplantation (LTx). In contrast to IL-1 beta, IL-1ra, TNF-alpha, and IL-6 showed a significant elevation in relation to the early phase after reperfusion, while TNF-alpha displayed a high grade of scatter. In addition, IL-1ra levels were significantly elevated during the anhepatic phase. Maximum serum levels were found at 15 min after reperfusion, 120 min after reperfusion, and on the 1st postoperative day, respectively. Serum levels decreased considerably at 24 h and 7 days after reperfusion. The comparative monitoring of systemic cytokine and cytokine antagonist levels, in particular the liberation of IL-1ra and IL-6 may provide useful parameters for the development of new liver preservation theories for LTx.     

Olfactory memory and maternal behaviour-induced changes in c-fos and zif/268 mRNA expression in the sheep brain

In sheep maternal behaviour and the formation of the selective olfactory, ewe/lamb bond are induced by feedback to the brain from stimulation of the vagina and cervix during parturition. In the present study, we have used in situ hybridization histochemistry to quantify changes in cellular expression of two immediately-early genes, c-fos and zif/268, in order to identify activated brain regions during the induction of maternal behaviour and olfactory bonding as well as regions where plastic changes are occurring during with the formation of the olfactory memory associated with bonding. Three different treatment groups were used. One group gave birth normally, became maternal and were allowed to interact with their lambs for 30 min. A second group received exogenous treatment with oestradiol and progesterone to induce lactation and then received a 5-min period of artificial stimulation of the vagina and cervix (VCS) which reliably induces maternal behaviour but could not interact with lambs. A final control group received exogenous hormone treatment but no VCS or interaction with lambs. Compared to the control group, post-partum animals and animals that had received VCS showed increased c-fos expression in a number of cortical regions (cingulate, entorhinal and somatosensory), the mediodorsal thalamic nucleus and the lateral habenula, the limbic system (bed nucleus of the stria terminalis, lateral septum, medial arnygdala, dentate gyrus and the CA3 region of the hippocampus) and the hypothalamus (medial preoptic area, mediobasal hypothalamus, paraventricular nucleus, supraoptic nucleus and periventricular complex). The group that gave birth and had contact with their lambs for 30 min had significantly enhanced c-fos mRNA expression in the cingulate cortex compared to those receiving VCS and additionally showed significantly increased c-fos mRNA expression in olfactory processing regions (olfactory bulb, piriform cortex and orbitofrontal cortex). Expression of zif/268 was significantly increased in the entorhinal cortex, orbitofrontal cortex and dentate gyrus of the parturition group compared to either the control or the VCS alone groups. These results show a clear differentiation between neural substrates controlling the expression of maternal behaviour and those involved in the olfactory memory process associated with selective recognition of offspring although at the level of the hippocampus and cingulate cortex there may be some degree of overlap. Alterations in zif/268 at tertiary processing sites for olfactory information (orbitofrontal cortex) and the entorhinal cortex and dentate gyrus may reflect plastic changes occurring during the early stages of olfactory memory formation.     

Localization of mRNA for inflammatory cytokines in radicular cyst tissue by in situ hybridization, and induction of inflammatory cytokines by human gingival fibroblasts in response to radicular cyst contents

