Search

Natural killer (NK) cells mediate innate host defense against microbial infection and cancer. Hypoxia and low glucose are characteristic for these tissue lesions but do not affect early interferon (IFN) γ and CC chemokine release by interleukin 15 (IL-15) primed human NK cells in vitro. Hypoxia inducible factor 1α (HIF-1α) mediates cellular adaption to hypoxia. Its production is supported by mechanistic target of rapamycin complex 1 (mTORC1) and signal transducer and activator of transcription 3 (STAT3). We used chemical inhibition to probe the importance of mTORC1 and STAT3 for the hypoxia response and of STAT3 for the cytokine response in isolated and IL-15 primed human NK cells. Cellular responses were assayed by magnetic bead array, RT-PCR, western blotting, flow cytometry, and metabolic flux analysis. STAT3 but not mTORC1 activation was essential for HIF-1α accumulation, glycolysis, and oxygen consumption. In both primed normoxic and hypoxic NK cells, STAT3 inhibition reduced the secretion of CCL3, CCL4 and CCL5, and it interfered with IL-12/IL-18 stimulated IFNγ production, but it did not affect cytotoxic granule degranulation up on target cell contact. We conclude that IL-15 priming promotes the HIF-1α dependent hypoxia response and the early cytokine response in NK cells predominantly through STAT3 signaling.

Natural Killer (NK) cells mediate innate immunity against cancer and intracellular infection, at that, operating in often oxygen-deprived environments. We performed a microarray experiment with a 2×2 factorial design to profile gene expression in human NK cells (Velasquez et al., 2016) [1]. In this experiment, NK cells from 5 healthy volunteers were primed or not for 6 h with the survival factor and inflammatory cytokine interleukin 15 (IL-15) under hypoxic or normoxic culture conditions (20 samples in total). Here, we provide details on the culture setup that govern the actual O2 partial pressure (pO2) experienced by the cells, as well as on the RNA extraction procedure used, which we optimized from commercial spin column protocols to obtain highly concentrated total RNA. We present a quality control analysis of the normalized microarray data, as well as overviews for differentially regulated genes. These data provide insights into NK cell transcriptional responses to immune stimulation under physiologically relevant low oxygen conditions. This dataset is deposited in the Gene Expression Omnibus database (accession number GSE70214).

Timely and reliable distinction of sepsis from non-infectious systemic inflammatory response syndrome (SIRS) supports adequate antimicrobial therapy and saves lives but is clinically challenging. Blood transcriptional profiling promises to deliver insights into the pathomechanisms of SIRS and sepsis and to accelerate the discovery of urgently sought sepsis biomarkers. However, suitable reference genes for normalizing gene expression in these disease conditions are lacking. In addition, variability in blood leukocyte subtype composition complicates gene profile interpretation. Here, we aimed to identify potential reference genes in natural killer (NK) cells and granulocytes from patients with SIRS and sepsis on intensive care unit (ICU) admission. Discovery by a two-step probabilistic selection from microarray data followed by validation through branched DNA assays in independent patients revealed several candidate reference genes in NK cells including AKIRIN1, PPP6R3, TAX1BP1, and ADRBK1. Initially, no candidate genes could be validated in patient granulocytes. However, we determined highly similar AKIRIN1 expression also in SIRS and sepsis granulocytes and no change by in vitro LPS challenge in granulocytes from healthy donors. Inspection of external neutrophil transcriptome datasets further support unchanged AKIRIN1 expression in human systemic inflammation. As a potential new reference gene in NK cells and granulocytes in infectious and inflammatory diseases, AKIRIN1 may improve our pathomechanistic understanding of SIRS and sepsis and help identifying new sepsis biomarkers.

