Stefanie Endesfelder

IL-22 Induces Lipopolysaccharide-Binding Protein in Hepatocytes: A Potential Systemic Role of IL-22 in Crohn’s Disease

Crohn′s disease (CD) is a common, chronic, inflammatory bowel disease characterized by intestinal infiltration of activated immune cells and distortion of the intestinal architecture. In this study, we demonstrate that IL-22, a cytokine that is mainly produced by activated Th1 and Th17 cells, was present in high quantities in the blood of CD patients in contrast to IFN-γ and IL-17. In a mouse colitis model, IL-22 mRNA expression was elevated predominantly in the inflamed intestine but also in the mesenteric lymph nodes. IL-22BP, the soluble receptor for IL-22, demonstrated an affinity to IL-22 that was at least 4-fold higher than its membrane-bound receptor, and its strong constitutive expression in the intestine and lymph nodes was decreased in the inflamed intestine. To investigate the possible role of systemic IL-22 in CD, we then administered IL-22 to healthy mice and found an up-regulation of LPS-binding protein (LBP) blood levels reaching concentrations known to neutralize LPS. This systemic up-regulation was associated with increased hepatic but not renal or pulmonary LBP mRNA levels. IL-22 also enhanced the secretion of LBP in human primary hepatocytes and HepG2 hepatoma cells in vitro. This increase was mainly transcriptionally regulated and synergistic with that of other LBP inducers. Finally, elevated LBP levels were detected in CD patients and the mouse colitis model. These data suggest that systemic IL-22 may contribute to the prevention of systemic inflammation provoked by LPS present in the blood of CD patients through its induction of hepatic LBP.

NMDA antagonist inhibits the extracellular signal-regulated kinase pathway and suppresses cancer growth

Glutamate antagonists limit the growth of human cancers in vitro. The mechanism of anticancer action of NMDA antagonists is not known, however. In this article, we report that the NMDA antagonist dizocilpine inhibits the extracellular signal-regulated kinase 1/2 pathway, an intracellular signaling cascade that is activated by growth factors and controls the proliferation of cancer cells. Dizocilpine reduces the phosphorylation of cAMP-responsive element binding protein, suppresses the expression of cyclin D1, up-regulates the cell cycle regulators and tumor suppressor proteins p21 and p53, and increases the number of lung adenocarcinoma cells in the G2 and S phases of the cell cycle. Silencing of the tumor suppressor protein p21 abolishes antiproliferative action of dizocilpine. Consistent with inhibition of the extracellular signal-regulated kinase 1/2-signaling cascade, dizocilpine reverses the stimulation of proliferation induced by epidermal, insulin, and basic fibroblast growth factors in lung adenocarcinoma cells. Furthermore, dizocilpine prolongs the survival of mice with metastatic lung adenocarcinoma and slows the growth of neuroblastoma and rhabdomyosarcoma in mice. These findings reveal the mechanism of antiproliferative action of dizocilpine and indicate that it may be useful in the therapy of human cancers.

IL-19 and IL-20: two novel cytokines with importance in inflammatory diseases

IL-19 and IL-20 are two cytokines that were discovered in 2000 and 2001, respectively. Based on the structure and location of their genes, their primary and secondary protein structures and the used receptor complexes, they were classified with IL-10, IL-22, IL-24, IL-26, IL-28 and IL-29 in the IL-10 family of cytokines, and form a subgroup with IL-24 within this family. IL-19 and IL-20 are produced by monocytes as well as non-immune tissue cells under inflammatory conditions. IL-19 and IL-20 act via a receptor complex that consists of the IL-20R1 and IL-20R2 chains. IL-20 is additionally able to signal via a second receptor complex (IL-22R1/IL-20R2). It is controversial whether or not IL-19 and IL-20 regulate the function of immune cells. However, the expression of their receptors aliments the perception that the cells of the skin, lungs and reproductive organs as well as various glands are major targets of these mediators. Results from animal experiments and massively increased expression of these mediators in human inflamed tissues support the assumption that they play an important role in the pathogenesis of a few inflammatory diseases. For this reason, the authors have reviewed the facts known at present regarding these cytokines and postulate that IL-19 and IL-20 are pharmacologically interesting distal elements of an inflammatory cascade.