The expression of mRNA encoding the inflammatory cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha(TNF-alpha) have been examined in radicular cysts by in situ hybridization. Furthermore, the biological activity of the contents of radicular cysts (RCC) has been assayed by adding extracts of RCC to cultured human gingival fibroblasts (HGFs) and analyzing the culture medium for the release of inflammatory cytokines. In the epithelial layer, keratinocytes expressed all cytokine mRNAs examined at various levels. Basal layer cells expressed mRNA for each cytokine. In the subepithelial granulation tissue of the cysts, fibroblasts and macrophages expressed mRNA for IL-6, IL-8, IL-1beta and TNF-alpha mRNA at varying levels; especially clear expression of TNF-alpha and IL-1beta mRNA was detected on macrophages. The infiltrating lymphoid cells, largely composed of T cells and plasma cells, expressed these cytokine mRNAs, especially those encoding IL-6 and IL-8, at various levels. In vitro analysis indicated dose-dependent release of both IL-6 and IL-8 by HGFs in response to RCC. After heating to 100 degrees C for 10 min, RCC almost completely failed to stimulate IL-6 release from HGFs. Furthermore, anti-IL-1beta antibody (neutralization test) did not prevent the stimulation of IL-6 release by RCC. Significant amounts of IL-6 and IL-8 were detected in RCC in two cases, and a trace amount of IL-1beta was detected in one case. This study demonstrated the wide expression of mRNA encoding inflammatory cytokines in radicular cyst tissues, and RCC itself was capable of stimulating IL-6 and IL-8 production from HGFs.     

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.     

Prolongation and enhancement of postischemic c-fos expression after fasting

BACKGROUND AND PURPOSE: A rapid but transient expression of c-fos after cerebral ischemia has been extensively documented. However, the mechanism of this induction and whether induction of c-fos is neuroprotective or detrimental to the brain after ischemia is presently not clear. Fasting before transient cerebral ischemia has been shown to reduce delayed neuronal necrosis and infarct volume. The purpose of the present study was to examine the effect of preischemic fasting for 24 hours on the expression of c-fos after transient focal cerebral ischemia. METHODS: Focal cerebral ischemia was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries for 60 minutes. Male Long-Evans rats weighting 250 to 300 g were randomly divided into two groups: fed (control group) and food deprived for 24 hours (fasted group) before ischemic surgery. Infarct volumes were measured on the basis of triphenyltetrazolium chloride-delineated infarct areas, and plasma glucose levels were determined by the glucose oxidase method. Temporal and spatial expression of c-fos was assessed by Northern blot analysis, in situ hybridization, and immunohistochemistry. RESULTS: Fasting for 24 hours before 60 minutes of ischemia resulted in a 26.6% decrease in preischemic plasma glucose levels and a 74.5% reduction in infarct volumes in the fasted group compared with the control group. A rapid but transient induction of c-fos mRNA was observed in the ischemic cortex in control animals after 60 minutes of ischemia. Fasting not only prolonged but also enhanced the intensity of c-fos expression in the ischemic cortex. Regional c-fos expression was also different between these two groups. CONCLUSIONS: The results support the contention that c-fos expression may be compatible with its purported neuroprotective role in selected experimental paradigms. The signaling mechanisms underlying the effect of fasting and subsequent lowering of plasma glucose levels on postischemic c-fos expression remain to be explored.     

Enhanced IL-1 beta and tumor necrosis factor-alpha release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure

Idiopathic pulmonary fibrosis (IPF) and asbestosis are fibrotic interstitial lung diseases characterized by alveolar wall fibrosis with accumulation of extracellular matrix, interstitial remodeling, and increased numbers of activated alveolar macrophages. Animal models and in vitro studies have shown that macrophage cytokines, namely IL-1 beta and TNF-alpha, play significant roles in the development of fibrosis. We found significant increases for TNF-alpha release in both diseases (p < 0.01) and a significant increase for IL-1 beta release in asbestosis compared to normal controls (p < 0.01). Also, the mRNA expression of these cytokines was increased in alveolar macrophages from patients with IPF or asbestosis compared with normals. The level of TNF-alpha release in macrophage supernatants correlated with the number of neutrophils per milliliter bronchoalveolar lavage fluid returned. Chrysotile, crocidolite, amosite asbestos, and silica stimulated IL-1 beta and TNF-alpha release and up-regulated their respective mRNA in macrophages or monocytes. To evaluate the role of IL-1 beta and TNF-alpha in the accumulation of extracellular matrix, we studied collagen types I and III and fibronectin gene expression in human diploid lung fibroblasts after short term (2 h) serum-free exposure to recombinant cytokines. Both cytokines up-regulated these genes 1.5- to 3.6-fold. These cytokines have the potential to influence the remodeling and fibrosis observed in the lower respiratory tract in IPF and asbestosis.     