Natural killer (NK) cells induce apoptosis in infected and transformed cells and are important producers of immunoregulatory cytokines. Therefore, they operate under low oxygen conditions (hypoxia) in inflammatory and tumor environments. In vitro studies of NK cells are, however, commonly performed in ambient air (normoxia). We used global gene expression profiling to evaluate changes in transcriptional pathways in primary human NK cells following short term culture under hypoxia compared with normoxia and in response to interleukin 15 (IL-15) priming using a 2 × 2 factorial design. The largest contrasts observed were priming dependences for associations between hypoxia and the hypoxia-inducible factor (Hif) 1 signaling and glycolysis pathways. RT-PCR confirmed positive synergistic hypoxia/IL-15 interactions for genes of key regulatory and metabolic enzymes. IL-15 primes NK cells for effector functions, which were recently demonstrated to depend on glycolytic switching. We did not, however, observe important increases in glycolytic flux through hypoxia and priming alone. Chemical Hif-1α inhibition suggested equal importance of this transcription factor for glycolysis and energy production under normoxia and hypoxia. Hypoxia promoted secretion of CC chemokines Ccl3/4/5 and macrophage migration inhibitory factor. Unexpectedly, hypoxia also stimulated migration of NK cells through the extracellular matrix and shifted amounts of susceptible leukemia target cells toward late apoptosis in a cell killing assay. We conclude that short term hypoxia supports these activities by positively interacting with NK cell priming at the level of glycolytic gene transcription. Hypoxic conditioning of NK cells may thus benefit their use in cell-based immunotherapy of cancer.

Objective: Endothelial damage and detachment of endothelial cells are known to occur in septic patients. Thus, recruitment of circulating enclothelial progenitor cells (cEPCs) to these lesions might have a beneficial effect on the clinical course in septic patients. Therefore, we were interested in whether EPCs, detected by flow cytometry, are increasingly mobilized during sepsis and if this mobilization is associated with clinical outcome.Design: Prospective, nonrandomized study.Setting. Intensive care unit of a university hospital.Patients: Patients with (n = 32) and without (n = 15) sepsis and healthy volunteers (n = 15).Interventions: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and cEPCs were characterized by three-color fluorescence flow cytometry using antibodies against CD133, CD34, and vascular endothelial growth factor receptor-2. Serum concentrations of vascular endothelial growth factor, granulocyte macrophage-colony stimulating factor, and erythropoietin were determined by enzyme-linked immunosorbent assay. Severity of sepsis was assessed according to Acute Physiology and Chronic Health Evaluation II scoring.Measurements and Main Results: In septic patients, the number of cEPCs was significantly higher than in nonseptic intensive care unit patients (p Conclusions: Our data suggest that cEPC enumeration in peripheral blood of septic patients might be a valuable marker to assess the clinical outcome in these patients.

Zahlreiche In-vitro- und In-vivo-Studien belegen, dass Dopamin neben seinen hamodynamischen Effekten eine Reihe immunmodulatorischer Wirkungen induziert. Dopamin reduziert die Synthese proinflammatorischer und induziert die Synthese antiinflammatorischer Mediatoren. Dopamin hemmt die Synthese neurohypophysarer Hormone und hemmt die Zellproliferation sowie die Thrombozytenaggregation. Es reduziert die Phagozytoseaktivitat neutrophiler Granulozyten und induziert Apoptose. Bei hohen Dopaminserumkonzentrationen, wie sie bei einer Sepsis durch vermehrte endogene Synthese, zusatzliche exogene Applikation und verringerte Clearance erreicht werden, konnten diese Effekte zu relevanten Veranderungen pathophysiologischer Ablaufe fuhren. Um die Bedeutung von Dopamin fur die zellulare und humorale Immunantwort von Patienten mit Sepsis hervorzuheben, sind in dieser Ubersicht die speziellen Wirkungen von Dopamin zusammengefasst und die zugrunde liegenden Mechanismen dargestellt.

OBJECTIVE In the pathogenesis of septic shock, caused by either bacterial toxins or trauma, increased production of multiple proinflammatory mediators, such as phospholipase A(2) (PLA(2)), cytokines, and chemokines, is known to be of major importance. The present study was undertaken to investigate the influence of a newly designed extracellular PLA(2) inhibitor (ExPLI) on synthesis of proinflammatory mediators and mortality rate in a rat sepsis model. DESIGN Prospective, randomized animal study. SETTING Experimental laboratory. SUBJECTS Male Wistar-rats weighing 200-300 g. INTERVENTIONS Mortality was induced by intraperitoneal bolus administration of lipopolysaccharide 15 mg/kg in 22 rats that were pretreated with NaCl or ExPLI (150 mg/kg). Furthermore, nine rats received a sublethal bolus of lipopolysaccharide (7.5 mg/kg) and nine rats received lipotechoic acid (8 mg/kg) simultaneously with or after ExPLI administration. Blood samples were collected from these rats, and cytokine concentrations were assessed by enzyme-linked immunosorbent assay. Lung and kidney were removed for RNA isolation and immunohistological analysis. MEASUREMENTS AND MAIN RESULTS ExPLI treatment significantly reduced lipopolysaccharide-induced mortality of rats (90.9 and 36.4%, p