Interaction of inflammation and hyperoxia in a rat model of neonatal white matter damage.

Intrauterine infection and inflammation are major reasons for preterm birth. The switch from placenta-mediated to lung-mediated oxygen supply during birth is associated with a sudden rise of tissue oxygen tension that amounts to relative hyperoxia in preterm infants. Both infection/inflammation and hyperoxia have been shown to be involved in brain injury of preterm infants. Hypothesizing that they might be additive or synergistic, we investigated the influence of a systemic lipopolysaccharide (LPS) application on hyperoxia-induced white matter damage (WMD) in newborn rats. Three-day-old Wistar rat pups received 0.25 mg/kg LPS i.p. and were subjected to 80% oxygen on P6 for 24 h. The extent of WMD was assessed by immunohistochemistry, western blots, and diffusion tensor (DT) magnetic resonance imaging (MRI). In addition, the effects of LPS and hyperoxia were studied in an in vitro co-culture system of primary rat oligodendrocytes and microglia cells. Both noxious stimuli, hyperoxia, and LPS caused hypomyelination as revealed by western blot, immunohistochemistry, and altered WM microstructure on DT-MRI. Even so, cellular changes resulting in hypomyelination seem to be different. While hyperoxia induces cell death, LPS induces oligodendrocyte maturity arrest without cell death as revealed by TUNEL-staining and immunohistological maturation analysis. In the two-hit scenario cell death is reduced compared with hyperoxia treated animals, nevertheless white matter alterations persist. Concordantly with these in vivo findings we demonstrate that LPS pre-incubation reduced premyelinating-oligodendrocyte susceptibility towards hyperoxia in vitro. This protective effect might be caused by upregulation of interleukin-10 and superoxide dismutase expression after LPS stimulation. Reduced expression of transcription factors controlling oligodendrocyte development and maturation further indicates oligodendrocyte maturity arrest. The knowledge about mechanisms that triggered hypomyelination contributes to a better understanding of WMD in premature born infants.

Prevention of hyperoxia-mediated pulmonary inflammation in neonatal rats by caffeine

In preterm human infants, briefly elevated concentrations of oxygen are associated with a prolonged increase in blood chemokine concentrations and the development of bronchopulmonary dysplasia (BPD). Caffeine given to preterm infants for the prevention or treatment of apnoea has been shown to reduce the rate of BPD. We tested the hypotheses that infant rats exposed to a combination of caffeine and hyperoxia would be less susceptible to lung injury than those exposed to hyperoxia alone and that caffeine decreases the pulmonary tissue expression of chemokines and leukocyte influx following hyperoxia. Using 6-day-old rat pups, we demonstrated that 24 h of 80% oxygen exposure caused pulmonary recruitment of neutrophils and macrophages. High levels of oxygen upregulated the expression of: the CXC chemokines, cytokine-induced neutrophil chemoattractant-1 and macrophage inflammatory protein-2; the CC-chemokine monocyte chemoattractant protein-1; the pro-inflammatory cytokines tumour necrosis factor-&agr; and interleukin-6, as measured by realtime PCR after the administration of caffeine (10 mg·kg−1 body weight); and attenuated chemokine and cytokine upregulation, as well as the influx of CD11b+, ED-1+ and myeloperoxidase+ leukocytes. These experiments suggest that protective effects of caffeine in the neonatal lung are mediated, at least in part, by reduction of pulmonary inflammation.

Erythropoietin attenuates hyperoxia-induced oxidative stress in the developing rat brain.

