Brain interleukin-1beta and tumor necrosis factor-alpha are involved in lipopolysaccharide-induced delayed rectal allodynia in awake rats

Recently, we have developed a model of delayed (12 h) increase in sensitivity (allodynia) to rectal distension (RD) induced by intraperitoneal lipopolysaccharide (LPS) in awake rats. Thus, we examined whether central interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are involved in LPS response. Abdominal contractions (criterion of visceral pain) were recorded in rats equipped with intramuscular electrodes. RDs were performed at various times after pharmacological treatments. RD induced abdominal contractions from a threshold volume of distension of 0.8 ml. At lowest volume (0.4 ml), this number was significantly increased 12 h after LPS. Intracerebroventricular (i.c.v.) injection of IL-1 receptor antagonist, IL-1beta converting enzyme inhibitor or recombinant human TNF-alpha soluble receptor reduced LPS-induced increase of abdominal contractions at 0.4 ml volume of distension. When injected i.c.v., recombinant human IL-1beta and recombinant bovine TNF-alpha reproduced LPS response at 9 and 12 h and at 6 and 9 h, respectively. These data suggest that IL-1beta and TNF-alpha act centrally to induce delayed rectal hypersensitivity and that central release of these cytokines is responsible of LPS-induced delayed (12 h) rectal allodynia.     

Mechanisms of tumor necrosis factor-α (TNF-α) hyperalgesia

Activation of immune cells by pathogens induces the release of a variety of proinflammatory cytokines, including IL-1β and TNF-α. Previous studies using IL-1β have demonstrated that this cytokine can alter brain function, resulting in a variety of ‘illness responses’ including increased sleep, decreased food intake, fever, etc. We have recently demonstrated that i.p. IL-1β also produces hyperalgesia and that this hyperalgesia (as well as most illness responses) is mediated via activation of subdiaphragmatic vagal afferents. The present series of studies were designed to provide an initial examination of the generality of proinflammatory cytokine-induced hyperalgesia by examining the effects of i.p. TNF-α on pain responsivity. These studies demonstrate that: (a) i.p. TNF-α produces dose-dependent hyperalgesia as measured by the tailflick test, (b) this hyperalgesia is mediated via the induced release of IL-1β, (c) hyperalgesia is mediated via activation of subdiaphragmatic vagal afferents, and (d) the effects of subdiaphragmatic vagotomy cannot be explained by a generalized depression of neural excitability.     

Brain-IL-1β induces local inflammation but systemic anti-inflammatory response through stimulation of both hypothalamic–pituitary–adrenal axis and sympathetic nervous system

It is well established that systemic inflammation induces a counter-regulatory anti-inflammatory response particularly resulting in deactivation of monocytes/macrophages. However, recently we demonstrated a systemic anti-inflammatory response without preceding signs of systemic inflammation in patients with brain injury/surgery and release of cytokines into the cerebrospinal fluid (CSF). In order to analyze the mechanisms and pathways of systemic immunodepression resulting from sterile cerebral inflammation we established an animal model using continuous intra-cerebroventricular (i.c.v.) or intra-hypothalamic (i.h.) infusion of rat recombinant (rr) tumor necrosis factor (TNF)-α and interleukin (IL)-1β for 48 h. Controls received intra-venous (i.v.) cytokine administration. Interestingly, i.c.v. and i.h. infusion of IL-1β but not TNF-α produced distinct signs of central nervous system (CNS) inflammation. Correspondingly, i.c.v. infusion of IL-1β particularly diminished the TNF-α but increased the IL-10 concentration in whole blood cultures after endotoxin stimulation. All parameters normalized within 48 h after termination of the infusion. Blocking the hypothalamic–pituitary–adrenal (HPA) axis by hypophysectomy (HPX) led to complete recovery of the diminished TNF-α concentration and temporarily inhibited the IL-10 increase. Blocking the sympathetic nervous system (SNS) transmission by application of the β₂-adrenoreceptor antagonist propranolol not only inhibited the increase but further downregulated the endotoxin induced IL-10 concentration in the media of whole blood cell cultures, whereas the TNF-α decrease was only partially prevented. Interestingly, HPX and propranolol also diminished the cell invasion into the CSF. In summary, activation of both the HPA axis and the SNS plays an important role in systemic anti-inflammatory response resulting from cytokines in brain and cerebral inflammation.     

The expression of proinflammatory cytokine mRNA in the sciatic–tibial nerve of ischemia–reperfusion injury

We evaluated the proinflammatory cytokines, TNF-α and IL-1β, mRNA expression in the rat sciatic and tibial nerves following ischemia–reperfusion (IR) injury, using competitive RT–PCR, to explore the role of cytokines in IR injury. The expressions of both TNF-α and IL-1β mRNA were related to severity of ischemia and occurred with reperfusion rather than ischemia alone. TNF-α gene expression peaked at 24 h of reperfusion, while that of IL-1β peaked at 12 h. These data support the notion that the proinflammatory cytokines TNF-α and IL-1β are involved in the inflammatory response of IR injury to the peripheral nervous system and may be involved in the pathophysiology of ischemic fiber degeneration.     

