Polymorphonuclear Neutrophils Adhesion Research Articles

Effect of Lonomia obliqua Venom on Human Neutrophils.

The significant incidence of deforestation in South America culminates in the contact of humans with typical forests species. Among these species, one may highlight Lonomia obliqua caterpillar, which, when touched by humans, can poison them through their bristles. Therefore, better acknowledging the mechanisms involved in envenomation caused by Lonomia obliqua caterpillar bristle extract (LOCBE) may contribute to further treatments. Recently, we demonstrated that LOCBE induces a pro-inflammatory profile in endothelial cells; thus, we decided to investigate the effects of LOCBE on human polymorphonuclear neutrophils (PMN), which are the first leukocytes that migrate to the inflammatory focus. Our results showed that treatment with LOCBE induced PMN chemotaxis together with alterations in actin cytoskeleton and focal adhesion kinase (FAK) activation, favoring migration. Concurrently, LOCBE induced PMN adhesion to matrix proteins, such as collagen IV, fibronectin, and fibrinogen. Moreover, we observed that LOCBE attenuated PMN apoptosis and increased reactive oxygen species (ROS) production together with nuclear factor kB (NF-κB) activation—a redox-sensitive transcription factor—as well as interleukin (IL)-1β and IL-8 release. We call attention to the ROS-dependent effect of LOCBE on increased cell migration once an antioxidant treatment reverted it. In summary, we report that LOCBE activates PMN, inducing pro-inflammatory responses modulated by ROS.

IL1B triggers inflammatory cytokine production in bovine oviduct epithelial cells and induces neutrophil accumulation via CCL2.

The oviduct is essential for reproduction. We previously showed that oviduct epithelial cells (OECs) isolated from aged cows expressed higher levels of inflammatory cytokines, including interleukin (IL) 1A and IL1B. In addition, aging is associated with tissue dysfunction and cellular senescence via a senescence-associated secretory phenotype (SASP) and immune cell accumulation. We investigated whether IL1A or IL1B causes SASP production, cellular senescence, and inflammatory responses in bovine OECs. The OECs were isolated from bovine oviducts from young (mean 50.3months) and aged cows (mean 157.0months) and cultured. Treatment with IL1A or IL1B induced SASP production (IL8, IL6, TNFA, and CCL2) and mRNA expression of cell adhesion molecules in bovine OECs, but both IL1s did not induce cellular senescence in OECs and migration of polymorphonuclear neutrophils (PMNs). Cultured medium of OECs treated with IL1s, especially IL1B, dramatically induced PMN migration. Treatment with the CCL2 inhibitor, but not IL8 or its receptor CXCR2 inhibitors, significantly reduced immune cell migration in IL1B-treated OEC-cultured medium. Treatment with IL1B increased PMN adhesion to OECs, resulting in further SASP production in OECs due to a PMN-OEC interaction. We suggest that senescence-associated IL1s cause SASP production in bovine OECs and CCL2 induced by IL1B is essential for the migration of immune cells to OECs. Specifically, IL1B regulates PMN migration and adhesion to bovine OECs, and PMNs accelerate inflammatory cytokine production from bovine OECs via a direct interaction. These phenomena may contribute to chronic oviductal inflammation, resulting in subfertility.

Gut-lymph-lung pathway mediates sepsis-induced acute lung injury.

This review attempts to unveil the possible mechanisms underlying how gut lymph affects lung and further gives rise to acute respiratory distress syndrome, as well as potential interventional targets under the condition of ischemia-reperfusion injury. We searched electronic databases including PubMed, MEDLINE, Cochrane Central Register of Controlled Trials, Google Scholar, Web of Science, and Embase to identify relevant literatures published up to December 2019. We enrolled the literatures including the Mesh Terms of “gut lymph or intestinal lymph and acute lung injury or acute respiratory distress syndrome.” Gut is considered to be the origin of systemic inflammation and the engine of multiple organ distress syndrome in the field of critical care medicine, whereas gut lymph plays a pivotal role in initiation of ischemia-reperfusion injury-induced acute respiratory distress syndrome. In fact, in the having been established pathologic model of sepsis leading to multiple organ dysfunction named by Gut Lymph theory, a variety of literatures showed the position and role of changes in gut lymph components in the initiation of systemic inflammatory response, which allows us to screen out potential intervention targets to pave the way for future clinic and basic research.

