Jitske Zijlstra

β2-Adrenergic activation enhances interleukin-8 production by human monocytes

Interleukin-8 (IL-8) is an important cytokine in inflammatory processes by functioning as a chemoattractant and as an activator of oxygen metabolism. In the present study we demonstrate that β2-adrenergic agonists potentiate the lipopolysaccharide (LPS) and IL-1 induced production of IL-8 by human monocytes. In addition, β2-adrenergic activation enhances IL-8 release and mRNA expression for IL-8 in the human monocytic cell line U937. β2-adrenergic activation of these cells also results in enhanced production of the anti-inflammatory cytokine IL-10. However, IL-10 is not involved in the regulation of IL-8 production. The effect of the β2-adrenergic agonist on IL-8 production is presumably mediated via increased cAMP formation, since it can be mimicked by the cAMP analogue dibutyryl-cAMP (db-cAMP). We conclude that enhancement of IL-8 production is one of the pathways via which β2-adrenergic agonists such as catecholamines can influence inflammatory responses.

MicroRNA-124 as a novel treatment for persistent hyperalgesia

BackgroundChronic pain is often associated with microglia activation in the spinal cord. We recently showed that microglial levels of the kinase G protein–coupled receptor kinase (GRK)2 are reduced in models of chronic pain. We also found that mice with a cell-specific reduction of around 50% in GRK2 level in microglia/macrophages (LysM-GRK2+/− mice) develop prolonged inflammatory hyperalgesia concomitantly with ongoing spinal microglia/macrophage activation. The microRNA miR-124 is thought to keep microglia/macrophages in brain and spinal cord in a quiescent state. In the present study, we investigated the contribution of miR-124 to regulation of hyperalgesia and microglia/macrophage activation in GRK2-deficient mice. In addition, we investigated the effect of miR-124 on chronic inflammatory and neuropathic pain in wild-type (WT) mice.MethodsHyperalgesia was induced by intraplantar IL-1β in WT and LysM-GRK2+/− mice. We determined spinal cord microglia/macrophage miR-124 expression and levels of pro-inflammatory M1 and anti-inflammatory M2 activation markers. The effect of intrathecal miR-124 treatment on IL-1β-induced hyperalgesia and spinal M1/M2 phenotype, and on carrageenan-induced and spared nerve injury-induced chronic hyperalgesia in WT mice was analyzed.ResultsTransition from acute to persistent hyperalgesia in LysM-GRK2+/− mice is associated with reduced spinal cord microglia miR-124 levels. In our LysM-GRK2+/− mice, there was a switch towards a pro-inflammatory M1 phenotype together with increased pro-inflammatory cytokine production. Intrathecal administration of miR-124 completely prevented the transition to persistent pain in response to IL-1β in LysM-GRK2+/− mice. The miR-124 treatment also normalized expression of spinal M1/M2 markers of LysM-GRK2+/− mice. Moreover, intrathecal miR-124 treatment reversed the persistent hyperalgesia induced by carrageenan in WT mice and prevented development of mechanical allodynia in the spared nerve injury model of chronic neuropathic pain in WT mice.ConclusionsWe present the first evidence that intrathecal miR-124 treatment can be used to prevent and treat persistent inflammatory and neuropathic pain. In addition, we show for the first time that persistent hyperalgesia in GRK2-deficient mice is associated with an increased ratio of M1/M2 type markers in spinal cord microglia/macrophages, which is restored by miR-124 treatment. We propose that intrathecal miR-124 treatment might be a powerful novel treatment for pathological chronic pain with persistent microglia activation.

Targeting the p53 pathway to protect the neonatal ischemic brain

To investigate whether inhibition of mitochondrial p53 association using pifithrin‐μ (PFT‐μ) represents a potential novel neuroprotective strategy to combat perinatal hypoxic‐ischemic (HI) brain damage.

IL-1β signaling is required for mechanical allodynia induced by nerve injury and for the ensuing reduction in spinal cord neuronal GRK2