Intercellular adhesion molecule-1 expression induced by interleukin (IL)-1β or an IL-1β fragment is blocked by an IL-1 receptor antagonist and a soluble IL-1 receptor

The effects of a recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) and a recombinant human soluble IL-1 receptor (sIL-1R) on cytokine-induced intercellular adhesion molecule-1 (ICAM-1) expression in a human glioblastoma cell line and a neuroblastoma cell line were determined. Cells were incubated with IL-1β, tumor necrosis factor (TNF)α and interferon (IFN)γ. Cells were also tested under identical conditions with an IL-1β synthetic peptide fragment (IL-1β_208–240) previously shown to possess biological activity. IL-1β, TNFα and IFNγ potentiated ICAM-1 expression in both cell lines in a dose-related manner. The IL-1β_208–240 fragments, corresponding to the rabbit, rat and human sequences, enhanced ICAM-1 expression in glioblastoma cells at high doses. ICAM-1 expression induced by IL-1β, rabbit IL-1β_208–240 and human IL-1β_208–240 was blocked by the IL-1ra, while TNFα- and IFNγ-induced ICAM-1 expression were not. ICAM-1 expression induced by IL-1β and human IL-1β_208–240 was also blocked by the sIL-1R. Our findings suggest that IL1β_208–240 acts as an IL-1β agonist in enhancing ICAM-1 expression in vitro and that this effect is receptor-mediated.     

The effect of interferon β-1b on lymphocyte-endothelial cell adhesion

Interferon β-1b (IFNβ-1b) (Betaseron®) has been recently approved forfor treatment of multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). The mechanism of action of IFNβ-1b is not understood, but its effect in reducing gadolinium enhanced MRI lesions suggest an effect at the blood brain barrier (BBB). Thus the objective of this study is to examine the effect of IFNβ-1b treatment of endothelial cells (EC) on lymphocyte-EC adhesion, and on the expression of the adhesion molecules (AM) ICAM-1, VCAM and E-selectin induced by IFN-γ, TNF-α, or IL-1β. Primary cultures of human umbilical vein EC (HUVEC) were used which under basal conditions expressed low levels of ICAM-1 but not VCAM or E-selectin. IFNβ-1b (1–1000 IU/ml) had minimal effect on basal expression of AM on HUVEC, but AM could be substantially upregulated by IFN-γ, IL-1β or TNF-α which was associated with a parallel increase in lymphocyte-EC adhesion. The effect of IFNβ-1b on AM expression induced by IFN-γ, IL-1β or TNF-α was slightly additive, and was associated with a modest increase in lymphocyte-EC adhesion. In contrast TGF-β, shown previously to downregulate lymphocyte-EC adhesion, inhibited this adhesion in our experiments. It is concluded that IFN-β does not downregulate the inducible expression of ICAM-1, VCAM or E-selectin on HUVEC and does not inhibit the adhesion of lymphocytes to HUVEC. These findings have implications on the mechanism of action of IFNβ-1b in MS.     

Transforming growth factor-β1 exhibits delayed gene expression following focal cerebral ischemia

Transforming growth factor-β1 (TGF-β1) is a pleiotropic peptide growth factor. The expression of TGF-β1 mRNA in the focal ischemic cortex of rats was studied by means of Northern hybridization. A moderately low level of constitutively expressed TGF-β1 mRNA was detected following ohm surgery or in the contralateral (nonlschemic) cortex. A significant increase of TGF-β1 mRNA level in the ischemic cortex was observed at 2 days (3.2-fold increase compared to sham-operated animals, p < 0.01, N = 4) following permanent occlusion of the middle cerebral artery (PMCAO). The elevated TGF-μ1 mRNA expression was plateaued for up to 16 days (3–6-fold increase, p < 0.01) following PMCAO- This temporal profile for TGF-β1 mRNA expression in focal stroke was significantly delayed compared to that of TNF-α, IL-1β and IL-6 MRNA expressions as demonstrated previously which peaked at 12 h and decreased to almost basal levels by S days following PMCAO. Interestingly, the TGF-β1 mRNA expression profile was remarkably parallel with that of monocyte/macrophage accumulation in the ischemic cortex, as well as with the increased formation of extracellular matrix in the focal ischemic brain. These data suggest that TGF-β1 may play a role in anti-inflammatory process and in tissue remodeling following ischernic brain injury.     