Polymorphonuclear Neutrophils Play a Decisive Role for Brain Injury and Neurological Recovery Poststroke.

Polymorphonuclear Neutrophils Play a Decisive Role for Brain Injury and Neurological Recovery Poststroke.

Nucleotide-binding oligomerization domain 1 (NOD1) modulates liver ischemia reperfusion through the expression adhesion molecules

In liver transplantation, organ shortage leads to the use of marginal grafts that are more susceptible to ischemia-reperfusion (IR) injury. We identified nucleotide-binding oligomerization domain 1 (NOD1) as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in IR. Herein, we aimed to elucidate the role of NOD1 in IR injury, particularly focusing on its effects on the endothelium and hepatocytes. Nod1 WT and KO mice were treated with NOD1 agonists and subjected to liver IR. Expression of adhesion molecules was analyzed in total liver, isolated hepatocytes and endothelial cells. Interactions between PMNs and hepatocytes were studied in an ex vivo co-culture model using electron microscopy and lactate dehydrogenase levels. We generated NOD1 antagonist-loaded nanoparticles (np ALINO). NOD1 agonist treatment increased liver injury, PMN tissue infiltration and upregulated ICAM-1 and VCAM-1 expression 20 hours after reperfusion. NOD1 agonist treatment without IR increased expression of adhesion molecules (ICAM-1, VCAM-1) in total liver and more particularly in WT hepatocytes, but not in Nod1 KO hepatocytes. This induction is dependent of p38 and ERK signaling pathways. Compared to untreated hepatocytes, a NOD1 agonist markedly increased hepatocyte lysis in co-culture with PMNs as shown by the increase of lactate dehydrogenase in supernatants. Interaction between hepatocytes and PMNs was confirmed by electron microscopy. In a mouse model of liver IR, treatment with np ALINO significantly reduced the area of necrosis, aminotransferase levels and ICAM-1 expression. NOD1 regulates liver IR injury through induction of adhesion molecules and modulation of hepatocyte-PMN interactions. NOD1 antagonist-loaded nanoparticles reduced liver IR injury and provide a potential approach to prevent IR, especially in the context of liver transplantation. Nucleotide-binding oligomerization domain 1 (NOD1) is as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in ischemia-reperfusion. Here, we show that the NOD1 pathway targets liver adhesion molecule expression on the endothelium and on hepatocytes through p38 and ERK signaling pathways. The early increase of adhesion molecule expression after reperfusion emphasizes the importance of adhesion molecules in liver injury. In this study we generated nanoparticles loaded with NOD1 antagonist. These nanoparticles reduced liver necrosis by reducing PMN liver infiltration and adhesion molecule expression.

Binding of intercellular adhesion molecule 1 to β2-integrin regulates distinct cell adhesion processes on hepatic and cerebral endothelium.

Flowing polymorphonuclear neutrophils (PMNs) are forced to recruit toward inflamed tissue and adhere to vascular endothelial cells, which is primarily mediated by the binding of β2-integrins to ICAM-1. This process is distinct among different organs such as liver and brain; however, the underlying kinetic and mechanical mechanisms regulating tissue-specific recruitment of PMNs remain unclear. Here, binding kinetics measurement showed that ICAM-1 on murine hepatic sinusoidal endothelial cells (LSECs) bound to lymphocyte function-associated antigen-1 (LFA-1) with higher on- and off-rates but lower effective affinity compared with macrophage-1 antigen (Mac-1), whereas ICAM-1 on cerebral endothelial cells (BMECs or bEnd.3 cells) bound to LFA-1 with higher on-rates, similar off-rates, and higher effective affinity compared with Mac-1. Physiologically, free crawling tests of PMN onto LSEC, BMEC, or bEnd.3 monolayers were consistent with those kinetics differences between two β2-integrins interacting with hepatic sinusoid or cerebral endothelium. Numerical calculations and Monte Carlo simulations validated tissue-specific contributions of β2-integrin-ICAM-1 kinetics to PMN crawling on hepatic sinusoid or cerebral endothelium. Thus, this work first quantified the biophysical regulation of PMN adhesion in hepatic sinusoids compared with cerebral endothelium.

