Case Report: Durable therapy response to Osimertinib in rare EGFR Exon 18 mutated NSCLC.

Up to 20% of all non-small cell lung cancer patients harbor tumor specific driver mutations that are effectively treated with tyrosine kinase inhibitors. However, for the rare EGFR deletion-insertion mutation of exon 18, there is very little evidence regarding the effectiveness of tyrosine kinase inhibitors. A particular challenge for clinicians in applying tyrosine kinase inhibitors is not only diagnosing a mutation but also interpreting rare mutations with unclear therapeutic significance. Thus, we present the case of a 65-year-old Caucasian male lung adenocarcinoma patient with an EGFR Exon 18 p.Glu709_Thr710delinsAsp mutation of uncertain therapeutic relevance. This patient initially received two cycles of standard platinum-based chemotherapy without any therapeutic response. After administration of Osimertinib as second line therapy, the patient showed a lasting partial remission for 12 months. Therapy related toxicities were limited to mild thrombocytopenia, which ceased after dose reduction of Osimertinib. To our knowledge, this is the first report of effective treatment of this particular mutation with Osimertinib. Hence, we would like to discuss Osimertinib as a viable treatment option in EGFR Exon 18 p.Glu709_Thr710delinsAsp mutated lung adenocarcinoma.

Targeting CREB-binding protein overrides LPS induced radioresistance in non-small cell lung cancer cell lines.

Non-small cell lung cancer (NSCLC) has a very poor prognosis even when treated with the best therapies available today often including radiation. NSCLC is frequently complicated by pulmonary infections which appear to impair prognosis as well as therapy, whereby the underlying mechanisms are still not known. It was investigated here, whether the bacterial lipopolysaccharides (LPS) might alter the tumor cell radiosensitivity. LPS were found to induce a radioresistance but solely in cells with an active TLR-4 pathway. Proteome profiling array revealed that LPS combined with irradiation resulted in a strong phosphorylation of cAMP response element-binding protein (CREB). Inhibition of CREB binding protein (CBP) by the specific inhibitor ICG-001 not only abrogated the LPS-induced radioresistance but even led to an increase in radiosensitivity. The sensitization caused by ICG-001 could be attributed to a reduction of DNA double-strand break (DSB) repair. It is shown that in NSCLC cells LPS leads to a CREB dependent radioresistance which is, however, reversible through CBP inhibition by the specific inhibitor ICG-001. These findings indicate that the combined treatment with radiation and CBP inhibition may improve survival of NSCLC patients suffering from pulmonary infections.

Lipoteichoic acids from Staphylococcus aureus stimulate proliferation of human non-small-cell lung cancer cells in vitro.

Pulmonary infections are frequent complications in lung cancer and may worsen its outcome and survival. Inflammatory mediators are suspected to promote tumor growth in non-small-cell lung cancer (NSCLC). Hence, bacterial pathogens may affect lung cancer growth by activation of inflammatory signalling. Against this background, we investigated the effect of purified lipoteichoic acids (LTA) of Staphylococcus aureus (S. aureus) on cellular proliferation and liberation of interleukin (IL)-8 in the NSCLC cell lines A549 and H226. A549 as well as H226 cells constitutively expressed TLR-2 mRNA. Even in low concentrations, LTA induced a prominent increase in cellular proliferation of A549 cells as quantified by automatic cell counting. In parallel, metabolic activity of A549 cells was enhanced. The increase in proliferation was accompanied by an increase in IL-8 mRNA expression and a dose- and time-dependent release of IL-8. Cellular proliferation as well as the release of IL-8 was dependent on specific ligation of TLR-2. Interestingly, targeting IL-8 by neutralizing antibodies completely abolished the LTA-induced proliferation of A549 cells. The pro-proliferative effect of LTA could also be reproduced in the squamous NSCLC cell line H226. In summary, LTA of S. aureus induced proliferation of NSCLC cell lines of adeno- and squamous cell carcinoma origin. Ligation of TLR-2 followed by auto- or paracrine signalling by endogenously synthesized IL-8 is centrally involved in LTA-induced tumor cell proliferation. Therefore, pulmonary infections may exert a direct pro-proliferative effect on lung cancer growth.