Interleukin-1β-induced brain injury in the neonatal rat can be ameliorated by α-phenyl-n-tert-butyl-nitrone

To examine the possible role of inflammatory cytokines in mediating perinatal brain injury, we investigated effects of intracerebral injection of interleukin-1beta (IL-1β) on brain injury in the neonatal rat and the mechanisms involved. Intracerebral administration of IL-1β (1 μg/kg) resulted in acute brain injury, as indicated by enlargement of ventricles bilaterally, apoptotic death of oligodendrocytes (OLs) and loss of OL immunoreactivity in the neonatal rat brain. IL-1β also induced axonal and neuronal injury in the cerebral cortex as indicated by elevated expression of β-amyloid precursor protein, short beaded axons and dendrites, and loss of tyrosine hydroxylase-positive neurons in the substantia nigra and the ventral tegmental areas. Administration of α-phenyl-n-tert-butyl-nitrone (PBN, 100 mg/kg i.p.) immediately after the IL-1β injection protected the brain from IL-1β-induced injury. Protection of PBN was linked with the attenuated oxidative stress induced by IL-1β, as indicated by decreased elevation of 8-isoprostane content and by the reduced number of 4-hydroxynonenal or malondialdehyde or nitrotyrosine-positive cells following IL-1β exposure. PBN also attenuated IL-1β-stimulated inflammatory responses as indicated by the reduced activation of microglia. The finding that IL-1β induced perinatal brain injury was very similar to that induced by lipopolysaccharide (LPS), as we previously reported and that PBN was capable to attenuate the injury induced by either LPS or IL-1β suggests that IL-1β may play a critical role in mediating brain injury associated with perinatal infection/inflammation.     

Changes in expressions of proinflammatory cytokines IL-1β, TNF-α and IL-6 in the brain of senescence accelerated mouse (SAM) P8

The senescence-accelerated mouse (SAM) is known to be a murine model for accelerated aging. The SAMP8 strain shows age-related deterioration of learning and memory at an earlier age than control mice (SAMR1). In the present study, we investigated the changes in expressions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the brain of SAMP8. In the hippocampus of 10 months old SAMP8, the expression of IL-1 mRNA was significantly elevated in comparison with that of SAMR1. In both strains of SAMs, increases in IL-1β protein in the brain were observed at 10 months of age compared with 2 and 5 months. The only differences found between the strain in protein levels were at 10 months and were elevations in IL-1β in the hippocampus and hypothalamus, and in TNF-α and IL-6 in the cerebral cortex and the hippocampus in SAMP8 as compared with SAMR1. However, lipopolysaccharide-induced increases in the expression of these cytokines in brain did not differ between SAMP8 and SAMR1. Increases in expression of proinflammatory cytokines in the brain may be involved in the age-related neural dysfunction and/or learning deficiency in SAMP8.     

Interferon β-1a downregulates TNFα-induced intercellular adhesion molecule 1 expression on brain microvascular endothelial cells through a tyrosine kinase-dependent pathway

TNFα (100 U/ml, 24 h) upregulated intercellular adhesion molecule 1 (ICAM1) expression on brain microvascular endothelial cell (BMEC) culture. The tyrosine kinase (TK) inhibitor genestein (100 μg/ml), the protein kinase C (PKC) inhibitor staurosporin (1 nM), and interferon (IF) β-1a (1000 U/ml) antagonized TNFα effect. When an ineffective dose of IFβ-1a (100 U/ml) was challenged with ineffective doses of either genestein (10 μg/ml) or staurosporin (0.1 nM), the combination IFβ-1a–genestein significantly reduced TNFα-induced ICAM1 expression whereas IFβ-1a–staurosporin did not. These findings indicate that a TK- rather than a PKC-dependent mechanism is involved in the modulation of TNFα response by IFβ-1a on BMECs.     

Blockade of angiotensin II AT1 receptors inhibits pressor action of centrally administered interleukin-1β in Sprague Dawley rats

The aim of the present study was to evaluate the influence of intraventricular administration of recombinant rat interleukin-1β (IL-1β) on regulation of resting blood pressure and heart rate and to test the hypothesis that the brain angiotensinergic system is involved in regulation of hemodynamic parameters by centrally applied IL-1β. The experiments were performed on Sprague Dawley rats, assigned to three series of experiments. In series 1 (control, n = 6), mean arterial pressure (MAP) and heart rate (HR) were recorded for 15 min under baseline conditions. This was followed by infusion of saline (0.9% sterile NaCl 5 μL/h) into the left cerebral ventricle (LCV). Measurements were continued during the next 60 min. In series 2 (n = 6) and 3 (n = 6) the experimental design was similar, except that in series 2 the animals were LCV infused with saline containing IL-1β (100 ng/h) and in series 3 with saline containing IL-1β (100 ng/h) and angiotensin type 1 (AT1) receptors antagonist (Losartan, 10 μg/h). LCV infusion of saline alone did not influence MAP and HR while administration of IL-1β elicited significant increase in MAP, but not in HR. The pressor effect was absent during combined infusion of IL-1β and Losartan. Results of the study provide evidence that centrally administered IL-1β exerts pressor effect, and reveal that this effect is mediated by stimulation of the brain angiotensin system and requires activation of AT1 receptors.     