Coronin 1A, a novel player in integrin biology, controls neutrophil trafficking in innate immunity

AbstractTrafficking of polymorphonuclear neutrophils (PMNs) during inflammation critically depends on the β2 integrins lymphocyte function–associated antigen 1 (LFA-1) (CD11a/CD18) and macrophage-1 antigen (CD11b/CD18). Here, we identify coronin 1A (Coro1A) as a novel regulator of β2 integrins that interacts with the cytoplasmic tail of CD18 and is crucial for induction of PMN adhesion and postadhesion events, including adhesion strengthening, spreading, and migration under flow conditions. Transition of PMN rolling to firm adhesion critically depends on Coro1A by regulating the accumulation of high-affinity LFA-1 in focal zones of adherent cells. Defective integrin affinity regulation in the genetic absence of Coro1A impairs leukocyte adhesion and extravasation in inflamed cremaster muscle venules in comparison with control animals. In a Helicobacter pylori mouse infection model, PMN infiltration into the gastric mucosa is dramatically reduced in Coro1A−/− mice, resulting in an attenuated gastric inflammation. Thus, Coro1A represents an important novel player in integrin biology, with key functions in PMN trafficking during innate immunity.

The Endothelin Receptor Etb Plays a Crucial Role for Recruitment of Neutrophils to the Vascular Wall in Sickle Cell Disease

Introduction: Although Sickle Cell Disease (SCD) is due to abnormal hemoglobin, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), play a key role in the pathophysiology of the disease, particularly in the vaso-occlusive events. Adhesion of PMNs to activated endothelium is critical in SCD and might contribute to vaso-occlusion; thus targeting PMN interactions with the endothelium may represent a good opportunity for new therapeutics. We looked for candidate mediators that may be involved in PMN activation and recruitment in tissues. We focused on endothelin-1 (ET-1) since (i) high levels of cytokines have been reported in SCD patients; (ii) we previously demonstrated that ET-1 receptors blockade in the SAD mouse model of SCD leads to reversal or prevention of vaso-occlusive events, nitrative stress, kidney and lung damage, and even death. While demonstrating that ET receptor (ETR) antagonism inhibited a tonic ET-1-dependent vasoconstriction during experimental vaso-occlusive crisis, we observed unexpected inhibition of PMN recruitment in lungs and kidneys. We postulated that activation of ETRs might stimulate a pathogenic proinflammatory role for PMNs in SCD.Methods: In the present study, we combinedintravital videomicroscopy of the cremaster muscle microcirculation in SAD mice and quantitative microfluidic fluorescence microscopy of human blood to investigate the involvement of the ETRs in the interaction between neutrophils and endothelial cells.Results: Experiments performed both on SAD mice and SCD patients indicate that blocking ETRs hinders PMN recruitment to endothelial cells in inflammatory conditions (Fig.1). In SAD mice, we showed that the ETRs are involved at several steps of PMN microvascular recruitment. Rolling adhesion involves the ETB receptor only; firm adhesion and post-adhesive dynamic events with transmigration involve both the ETA and the ETB receptors. Inhibition experiments performed with the highly selective BQ788 antagonist (specific for ETB receptor) provide potent anti-inflammatory action in SAD mice. In SCD patients and healthy volunteers, we evidenced that human PMNs display functional ETB receptors that trigger a downstream calcium signaling leading to enhanced adhesion to endothelial cells. Furthermore, we investigated the expression of preproET-1 mRNA and the secretion of ET-1 in human PMNs from SCD patients and healthy controls and we highlighted the ability of PMNs to produce and secrete ET-1. We also found an unexpected stimulatory role for the ETB receptor in PMN adhesion to endothelial cells in laminar flow conditions. Last, we showed that this abnormal adhesion involves ETB receptors on both endothelial cells and PMNs.Conclusion: SAD mice and SCD patients both provide consistent evidence for a powerful anti-adhesion role of ETB blockade. We emphasize that our work is the first to unravel the impact of the ET receptors on the different phases of PMN-endothelial interaction, further complementing early evidence that endothelins are chemoattractants for neutrophils in vitro. Our findings involving a potent contribution of the ETB receptor to vascular inflammation are novel as well as the fact that this pathogenic phenomenon is found in sickle but not in normal mice. Overall, the aforementioned findings indicated that human neutrophils display functional ETB receptors with calcium signaling capability, and we confirmed that human PMNs synthesize ET-1 that may be involved in autocrine and paracrine pathophysiological actions. Thus, the ET-1- ETB axis should be considered a cytokine-like potent proinflammatory pathway in SCD. If endothelin receptors antagonists prove safe and effective for preventing or treating acute vasoocclusive events in the clinical setting, they should include anti- ETB potency and may provide major benefits for lung and renal integrity, quality of life, and survival of SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