Oxygen toxicity contributes to the pathogenesis of adverse neurological outcome in survivors of preterm birth in clinical studies. In infant rodent brains, hyperoxia triggers widespread apoptotic neurodegeneration, induces pro-inflammatory cytokines and inhibits growth factor signaling cascades. Since a tissue-protective effect has been observed for recombinant erythropoietin (rEpo), we hypothesized that rEpo would influence hyperoxia-induced oxidative stress in the developing rat brain. The aim of this study was to investigate the level of glutathione (reduced and oxidized), lipid peroxidation and the expression of heme oxygenase-1 (HO-1) and acetylcholinesterase (AChE) after hyperoxia and rEpo treatment. Six-day-old Wistar rats were exposed to 80% oxygen for 2-48 h and received 20,000 IU/kg rEpo intraperitoneally (i.p.). Sex-matched littermates kept under room air and injected with normal saline or rEpo served as controls. Treatment with rEpo significantly reduced hyperoxia-induced upregulation of oxidized glutathione (GSSG) and malondialdehyde, a product of lipid breakdown, whereas reduced glutathione (GSH) was upregulated by rEpo. In parallel, hyperoxia-treated immature rat brains revealed rEpo-suppressible upregulation of synaptic AChE-S as well as of the stress-inducible AChE-R variant, together predicting rEpo-protected cholinergic signaling and restrained inflammatory reactions. Furthermore, treatment with rEpo induced upregulation of HO-1 on mRNA, protein and activity level in the developing rat brain. Our results suggest that rEpo generates its protective effect against oxygen toxicity by a reduction of diverse oxidative stress parameters and by limiting the stressor-inducible changes in both HO-1 and cholinergic functions.

A critical role for Fas/CD‐95 dependent signaling pathways in the pathogenesis of hyperoxia‐induced brain injury

Prematurely born infants are at risk for development of neurocognitive impairment in later life. Oxygen treatment has been recently identified as a trigger of neuronal and oligodendrocyte apoptosis in the developing rodent brain. We investigated the role of the Fas death receptor pathway in oxygen‐triggered developmental brain injury.

Caffeine protects neuronal cells against injury caused by hyperoxia in the immature brain

Caffeine administered to preterm infants has been shown to reduce rates of cerebral palsy and cognitive delay, compared to placebo. We investigated the neuroprotective potential of caffeine for the developing brain in a neonatal rat model featuring transient systemic hyperoxia. Using 6-day-old rat pups, we found that after 24 and 48h of 80% oxygen exposure, apoptotic (TUNEL(+)) cell numbers increased in the cortex, hippocampus, and central gray matter, but not in the hippocampus or dentate gyrus. In the dentate gyrus, high oxygen exposure led to a decrease in the number of proliferating (Ki67(+)) cells and the number of Ki67(+) cells double staining for nestin (immature neurons), doublecortin (progenitors), and NeuN (mature neurons). Absolute numbers of nestin(+), doublecortin(+), and NeuN(+) cells also decreased after hyperoxia. This was mirrored in a decline of transcription factors expressed in immature neurons (Pax6, Sox2), progenitors (Tbr2), and mature neurons (Prox1, Tbr1). Administration of a single dose of caffeine (10mg/kg) before high oxygen exposure almost completely prevented these effects. Our findings suggest that caffeine exerts protection for neonatal neurons exposed to high oxygen, possibly via its antioxidant capacity.

Minocycline protects the immature white matter against hyperoxia.

Poor neurological outcome in preterm infants is associated with periventricular white matter damage and hypomyelination, often caused by perinatal inflammation, hypoxia-ischemia, and hyperoxia. Minocycline has been demonstrated in animal models to protect the immature brain against inflammation and hypoxia-ischemia by microglial inhibition. Here we studied the effect of minocycline on white matter damage caused by hyperoxia. To mimic the 3- to 4-fold increase of oxygen tension caused by preterm birth, we have used the hyperoxia model in neonatal rats providing 24h exposure to 4-fold increased oxygen concentration (80% instead of 21% O2) from P6 to P7. We analyzed whether minocycline prevents activation of microglia and damage of oligodendroglial precursor cell development, and whether acute treatment of hyperoxia-exposed rats with minocycline improves long term white matter integrity. Minocycline administration during exposure to hyperoxia resulted in decreased apoptotic cell death and in improved proliferation and maturation of oligodendroglial precursor cells (OPC). Minocycline blocked changes in microglial morphology and IL-1β release induced by hyperoxia. In primary microglial cell cultures, minocycline inhibited cytokine release while in mono-cultures of OPCs, it improved survival and proliferation. Long term impairment of white matter diffusivity in MRI/DTI in P30 and P60 animals after neonatal hyperoxia was attenuated by minocycline. Minocycline protects white matter development against oxygen toxicity through direct protection of oligodendroglia and by microglial inhibition. This study moreover demonstrates long term benefits of minocycline on white matter integrity.

Erythropoietin Modulates Autophagy Signaling in the Developing Rat Brain in an In Vivo Model of Oxygen-Toxicity

Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo). Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg) proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.