Cytokine patterns in tuberculous and sarcoid granulomas: correlations with histopathologic features of the granulomatous response

Cytokines play an important role in granuloma formation, but the extent that cytokine profiles are similar in different granulomatous diseases and whether differences in the histopathologic features of the granulomatous response results from differences in cytokine production have not been evaluated. To investigate these questions, we used RT-PCR to quantify the expression of mRNAs coding for 16 cytokines in granulomatous lymph nodes from patients with tuberculosis and sarcoidosis and from control tissues, and we sought correlations between the level of expression of these cytokines and the histopathologic features of the granulomas. Expression of mRNAs coding for a number of cytokines (IL-1beta, IFN-gamma, TNF-alpha, granulocyte-macrophage (GM)-CSF, IL-12 (p40), and lymphotoxin-beta) was increased in tuberculous and sarcoid granulomas compared with that of control tissues. All sarcoid granulomas were shown to express a Th1 pattern of cytokine mRNAs, while tuberculous lymph nodes expressed either a Th1 or a Th0 profile. GM-CSF and lymphotoxin-beta mRNAs were more abundant in sarcoid than in tuberculous granulomas, whereas IL-8 mRNA was strongly expressed only in tuberculous lymph nodes. Strong expression of GM-CSF, TNF-alpha, and IL-8 by granulomas was shown to be correlated, respectively, with the presence of florid granulomatous lesions, the absence of central necrosis, and the presence of neutrophil infiltration. These results demonstrate that the formation of tuberculous and sarcoid granulomas in humans is associated with the expression of characteristic cytokine profiles and indicate that the expression of certain cytokines is associated with the development of specific pathologic features in the resulting granulomas.     

Serotonin depletion exacerbates changes in striatal gene expression following quinolinic acid injection

Controversy exists as to whether serotonin (5-HT) plays a neuroprotective role during brain injury. We sought to determine if prior 5-HT depletion alters gene expression patterns normally associated with NMDA receptor-mediated excitotoxicity of the rodent striatum. Adult male Sprague-Dawley rats were treated systemically with saline or p-chlorophenylalanine (pCPA, 350 mg/kg) to block 5-HT synthesis. After 3 days, these rats received unilateral injection (1 microliter) of quinolinic acid (QA, 40 micrograms in 0.1 M phosphate buffered saline, pH 7.4) or saline vehicle directly into the anterior striatum. All rats were sacrificed 6 or 48 h later. Striatal tissues containing the saline or QA injection site were subjected to Northern analysis of preprotachykinin (PPT), preproenkephalin (PPE), and zif/268 mRNAs, as well as HPLC-EC detection of monoamines. At the time of the intrastriatal injection, 5-HT levels were depleted greater than 95% by pCPA as compared to saline controls. At 48 h post-QA injection, PPT and PPE mRNAs were markedly reduced within the striatal lesion site of saline/QA and pCPA/QA groups with respect to their contralateral uninjected control sides. In the pCPA/QA group, striatal PPE and PPT mRNA levels were further reduced as compared to the saline/QA group with PPE mRNA reductions reaching statistical significance at 95% (ANOVA with Scheffe F-test). Exacerbation of the excitotoxic lesion in the 5-HT depleted rat was further exemplified by a larger increase in zif/268 mRNA measured at 6 h post-intrastriatal injection in the pCPA/QA group as compared to saline/QA animals (P < 0.05 by ANOVA with Scheffe F-test). These results suggest that 5-HT depletion may adversely affect neuronal survival following intrastriatal QA exposure and lend support to the hypothesis that increasing 5-HT levels during NMDA receptor-mediated excitotoxicity may spare neurons destined to degenerate.