343 - CD11b/CD18 Contributes to Susceptibility of Ventricular Arrhythmias and Infarct Size

Introduction The role of polymorphonuclear neutrophils (PMN) in myocardial ischemia and reperfusion (I/R) is controversially discussed. The PMN surface protein CD11b/CD18 plays a critical role for PMN activation and adhesion. Using CD11b/CD18 deficient (CD11b-/-) mice we herein sought to further elucidate the effects of infiltrating PMN on ventricular remodeling and arrhythmogenesis in the context of myocardial I/R. Methods and Results Wildtype (WT) and CD11b-deficient (CD11b-/-) mice of C57BL/6J background were subjected to 40 minutes of myocardial ischemia by ligation of the left anterior descending artery following 6- or 48 hours of reperfusion (I/R). CD11b-/- resulted in significantly less myocardial myeloperoxidase release (MPO+ cells / field of view: WT: 308.4 ± 22.2 vs. CD11b-/-: 178.3 ± 13.1, p Conclusions Decreased myocardial PMN infiltration and oxidative protein modifactions caused by the absence of CD11b/CD18 suggests a clear role of PMN infiltration for maldadaptive left ventricular remodelling and arrhythmogenesis following myocardial I/R.

Nicotinic receptor involvement in regulation of functions of mouse neutrophils from inflammatory site

Participation of nicotinic acetylcholine receptors (nAChRs) in functioning of polymorphonuclear neutrophils (PMNs) isolated from inflammatory site of mice and expression of different nAChR subunits were studied. Nicotine and acetylcholine (ACh) modified respiratory burst induced by a chemotactic peptide N-formyl-MLF in neutrophils of male (but not female) mice. Antagonists of nAChRs α-cobratoxin (αCTX), α-conotoxins MII and [A10L]PnIA at concentrations of 0.01–5μM, 0.2μM and 1μM, respectively, eliminated nAChR agonist effects. ACh also affected adhesion of PMNs, this effect was also prevented by αCTX (100nM) and MII (1nM). Neutrophils of female mice after chronic nicotine consumption acquired sensitivity to nAChR agonists. Changes of free intracellular Ca2+ concentration in neutrophils under the action of nAChR ligands were analyzed. In cells with no Ca2+ oscillations and relatively low resting level of intracellular Ca2+, nicotine triggered Ca2+-spikes, the lag of the response shortened with increasing nicotine concentration. A nicotinic antagonist caramiphen strongly decreased the effect of nicotine. RT-PCR analysis revealed mRNAs of α2, α3, α4, α5, α6, α7, α9, β2, β3, and β4 nAChR subunits. Specific binding of [125I]-α-bungarotoxin was demonstrated. Thus in view of the effects and binding characteristics the results obtained suggest a regulatory role of α7, α3β2 or α6* nAChR types in specific functions of PMNs.

Renal cell carcinoma alters endothelial receptor expression responsible for leukocyte adhesion.

Renal cell carcinoma (RCC) escapes immune recognition. To elaborate the escape strategy the influence of RCC cells on endothelial receptor expression and endothelial leukocyte adhesion was evaluated. Human umbilical vein endothelial cells (HUVEC) were co-cultured with the RCC cell line, Caki-1, with and without tumor necrosis factor (TNF)-alpha. Intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial (E)-selectin, standard and variants (V) of CD44 were then analysed in HUVEC, using flow cytometry and Western blot analysis. To determine which components are responsible for HUVEC-Caki-1 interaction causing receptor alteration, Caki-1 membrane fragments versus cell culture supernatant were applied to HUVECS. Adhesion of peripheral blood lymphocytes (PBL) and polymorphonuclear neutrophils (PMN) to endothelium was evaluated by co-culture adhesion assays. Relevance of endothelial receptor expression for adhesion to endothelium was determined by receptor blockage. Co-culture of RCC and HUVECs resulted in a significant increase in endothelial ICAM-1, VCAM-1, E-selectin, CD44 V3 and V7 expression. Previous stimulation of HUVECs with TNF-alpha and co-cultivation with Caki-1 resulted in further elevation of endothelial CD44 V3 and V7 expression, whereas ICAM-1, VCAM-1 and E-selectin expression were significantly diminished. Since Caki-1 membrane fragments also caused these alterations, but cell culture supernatant did not, cell-cell contact may be responsible for this process. Blocking ICAM-1, VCAM-1, E-selectin or CD44 with respective antibodies led to a significant decrease in PBL and PMN adhesion to endothelium. Thus, exposing HUVEC to Caki-1 results in significant alteration of endothelial receptor expression and subsequent endothelial attachment of PBL and PMN.

