Stephen Poole

Interleukin-1β-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity

Inflammation causes the induction of cyclooxygenase-2 (Cox-2), leading to the release of prostanoids, which sensitize peripheral nociceptor terminals and produce localized pain hypersensitivity. Peripheral inflammation also generates pain hypersensitivity in neighbouring uninjured tissue (secondary hyperalgesia), because of increased neuronal excitability in the spinal cord (central sensitization), and a syndrome comprising diffuse muscle and joint pain, fever, lethargy and anorexia. Here we show that Cox-2 may be involved in these central nervous system (CNS) responses, by finding a widespread induction of Cox-2 expression in spinal cord neurons and in other regions of the CNS, elevating prostaglandin E2 (PGE2) levels in the cerebrospinal fluid. The major inducer of central Cox-2 upregulation is interleukin-1β in the CNS, and as basal phospholipase A2 activity in the CNS does not change with peripheral inflammation, Cox-2 levels must regulate central prostanoid production. Intraspinal administration of an interleukin-converting enzyme or Cox-2 inhibitor decreases inflammation-induced central PGE2 levels and mechanical hyperalgesia. Thus, preventing central prostanoid production by inhibiting the interleukin-1β-mediated induction of Cox-2 in neurons or by inhibiting central Cox-2 activity reduces centrally generated inflammatory pain hypersensitivity.

Spinal Glia and Proinflammatory Cytokines Mediate Mirror-Image Neuropathic Pain in Rats

Mirror-image allodynia is a mysterious phenomenon that occurs in association with many clinical pain syndromes. Allodynia refers to pain in response to light touch/pressure stimuli, which normally are perceived as innocuous. Mirror-image allodynia arises from the healthy body region contralateral to the actual site of trauma/inflammation. Virtually nothing is known about the mechanisms underlying such pain. A recently developed animal model of inflammatory neuropathy reliably produces mirror-image allodynia, thus allowing this pain phenomenon to be analyzed. In this sciatic inflammatory neuropathy (SIN) model, decreased response threshold to tactile stimuli (mechanical allodynia) develops in rats after microinjection of immune activators around one healthy sciatic nerve at mid-thigh level. Low level immune activation produces unilateral allodynia ipsilateral to the site of sciatic inflammation; more intense immune activation produces bilateral (ipsilateral + mirror image) allodynia. The present studies demonstrate that both ipsilateral and mirror-image SIN-induced allodynias are (1) reversed by intrathecal (peri-spinal) delivery of fluorocitrate, a glial metabolic inhibitor; (2) prevented and reversed by intrathecal CNI-1493, an inhibitor of p38 mitogen-activated kinases implicated in proinflammatory cytokine production and signaling; and (3) prevented or reversed by intrathecal proinflammatory cytokine antagonists specific for interleukin-1, tumor necrosis factor, or interleukin-6. Reversal of ipsilateral and mirror-image allodynias was rapid and complete even when SIN was maintained constantly for 2 weeks before proinflammatory cytokine antagonist administration. These results provide the first evidence that ipsilateral and mirror-image inflammatory neuropathy pain are created both acutely and chronically through glial and proinflammatory cytokine actions.

Interleukin-1β as a potent hyperalgesic agent antagonized by a tripeptide analogue

Interleukin-1 (IL-1) describes two inflammatory proteins1, IL-1α and IL-1β, produced by activated macrophages and other cell types2 and encoded by two genes. Their amino acid sequences have only 26% similarity1, but their biological activities are comparable, with a few exceptions3,4; indeed, both molecules appear to act at the same receptor5,6. As IL-1 releases prostaglandins7 which sensitize nociceptors in man and in experimental animals8, we tested IL-1α and IL-1β in rats for hyperalgesic (nociceptive) activity. Our results show that IL-1β given systemically is an extremely potent hyperalgesic agent with a probable peripheral site of action; IL-1α is ∼3,000 times less active than IL-1β. We have delineated the region of IL-1β mediating the hyperalgesic effect and developed an analgesic tripeptide analogue of IL-1β which antagonizes hyperalgesia evoked by IL-1β and by the inflammatory agent carrageenan.