Thermoregulation and hypothalamic histamine turnover modulated by interleukin-1β in rats

To clarify the involvement of hypothalamic histamine in thermogenic response provoked by high ambient temperature, or interleukin-1β (IL-1β), changes in rectal temperature and histamine turnover were investigated. Rectal temperature was maintained normally after exposure to high ambient temperature, but elevated by IL-1β. In spite of these different responses of body temperature, hypothalamic histamine turnover was increased in each treatment. The results suggest that hypothalamic histaminergic neurons are activated not only peripherally by high ambient temperature, but also centrally by IL-1β as endogenous pyrogen.     

In vivo Angiotensin II AT1 receptor blockade selectively inhibits LPS-induced innate immune response and ACTH release in rat pituitary gland

Systemic lipopolysaccharide (LPS) administration induces an innate immune response and stimulates the hypothalamic–pituitary–adrenal axis. We studied Angiotensin II AT₁ receptor participation in the LPS effects with focus on the pituitary gland. LPS (50 μg/kg, i.p.) enhanced, 3 h after administration, gene expression of pituitary CD14 and that of Angiotensin II AT_1A receptors in pituitary and hypothalamic paraventricular nucleus (PVN); stimulated ACTH and corticosterone release; decreased pituitary CRF₁ receptor mRNA and increased all plasma and pituitary pro-inflammatory factors studied.The AT₁ receptor blocker (ARB) candesartan (1 mg/kg/day, s.c. daily for 3 days before LPS) blocked pituitary and PVN AT₁ receptors, inhibited LPS-induced ACTH but not corticosterone secretion and decreased LPS-induced release of TNF-α, IL-1β and IL-6 to the circulation. The ARB reduced LPS-induced pituitary gene expression of IL-6, LIF, iNOS, COX-2 and IκB-α; and prevented LPS-induced increase of nNOS/eNOS activity. The ARB did not affect LPS-induced TNF-α and IL-1β gene expression, IL-6 or IL-1β protein content or LPS-induced decrease of CRF₁ receptors. When administered alone, the ARB increased basal plasma corticosterone levels and basal PGE₂ mRNA in pituitary.Our results demonstrate that the pituitary gland is a target for systemically administered LPS. AT₁ receptor activity is necessary for the complete pituitary response to LPS and is limited to specific pro-inflammatory pathways. There is a complementary and complex influence of the PVN and circulating cytokines on the initial pituitary response to LPS. Our findings support the proposal that ARBs may be considered for the treatment of inflammatory conditions.     

Upregulation and interaction of TNFα and gelatinases A and B in painful peripheral nerve injury

Chronic constriction injury (CCI) to peripheral nerve causes a painful neuropathy in association with a process of axonal degeneration and endoneural remodeling that involves macrophage recruitment and local increase in extracellular proteases and tumor necrosis factor alpha (TNF-α). Cell surface activation of TNF-α from its transmembrane precursor, as well as sequestration of TNF-α receptors II and I, is performed by the zinc-dependent endopeptidase family of matrix metalloproteinases (MMPs). Among TNF-α-converting MMPs, basal lamina degrading gelatinases are thought to play a role in sciatic nerve injury. In the present study, we determined the forms of TNF-α involved in the development of CCI neuropathy in rats, using Western blot analysis, and the temporal correlation of TNF-α and TNFRI protein profiles with gelatinases activity at the site of peripheral nerve injury. We observed two peaks in TNF-α protein during the first week of CCI that correspond to previously reported peaks in painful behavior. We propose that the first peak at 6 h post-CCI is due to the local expression of the cytotoxic transmembrane 26 kDa TNF-α protein released by resident Schwann cells, mast cells and macrophages. This peak in TNF-α protein expression corresponds to an increase in gelatinase B (MMP-9) activity, which is greatly upregulated as early as 3 h following CCI to rat sciatic nerve. The second peak occurs at 5 days post-CCI, and may represent TNF-α protein released by hematogenously recruited macrophages. This peak is marked by the increase in active soluble 17 kDa TNF-α and by gelatinase A (MMP-2) upregulation. These observations suggest that there is a pathogenic role for the TNF-α-converting function of MMP-2 in painful CCI neuropathy. We conclude that severe nerve injury induces MMPs, TNF-α and TNFRI, which interactively control the privileged endoneurial environment and the pathogenesis of the painful neuropathies associated with the macrophage-dependent processes of Wallerian degeneration.