Sphingosine-1-phosphate receptor 2 mediates endothelial cells dysfunction by PI3K-Akt pathway under high glucose condition

Endothelial dysfunction is believed the early stage of development of diabetic cardiovascular complications. Sphingosine-1-phosphate (S1P) regulates various biological activities by binding to sphingosine-1-phosphate receptors (S1PRs) including S1PR1-S1PR5. In the present study, the role of S1P receptors in S1P-induced human coronary artery endothelial cells (HCAECs) dysfunction under high glucose condition was investigated and the underlying mechanism was explored. S1PR1-S1PR5 mRNA levels were detected by quantitative Real-time PCR. NO level and polymorphonuclear neutrophils (PMN)-endothelial cells adhesion were measured by nitrate reductase and myeloperoxidase colorimetric method, respectively. Protein levels of endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1(ICAM-1), phosphatidylinositol 3-kinase (PI3K) and Akt were measured by Western blot analysis. S1PR2 were found the predominant S1P receptor expressed in HCAECs exposed to high glucose. NO level and eNOS activity were remarkably decreased, while PMN adhesion, VCAM-1 and ICAM-1 protein levels were increased significantly by S1P treatment in HCAECs exposed to high glucose and normal glucose. Blockage of S1PR2 with specific antagonist JTE-013 and small interfering RNA (siRNA) resulted in enhanced NO level and eNOS activity as well as decreased PMN adhesion, reduced protein levels of VCAM-1 and ICAM-1 induced by S1P. Furthermore, Phosphor-PI3K and phosphor-Akt level were markedly increased by S1PR2 blockade in S1P-treated cells exposed to high glucose, which were suppressed by PI3K inhibitor wortmannin. In conclusion, S1P/S1PR2 mediated endothelial dysfunction partly by inhibiting PI3K/Akt signaling pathway under high glucose condition. S1PR2 blockage could ameliorate endothelial dysfunction which might provide a potential therapeutic strategy for diabetic vascular complications.

Neutrophil interactions with epithelial-expressed ICAM-1 enhances intestinal mucosal wound healing.

A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. While epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 plays an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing.

Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.

Involvement of leucocyte/endothelial cell interactions in anorexia nervosa.

Anorexia nervosa is a common psychiatric disorder in adolescence and is related to cardiovascular complications. Our aim was to study the effect of anorexia nervosa on metabolic parameters, leucocyte-endothelium interactions, adhesion molecules and proinflammatory cytokines. This multicentre, cross-sectional, case-control study employed a population of 24 anorexic female patients and 36 controls. We evaluated anthropometric and metabolic parameters, interactions between leucocytes polymorphonuclear neutrophils (PMN) and human umbilical vein endothelial cells (HUVEC), proinflammatory cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and soluble cellular adhesion molecules (CAMs) including E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Anorexia nervosa was related to a decrease in weight, body mass index, waist circumference, systolic blood pressure, glucose, insulin and HOMA-IR, and an increase in HDL cholesterol. These effects disappeared after adjusting for BMI. Anorexia nervosa induced a decrease in PMN rolling velocity and an increase in PMN rolling flux and PMN adhesion. Increases in IL-6 and TNF-α and adhesion molecule VCAM-1 were also observed. This study supports the hypothesis of an association between anorexia nervosa, inflammation and the induction of leucocyte-endothelium interactions. These findings may explain, in part at least, the increased risk of vascular disease among patients with anorexia nervosa.