The role of cytokines in mediating effects of prenatal infection on the fetus: implications for schizophrenia

Maternal infections with bacterial or viral agents during pregnancy are associated with an increased incidence of schizophrenia in the offspring at adulthood although little is known about the mechanism by which maternal infection might affect fetal neurodevelopment. Exposure of pregnant rodents to the bacterial endotoxin, lipopolysaccharide (LPS), results in behavioral deficits in the adult offspring that are relevant to schizophrenia. It is however unknown whether these effects are due to the direct action of the inflammatory stimulus on the developing fetus, or due to secondary immune mediators (cytokines) activated at maternal/fetal sites. In this study we sought to elucidate the site of action of LPS, following a single intraperitoneal (i.p.) injection, in pregnant rats at gestation day 18. Animals received 5 μCi of iodinated LPS (125I-LPS) and its distribution was assessed in maternal/fetal tissues (1–8 h). In addition, induction of the inflammatory cytokines, TNF-α, IL-1β and IL-6, was measured in maternal/fetal tissues following maternal LPS challenge (0.05 mg/kg, i.p.) (2–8 h). 125I-LPS was detected in maternal tissues and placenta, but not the fetus. This distribution was accompanied by significant increases in TNF-α, IL-1β and IL-6 in maternal plasma and placenta, but not in fetal liver or brain. A significant increase in IL-1β was however detected in fetal plasma, possibly due to transfer from the maternal circulation or placenta. Collectively, these data suggest that effects of maternal LPS exposure on the developing fetal brain are not mediated by the direct action of LPS, but via indirect actions at the level of the maternal circulation or placenta.

“Cytokine Storm” in the Phase I Trial of Monoclonal Antibody TGN1412: Better Understanding the Causes to Improve PreClinical Testing of Immunotherapeutics

The CD28-specific mAb TGN1412 rapidly caused a life-threatening “cytokine storm” in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.

Proinflammatory cytokines oppose opioid-induced acute and chronic analgesia

Spinal proinflammatory cytokines are powerful pain-enhancing signals that contribute to pain following peripheral nerve injury (neuropathic pain). Recently, one proinflammatory cytokine, interleukin-1, was also implicated in the loss of analgesia upon repeated morphine exposure (tolerance). In contrast to prior literature, we demonstrate that the action of several spinal proinflammatory cytokines oppose systemic and intrathecal opioid analgesia, causing reduced pain suppression. In vitro morphine exposure of lumbar dorsal spinal cord caused significant increases in proinflammatory cytokine and chemokine release. Opposition of analgesia by proinflammatory cytokines is rapid, occurring < or =5 min after intrathecal (perispinal) opioid administration. We document that opposition of analgesia by proinflammatory cytokines cannot be accounted for by an alteration in spinal morphine concentrations. The acute anti-analgesic effects of proinflammatory cytokines occur in a p38 mitogen-activated protein kinase and nitric oxide dependent fashion. Chronic intrathecal morphine or methadone significantly increased spinal glial activation (toll-like receptor 4 mRNA and protein) and the expression of multiple chemokines and cytokines, combined with development of analgesic tolerance and pain enhancement (hyperalgesia, allodynia). Statistical analysis demonstrated that a cluster of cytokines and chemokines was linked with pain-related behavioral changes. Moreover, blockade of spinal proinflammatory cytokines during a stringent morphine regimen previously associated with altered neuronal function also attenuated enhanced pain, supportive that proinflammatory cytokines are importantly involved in tolerance induced by such regimens. These data implicate multiple opioid-induced spinal proinflammatory cytokines in opposing both acute and chronic opioid analgesia, and provide a novel mechanism for the opposition of acute opioid analgesia.

Circulating interleukin-6 mediates the febrile response to localised inflammation in rats

1 1 Interleukin (IL)‐6 is an important mediator of the host response to disease and has been proposed, largely based upon circumstantial evidence, as the principal endogenous circulating pyrogen responsible for activating CNS mechanisms in fever during infection and inflammation. In the present investigation, we studied the role of peripheral IL‐6 in fever and its relationship with IL‐1, itself an important endogenous pyrogen and a potent stimulus of IL‐6 production. 2 2 Injection of lipopolysaccharide (LPS) into a sterile, subcutaneous air pouch (i.po.) in rats evoked an increase in body temperature which peaked at 3 h, and which was abolished in animals pretreated (intraperitoneally) with IL‐6 antiserum. 3 3 The increase in body temperature was accompanied by a significant elevation in concentrations of (immunoreactive) IL‐1 and IL‐6 at the site of inflammation (pouch), but only IL‐6 in the circulation and cerebrospinal fluids. We propose that much of the circulating IL‐6 originates at the site of inflammation, since injection of human recombinant (hr)IL‐6 (i.po.) was detected (10 min after the injection) in the plasma using an ELISA specific for human IL‐6. 4 4 However, despite the relatively high concentration of IL‐6 injected (25 μg kg−1, i.po.), this cytokine had no effect on body temperature when injected alone, but did induce fever when co‐injected with a non‐pyrogenic dose (when given alone) of IL‐1β, and exacerbated the fever to a pyrogenic dose of IL‐1β. 5 5 The results from the present study demonstrate that IL‐6 is a circulating endogenous pyrogen during LPS‐induced fever, which acts in concert with IL‐1β at the local site of inflammation, before entering the circulation. Circulating IL‐6 can then activate CNS mechanisms resulting in the development of the febrile response during disease.