Acute respiratory distress syndrome (ARDS) is characterized by alterations in microvascular permeability. In ARDS secreted phospholipase A 2 (sPLA 2 ) IB and IIA are found to be highly upregulated. In this study, we therefore investigated the influence of exogenously added sPLA 2 -IB and sPLA 2 -IIA on the production of chemokines and adhesion molecules in lung microvascular endothelial cells (LMVEC). Treatment of LMVEC with sPLA 2 s resulted in a significant increase in the production of chemokines and adhesion molecules due to an increased expression of their mRNA and in an enhanced release of oleic acid. The upregulation of chemokines and adhesion molecules by LPS was stronger in the presence of sPLA 2 . Activation of NF-κB occurred upon stimulation with sPLA 2 . Moreover the MAPkinase pERK seems to be involved since a specific pERK inhibitor, e.g., U0126, but not a p38Kinase inhibitor, e.g., SB203580 prevented sPLA 2 -induced chemokine upregulation. Our data therefore suggest that LMVEC are a highly sensitive target for the direct action of extracellular sPLA 2 s.

1. In acute respiratory distress syndrome (ARDS) induced by endotoxins, a high production of inflammatory mediators by microvascular lung endothelial cells (LMVEC) can be observed. Activation of cells by endotoxins may result in elevated secretion of phospholipase A(2) (sPLA(2)) which is thought to contribute to tissue damage. The present study was undertaken to investigate the role of sPLA(2) in chemokine production in human lung microvascular endothelial cells (LMVEC) stimulated with the endotoxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA). In particular, we investigated the effects of sPLA(2) inhibitors, specifically, the extracellular PLA(2) inhibitors (ExPLIs), composed of N-derivatized phosphatidyl-ethanolamine linked to polymeric carriers, and LY311727, a specific inhibitor of non-pancreatic sPLA(2). 2. ExPLIs markedly inhibited LPS and LTA induced production and mRNA expression of the neutrophile attracting chemokines IL-8, Gro-alpha and ENA-78, as well as of the adhesion molecules ICAM-1 and E-selectin. Concomitantly, ExPLIs inhibited the LPS-induced activation of NF-kappaB by LPS but not its activation by TNF-alpha or IL-1. 3. Endotoxin mediated chemokine production in LMVEC seems not to involve PLA(2) activity, since LPS stimulation was not associated with activation of intracellular or secreted PLA(2). It therefore seems that the inhibitory effect of the ExPLIs was not due to their PLA(2) inhibiting capacity. This was supported by the finding that the LPS-induced chemokine production was not affected by the selective sPLA(2) inhibitor LY311727. 4. It is proposed that the ExPLIs may be considered a prototype of potent suppressors of specific endotoxin-induced inflammatory responses, with potential implications for the therapy of subsequent severe inflammation.

Recent experimental work suggests that circulating endothelial progenitor cells (cEPCs) are recruited to the angiogenic vascular system of malignant gliomas. Consequently, the level of cEPCs has been proposed as a novel biomarker for the diagnosis and monitoring of these lesions. The aim of the present study was to examine the level of cEPCs and the level of EPC mobilizing mediators in the blood of patients with malignant gliomas. The authors were also interested in whether a correlation could be observed between the level of cEPCs and the extent of glioma angiogenesis determined by conventional methods.Peripheral blood mononuclear cells from the whole blood of 12 patients with malignant gliomas (all glioblastomas multiforme [GBMs]), 10 with metastases to the brain, and 10 healthy volunteers were isolated using Ficoll density gradient centrifugation. The number of cEPCs was quantified by fluorescence-activated cell sorting analysis using antibodies against CD34, CD133, and VEGFR-2. Serum concentrations of VEGF and granulocyte-macrophage colony-stimulating factor (GM-CSF) were determined using the enzyme-linked immunosorbent assay. Histological analysis of tumor blood vessel density was performed by CD34 immunohistochemical staining.The number of cEPCs was significantly higher in patients with GBMs than in those with metastases (p0.04) or in the healthy volunteers (p0.004). The serum VEGF concentrations in patients with GBMs and metastases were significantly higher than in the healthy volunteers (p0.0001). Similar findings were observed for concentrations of GM-CSF. In addition, the patients in the GBM group with higher levels of cEPCs had significantly higher tumor blood vessel densities (1.71 +/- 1.17% of total area) compared with patients who had low levels of cEPCs (0.62 +/- 0.28% of total area; p0.02).Endothelial progenitor cells are increasingly mobilized in patients with malignant gliomas, and their levels correlate with tumor angiogenic activity. Therefore, the authors' results suggest that cEPCs may have the potential to serve as a novel biomarker for the identification of patients who would benefit from antiangiogenic therapy for GBM.