G408(P) Cytokine storm associated multi-organ failure with poor neurological outcome, during rituximab administration in a child with relapsed acute lymphoblastic leukaemia and ebv related lymphoproliferative disease

We present the case of a 6 year old boy, with relapsed Acute Lymphoblastic Leukaemia and subsequent matched unrelated bone marrow transplant. His Epstein Barr Virus (EBV) titres were significant 6 months post-transplant with evidence of EBV related lymphoproliferative disease. Following local protocols, Rituximab was used for therapy and within minutes of starting the infusion the patient suffered pyrexia, hypotension and seizures with subsequent multi-organ failure, due to a cytokine storm. Organ damage included fulminant hepatic failure, renal failure meriting dialysis, cardiac failure requiring inotropic support and a significant acute brain injury. There was significant neurological impairment with radiological and electrophysiological evidence of global brain cell damage. As a result of irreversible neurological injury, the focus of treatment was switched from curative to palliative. Cytokine storm is an immune mediated phenomenon, characterised by an overwhelming release of cytokines. This can produce a sepsis like response and may lead to multi-organ failure. Cytokines are normally produced by leucocytes as a response to an infective or inflammatory process and their main role is to induce leukopoeisis. Rituximab is an anti-CD20 monoclonal antibody that has been useful in treating EBV related lymphoproliferative disease. Rituximab triggers the rapid release of cytokines that may lead to a cytokine storm with maximum levels within 2 h from the start of the infusion. Cytokine storms are characterised by overproduction of immune mediators that in turn lead to cellular overactivation, increased endothelial permeability, polymorphonuclear neutrophil adhesion and migration. As there is lack of regulatory intervention, tissue congestion with activated leucocytes ensues which eventually causes parenchymal injury. Although we report multi-organ failure in this case, lungs and gut are the organs that are more commonly affected. The incidence of cytokine storm in paediatric oncology patients is not known. It has been associated with haemophagocytic lymphohistiocytosis, graft-versus-host disease following haemopoetic stem cell transplant and the use of certain monoclonal antibodies. As in this case, results of the cytokine storm can be catastrophic, despite early recognition.

Mechanosignal Transduction via NOX2 with Lung Ischemia Reperfusion: Lessons for Lung Transplant

I/R injury, a major cause of lung transplant failure, occurs predominantly due to recruitment of polymorphonuclear neutrophils (PMN) into the (donor) lung endothelium from the recipient's circulation post‐transplant surgery. Endothelial signaling is pivotal in driving the recruitment and adherence of PMN to the vascular wall. We used our past research, expertise and knowledge on pulmonary endothelial mechanosignaling (wherein we unraveled the cascade associated with ischemia to show a KATP closure induced endothelial NADPH oxidase 2 or NOX2 activation and reactive oxygen species, ROS, production) to evaluate how stop and restart of flow lead to PMN adherence. We show here (using in vitro, in situ and in vivo models of lung I/R) that endothelial NOX2 activation (as monitored by ROS production in vitro, in situ and oxidative damage in vivo) is linked to expression of adhesion molecules (CAMs) and to the recruitment of PMN. The mechanosignal can be blocked by preventing NOX2 activation either directly by blocking NOX2 assembly (with apocynin or with MJ33 that blocks the PLA2 activity of peroxiredoxin 6) or by blocking the upstream events (KATP channel); this has potential application in lung transplantation.

Levosimendan exerts anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro.

Levosimendan is a positive inotropic drug for the treatment of acute decompensated heart failure (HF). Clinical trials showed that levosimendan was particularly effective in HF due to myocardial infarction. Myocardial necrosis induces a strong inflammatory response, involving chemoattractants guiding polymorphonuclear neutrophils (PMN) into the infarcted myocardial tissue. Our aim was to examine whether levosimendan exhibits anti-inflammatory effects on human adult cardiac myocytes (HACM) and human heart microvascular endothelial cells (HHMEC). Cardiac myocytes and endothelial cells were stimulated with interleukin-1β (IL)-1β (200 U/ml) and treated with levosimendan (0.1-10 µM) for 2-48 hours. IL-1β strongly induced expression of IL-6 and IL-8 in HACM and E-selectin and intercellular adhesion molecule-1 (ICAM-1) in HHMEC and human umbilical vein endothelial cells (HUVEC). Treatment with levosimendan strongly attenuated IL-1β-induced expression of IL-6 and IL-8 in HACM as well as E-selectin and ICAM-1 in ECs. Levosimendan treatment further reduced adhesion of PMN to activated endothelial cells under both static and flow conditions by approximately 50 %. Incubation with 5-hydroxydecanoic acid, a selective blocker of mitochondrial ATP-dependent potassium channels, partly abolished the above seen anti-inflammatory effects. Additionally, levosimendan strongly diminished IL-1β-induced reactive oxygen species and nuclear factor-κB (NF-κB) activity through inhibition of S536 phosphorylation. In conclusion, levosimendan exhibits anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro. These findings could explain, at least in part, the beneficial effects of levosimendan after myocardial infarction.