Many neurotransmitters involved in pain perception transmit signals via G protein-coupled receptors (GPCRs). GPCR kinase 2 (GRK2) regulates agonist-induced desensitization and signaling of multiple GPCRs and interacts with downstream molecules with consequences for signaling. In general, low GRK2 levels are associated with increased responses to agonist stimulation of GPCRs. Recently, we reported that in mice with reduced GRK2 levels, inflammation-induced mechanical allodynia was increased. In addition, mice with impaired interleukin (IL)-1 beta signaling did not develop mechanical allodynia after L5 spinal nerve transection (SNT). We hypothesized that in the L5 SNT model mechanical allodynia would be associated with reduced neuronal GRK2 levels in the spinal cord dorsal horn and that IL-1 beta signaling would be required to induce both the decrease in GRK2 and mechanical allodynia. We show here that in wild type (WT) mice L5 SNT induces a bilateral decrease in neuronal GRK2 expression in the lumbar spinal cord dorsal horn, 1 and 2 weeks after L5 SNT. No changes in GRK2 were observed in the thoracic segments. Moreover, spinal cord GRK2 expression was not decreased in IL-1R(-/-) mice after L5 SNT. These data show that IL-1 beta signaling is not only required for the development of mechanical allodynia, but also to reduce neuronal GRK2 expression. These results suggest a functional relation between the L5 SNT-induced IL-1 beta-mediated decrease in GRK2 and development of mechanical allodynia.

Role of endogenous pro-enkephalin A-derived peptides in human T cell proliferation and monocyte IL-6 production

In this paper, we describe that met-enkephalin and/or enkephalin-containing intermediary peptides of the prohormone pro-enkephalin A are produced and secreted by human peripheral blood T cells and monocytes. The peptides are produced after stimulation with the mitogenic monoclonal antibodies anti-CD2.1/2.2 and anti-CD28. In monocytes, enkephalin synthesis was induced by stimulation with lipopolysaccharide. We demonstrate here that these immune cell-derived enkephalins play an important regulatory role in the immune response. By using an anti-sense oligonucleotide strategy we could block the production of enkephalins. Blockade of the production of met-enkephalin and enkephalin-containing intermediary peptides resulted in enhancement of the proliferative T cell response and inhibition of monocyte IL-6 secretion. In vitro reconstitution of the anti-sense treated cultures with synthetic met-enkephalin or the delta-type specific opioid receptor agonist deltorphin could reverse inhibition of monocyte IL-6 production, suggesting that endogenous enkephalins act via membrane opioid receptors. In contrast, addition of met-enkephalin or deltorphin to the anti-sense treated T cell cultures did not have any effect on T cell proliferation.

Mitochondrial JNK phosphorylation as a novel therapeutic target to inhibit neuroinflammation and apoptosis after neonatal ischemic brain damage

Neonatal encephalopathy is associated with high mortality and life-long developmental consequences. Therapeutic options are very limited. We assessed the effects of D-JNKi, a small peptide c-Jun N-terminal kinase (JNK) MAP kinase inhibitor, on neuroinflammation, mitochondrial integrity and neuronal damage in a neonatal rat model of ischemic brain damage. Hypoxic-ischemic (HI) brain injury was induced in postnatal-day 7 rats by unilateral carotid artery occlusion and hypoxia, and was followed by intraperitoneal D-JNKi treatment. We demonstrate here for the first time that a single intraperitoneal injection with D-JNKi directly after HI strongly reduces neonatal brain damage by >85% with a therapeutic window of at least 6h. D-JNKi treatment also restored cognitive and motor function as analyzed at 9weeks post-insult. Neuroprotective D-JNKi treatment inhibited phosphorylation of nuclear c-Jun (P-c-Jun), and consequently reduced activity of the AP-1 transcription factor and production of cerebral cytokines/chemokines as determined at 3 and 24h post-HI. Inhibition of P-c-Jun by D-JNKi is thought to be mediated via inhibition of the upstream phosphorylation of cytosolic and nuclear JNK and/or by preventing the direct interaction of phosphorylated (P-)JNK with c-Jun. Surprisingly, however, HI did not induce a detectable increase in P-JNK in cytosol or nucleus. Notably, we show here for the first time that HI induces P-JNK only in the mitochondrial fraction, which was completely prevented by D-JNKi treatment. The hypothesis that mitochondrial JNK activation is key to HI brain injury was supported by data showing that treatment of rat pups with SabKIM1 peptide, a specific mitochondrial JNK inhibitor, is also neuroprotective. Inhibition of HI-induced mitochondrial JNK activation was associated with preservation of mitochondrial integrity as evidenced by prevention of ATP loss and inhibition of lipid peroxidation. The HI-induced increase in apoptotic markers (cytochrome c release and caspase 3 activation) as analyzed at 24h post-HI were also strongly reduced by D-JNKi and the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL were upregulated. Neuroprotection was lost after repeated 0+3h D-JNKi treatment which was associated with complete inhibition of the second peak of AP-1 activity and disability to upregulate mitochondrial Bcl-2 and Bcl-xL. We show here for the first time that D-JNKi treatment efficiently protects the neonatal brain against ischemic brain damage and subsequent cognitive and motor impairment. We propose that inhibition of phosphorylation of mitochondrial JNK is a pivotal step in preventing early loss of mitochondrial integrity leading to reduced neuroinflammation and inhibition of apoptotic neuronal loss. Moreover we show the crucial role of upregulation of mitochondrial anti-apoptotic proteins to maintain neuroprotection.