Large quantitaties of inflammatory mediators are released during the course of endotoxaemia. These mediators in turn can stimulate the sympathetic nervous system (SNS) to release catecholamines, which ultimately regulate inflammation-associated impairment in tissue perfusion, myocardial impairment and vasodilatation. Treatment of sepsis is based on surgical and/or antibiotic therapy, appropriate fluid management and application of vasoactive catecholamines. With respect to the latter, discussions on the vasopressor of choice are still ongoing. Over the past decade dopamine has been considered the 'first line' vasopressor and is frequently used to improve organ perfusion and blood pressure. However, there is a growing body of evidence that dopamine has deleterious side effects; therefore, its clinical relevance seems to be more and more questionable. Nevertheless, it has not been convincingly demonstrated that other catecholamines are superior to dopamine in this respect. Apart from its haemodynamic action, dopamine can modulate immune responses by influencing the cytokine network. This leads to inhibition of expression of adhesion molecules, inhibition of cytokine and chemokine production, inhibition of neutrophil chemotaxis and disturbed T-cell proliferation. In the present review we summarize our knowledge of the immunomodulatory effects of dopamine, with an emphasis on the mechanisms by which these effects are mediated.

Inflammatory responses during sepsis are determined by leucocyte recruitment into inflamed tissues. Both chemokines and adhesion molecules are believed to be involved in this process. As fractalkine exists as transmembrane protein with cell adhesion properties and as soluble chemotactic factor, the present study was conducted to study the role of fractalkine, produced by microvascular and macrovascular endothelial cells, in neutrophil recruitment. Lung microvascular endothelial cells (LMVECs) stimulated with lipopolysaccharide, tumour necrosis factor-alpha or interleukin-1 (IL-1) produced much more fractalkine compared with the macrovascular human umbilical vein endothelial cells (HUVECs). No differences were found between microvascular endothelial cells of different organs. Chemotactic activity in supernatants was significantly stronger in stimulated LMVEC when compared with HUVEC. Although recombinant fractalkine induced migration of neutrophils, IL-8 and monocyte chemoattractant protein-1 were found to be more strictly required. In vivo fractalkine was strongly upregulated in septic lung and kidney. Our data suggest that fractalkine production per se does not explain the preference for inflammation in the lung of septic patients.

Large quantitaties of inflammatory mediators are released during the course of endotoxaemia. These mediators in turn can stimulate the sympathetic nervous system (SNS) to release catecholamines, which ultimately regulate inflammation-associated impairment in tissue perfusion, myocardial impairment and vasodilatation. Treatment of sepsis is based on surgical and/or antibiotic therapy, appropriate fluid management and application of vasoactive catecholamines. With respect to the latter, discussions on the vasopressor of choice are still ongoing. Over the past decade dopamine has been considered the 'first line' vasopressor and is frequently used to improve organ perfusion and blood pressure. However, there is a growing body of evidence that dopamine has deleterious side effects; therefore, its clinical relevance seems to be more and more questionable. Nevertheless, it has not been convincingly demonstrated that other catecholamines are superior to dopamine in this respect. Apart from its haemodynamic action, dopamine can modulate immune responses by influencing the cytokine network. This leads to inhibition of expression of adhesion molecules, inhibition of cytokine and chemokine production, inhibition of neutrophil chemotaxis and disturbed T-cell proliferation. In the present review we summarize our knowledge of the immunomodulatory effects of dopamine, with an emphasis on the mechanisms by which these effects are mediated.