Inhibition of neutrophil-dependent cytotoxicity for human endothelial cells by ACE inhibitors.

Angiotensin-converting enzyme inhibitors (ACEi) have immunomodulating properties and have been suggested to protect against endothelial injury, for example myocardial infarction and reperfusion injury. We tested whether two ACEi (captopril and enalapril), differing in a thiol group, protected human umbilical vein endothelial cells (HUVEC) from cytotoxicity induced by polymorphonuclear neutrophils (PMN) in vitro, when cells were activated by tumour necrosis factor-α (TNFα) or the arachidonate derivative lipoxin-A4 (LXA4 ), using separate cytotoxicity pathways. When (51) Cr labelled HUVEC were treated with captopril (0-500μm) or enalapril (0-100μm) for 2h and then activated by TNFα (100ng/ml) for 24h, a significant, dose-dependent reduction of (51) Cr release was observed. Similarly, captopril reduced (51) Cr release when LXA4 (0.1μm) was used to stimulate PMN for 4h. Among previously defined mechanisms of significance for the cytotoxic reaction, expression of ICAM-1, but not intracellular Ca(2+) changes in PMN or PMN adherence to HUVEC, were reduced by ACEi treatment. Moreover, both ACEi inhibited HUVEC surface expression of TNFα receptor I (but not II). Thus, these ACEi, particularly captopril, interfere with PMN-induced cytotoxicity for endothelial cells by modulating pro-inflammatory surface receptors, which is a novel effect that might be explored for further therapeutic approaches.

Regulatory mechanism of pyrrolidine dithiocarbamate is mediated by nuclear factor-κB and inhibits neutrophil accumulation in ARDS mice.

The aim of the present study was to investigate the regulatory mechanism of nuclear factor (NF)-κB on polymorphonuclear neutrophil (PMN) accumulation and the inflammatory response in lung tissues with acute respiratory distress syndrome (ARDS), as well as the therapeutic effect of pyrrolidine dithiocarbamate (PDTC). Mouse models of ARDS were established by intraperitoneal injection of lipopolysaccharide (LPS). BALB/c mice were divided into control, LPS and PDTC + LPS groups. The expression of PMN adhesion molecules, CD11b/CD18 and intercellular adhesion molecule-1 (ICAM-1), were detected by immunohistochemistry, while the protein expression levels of NF-κB p65 in the lung tissue were analyzed by western blot analysis. In addition, flow cytometry was used to investigate the apoptosis rate of PMNs in the bronchoalveolar fluid, and the expression levels of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α and myeloperoxidase (MPO) activity were also determined. Following an intraperitoneal injection of LPS, alveolar septum rupture, pulmonary interstitial hyperemia and PMN infiltration in the alveolar was observed. The protein expression of p65 in the pulmonary cytoplasm decreased, while the expression of p65 in the nucleus increased. The levels of IL-8, IL-1β and TNF-α increased and the high expression status was maintained for 24 h. As the time increased, CD11b/CD18 and ICAM-1 expression increased, as well as MPO activity, while the apoptosis of PMNs was delayed. Compared with the LPS group, the expression of p65 in the pulmonary cytoplasm and the PMN apoptosis rate increased following PDTC intervention, while the expression of p65 in the nucleus decreased, as well as the expression levels of the cytokines and MPO activity. Therefore, PDTC reduced the production of inflammatory cytokines via the NF-κB pathway, which reduced the activation of PMNs in the lung tissue and promoted PMN apoptosis.