Crucial role of neutrophils in the development of mechanical inflammatory hypernociception

Neutrophil migration is responsible for tissue damage observed in inflammatory diseases. Neutrophils are also implicated in inflammatory nociception, but mechanisms of their participation have not been elucidated. In the present study, we addressed these mechanisms in the carrageenan‐induced mechanical hypernociception, which was determined using a modification of the Randall‐Sellito test in rats. Neutrophil accumulation into the plantar tissue was determined by the contents of myeloperoxidase activity, whereas cytokines and PGE2 levels were measured by ELISA and radioimmunoassay, respectively. The pretreatment of rats with fucoidin (a leukocyte adhesion inhibitor) inhibited carrageenan‐induced hypernociception in a dose‐ and time‐dependent manner. Inhibition of hypernociception by fucoidin was associated with prevention of neutrophil recruitment, as it did not inhibit the hypernociception induced by the direct‐acting hypernociceptive mediators, PGE2 and dopamine, which cause hypernociception, independent of neutrophils. Fucoidin had no effect on carrageenan‐induced TNF‐α, IL‐1β, and cytokine‐induced neutrophil chemoattractant 1 (CINC‐1)/CXCL1 production, suggesting that neutrophils were not the source of hypernociceptive cytokines. Conversely, hypernociception and neutrophil migration induced by TNF‐α, IL‐1β, and CINC‐1/CXCL1 was inhibited by fucoidin, suggesting that neutrophils are involved in the production of direct‐acting hypernociceptive mediators. Indeed, neutrophils stimulated in vitro with IL‐1β produced PGE2, and IL‐1β‐induced PGE2 production in the rat paw was inhibited by the pretreatment with fucoidin. In conclusion, during the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, at least by mediating the release of direct‐acting hypernociceptive mediators, such as PGE2. Therefore, the blockade of neutrophil migration could be a target to development of new analgesic drugs.

Interleukin-10, interleukin-4, and transforming growth factor-β differentially regulate lipopolysaccharide-induced production of pro-inflammatory cytokines and nitric oxide in co-cultures of rat astroglial and microglial cells

The pro‐inflammatory cytokines interleukin‐1β (IL‐1β), IL‐6, tumor necrosis factor‐α (TNF‐α), and nitric oxide (NO) can be produced by activated glial cells and play a critical role in various neurological diseases. Using primary co‐cultures of rat microglial and astroglial cells, we investigated the effects of the anti‐inflammatory cytokines transforming growth factor‐β1 (TGF‐β1)/β2, IL‐4, and IL‐10 on the production of (pro‐) inflammatory mediators after stimulation of the cells with lipopolysaccharide (LPS; 0.1 μg/ml, 24 h). IL‐10 (10 and 100 ng/ml) and IL‐4 (5 and 50 U/ml) suppressed the LPS‐induced production of NO, IL‐6, and TNF‐α in a dose‐dependent manner, whereas TGF‐β1/β2 (2 and 20 ng/ml) only suppressed NO production. LPS‐induced levels of IL‐1β were suppressed by IL‐10, but not by IL‐4 and TGF‐β1/β2. Conversely, co‐incubation of the glial cells with LPS and antibodies to TGF‐β1/β2 selectively enhanced LPS‐induced NO production, whereas co‐incubation with antibody to IL‐10 enhanced LPS‐induced production of all pro‐inflammatory cytokines and NO. This finding strongly suggests that effective concentrations of TGF‐β1/β2 and IL‐10 are produced by LPS‐stimulated glial cell co‐cultures. Production of IL‐10 in these co‐cultures was confirmed by measurement of rat IL‐10 by radioimmunoassay. We conclude that anti‐inflammatory cytokines affect the production of inflammatory mediators in LPS‐activated co‐cultures of microglial and astroglial cells differentially. GLIA 30:134–142, 2000.

A simple sensitive bioassay for interleukin-1 which is unresponsive to 103 U/ml of interleukin-2

A subclone, NOB-1, of the mouse EL-4 line constitutively produces very little interleukin-2 but in response to interleukin-1 produces high concentrations of interleukin-2. Co-stimulation with mitogen, phorbol esters or calcium ionophores was not required. NOB-1 is not responsive to tumour necrosis factor alpha, tumour necrosis factor beta, interferon gamma and lipopolysaccharide. The NOB-1 line was used in conjunction with a CTLL line to detect less than 1 pg/ml interleukin-1. Rapid assay was performed by co-culturing the EL-4 cells with CTLL cells. By incorporating a pre-incubation step, followed by thorough washing of the EL-4 cells, responses to interleukin-1 were maintained, but interleukin-2 had no effect. The assay was used to detect interleukin-1 in serum samples and to evaluate neutralizing antisera to interleukin-1.