Mesenchymal stem cells restore cortical rewiring after neonatal ischemia in mice

A study was undertaken to investigate the effect of neonatal hypoxic‐ischemic (HI) brain damage and mesenchymal stem cell (MSC) treatment on the structure and contralesional connectivity of motor function‐related cerebral areas.

A role for G protein‐coupled receptor kinase 2 in mechanical allodynia

Inflammation and nerve injury can both induce mechanical allodynia via mechanisms involving the production of pro‐inflammatory cytokines and increased neuronal activity. Many neurotransmitters involved in pain signal via G protein‐coupled receptors (GPCRs). GPCR kinase (GRK)2 is a member of the GRK family that regulates agonist‐induced desensitization and signalling of GPCRs. Low intracellular GRK2 levels are associated with increased receptor signalling. The aim of this study was to investigate whether mechanical allodynia is associated with decreased spinal cord GRK2 expression and whether reduced GRK2 increases inflammation‐induced mechanical allodynia. Mechanical allodynia was induced in rats by chronic constriction injury of the sciatic nerve. After 2 weeks, neuronal GRK2 expression was decreased bilaterally in the superficial layers of the lumbar spinal cord dorsal horn. Moreover, interleukin‐1β significantly reduced GRK2 expression ex vivo in spinal cord slices. To investigate whether reduced GRK2 potentiates inflammation‐induced mechanical allodynia, we used GRK2+/– animals expressing decreased GRK2. At baseline, the threshold for mechanical stimulation did not differ between GRK2+/– and wild‐type mice. However, GRK2+/– animals were more sensitive to mechanical stimulation than wild‐type animals after intraplantar λ‐carrageenan injection. We propose cytokine‐induced down‐regulation of spinal cord neuronal GRK2 expression as a novel mechanism that contributes to increased neuronal signalling in mechanical allodynia.

Mechanical ventilation of healthy rats suppresses peripheral immune function

This study was designed to investigate the possible effect of injurious mechanical ventilation on peripheral immune function of healthy rats. Three ventilation strategies were compared: 1) low peak inspiratory pressure (PIP)/positive end-expiratory pressure (PEEP); 2) high PIP/PEEP; and 3) high PIP/zero PEEP (ZEEP). As a reference group, healthy, nonventilated, sham-operated, anaesthetised rats were used. After 4 h, rats were sacrificed and macrophage inflammatory protein (MIP)‐2 levels in lung and plasma were determined. Peripheral immune function was determined by measurement of splenic natural killer (NK) activity, mitogen-induced splenocyte proliferation and in vitro cytokine production. All immune measurements in the low PIP/PEEP group did not differ from the immune measurements in the reference group. High PIP strategies, irrespective of applied PEEP, enhanced MIP‐2 levels in lung and plasma. NK cell activity, mitogen-induced splenocyte proliferation and MIP‐2 and interleukin (IL)‐10 production significantly decreased after high PIP/PEEP ventilation. In the high PIP/ZEEP‐ventilated group, the decrease in splenocyte proliferation, MIP‐2 and IL‐10 production and NK cell activity was more pronounced and interferon‐γ production was also significantly lower than in the low PIP/PEEP group. These data show that high positive inspiratory pressure ventilation induces an inflammatory response in the lung, whereas at the same time the peripheral immune response is downregulated. Ventilator-induced peripheral immune suppression may contribute to poor outcome in acute respiratory distress syndrome patients.

Neonatal dexamethasone treatment induces long-lasting changes in T-cell receptor Vβ repertoire in rats

Glucocorticoids are frequently administered for the prevention of chronic lung disease in infants with respiratory distress syndrome. However, neonatal treatment may have consequences for immune functioning in the long-term. Here we demonstrate that neonatal glucocorticoid treatment has long-lasting effects on mRNA expression of several Vbeta genes within the CD4 and CD8 T cell subset in rats. Changes in the peripheral T cell Vbeta repertoire may be a consequence of altered intrathymic selection events in which corticosterone plays an important role. Indeed, here we show that neonatal glucocorticoid treatment affects corticosterone production by thymic epithelial cells during neonatal life. In conclusion, changes in T cell Vbeta repertoire after neonatal glucocorticoid treatment may contribute to altered immune reactivity in later life.