In cancer cell lines inhibition of SCF/c-Kit pathway leads to radiosensitization only when SCF is strongly over-expressed.

BACKGROUND AND PURPOSE: The SCF/c-Kit pathway is often overexpressed in human tumors leading to an enhanced tumorigenesis, proliferation and migration. It was now tested for NSCLC and prostate cancer cells growing in 2D and 3D whether the inhibition of this pathway can be used to achieve a significant radiosensitization and whether a respective biomarker may be identified. MATERIAL AND METHODS: Experiments were performed with different cancer cell lines (NSCLC: H23, H520, H226, H1975 and PrCa: DU145) growing either under 2D or 3D conditions. Expression of SCF and c-Kit was determined by RT-PCR and Western blot, SCF was knocked down by siRNA, c-Kit was inhibited by ISCK03 inhibitor and cell survival was determined by colony formation assay. RESULTS: There is a profound variation in the expression of both c-Kit and SCF with no association between each other. Neither levels did correlate with the respective cellular radiosensitivity determined for 2D or 3D with only a trend seen for SCF. Knock-down of SCF was generally found to result in no or only minor reduction of plating efficiency or cellular radioresistance. A significant reduction was only obtained for H520 cells characterized by an extreme over-expression of SCF. The inhibition of c-Kit by a specific inhibitor was also found to result only in minor radiosensitization. CONCLUSION: Generally, the SCF/c-Kit pathway does not have a dominant effect on both, cell survival and radioresponse and, as a consequence, knockdown of this pathway does not result in a strong effect on radioresistance, except when SCF is strongly over-expressed.

Cancer cell motility is affected through 3D cell culturing and SCF/c-Kit pathway but not by X-irradiation.

BACKGROUND AND PURPOSE: Success of radiotherapy is often limited by therapy resistance and metastasis resulting from cancer cell motility. It was tested in vitro whether this cancer cell motility is affected by growth condition, active SCF/c-Kit pathway or X-irradiation. MATERIALS AND METHODS: Cell motility was measured with BioCoat™ Matrigel™ invasion chamber using four different cancer cell lines (NSCLC: H23, H520, H226 and PrCa: DU145). Cells were grown in 2D or 3D, SCF was knocked down by siRNA and cells were irradiated with 2 or 6Gy. RESULTS: All cell lines except H520 showed a 2-3-fold increase in cell motility when grown in 3D. This effect was considered to result from the EMT-like change seen when cells were grown in 3D as indicated by the enhanced expression of vimentin and N-cadherin and reduction of E-cadherin. Just the opposite trends were found for H520 cells. Knockdown of SCF was found to result in reduced cell motility for both 2D and 3D. In contrast, X-irradiation did not modulate cell motility neither under 2D nor 3D. In line with this, X-irradiation did neither induce the expression of EMT-associated genes nor SCF. CONCLUSION: X-irradiation affects neither the expression of important EMT genes such as vimentin, E-cadherin and N-cadherin nor SCF/c-Kit signaling and, as a consequence, does not alter cell motility.

Interactions between neutrophils and non-small cell lung cancer cells: enhancement of tumor proliferation and inflammatory mediator synthesis.

The inflammatory tumor microenvironment plays a crucial role in tumor progression. In lung cancer, both bacterial infections and neutrophilia are associated with a poor prognosis. In this study, we characterized the effect of isolated human neutrophils on proliferation of the non-small cell lung cancer (NSCLC) cell line A549 and analyzed the impact of A549-neutrophil interactions on inflammatory mediator generation in naive and lipopolysaccharide (LPS)-exposed cell cultures. Co-incubation of A549 cells with neutrophils induced proliferation of resting and LPS-exposed A549 cells in a dose-dependent manner. In transwell-experiments, this effect was demonstrated to depend on direct cell-to-cell contact. This pro-proliferative effect of neutrophils on A549 cells could be attenuated by inhibition of neutrophil elastase activity, but not by oxygen radical neutralization. Correspondingly, neutrophil elastase secretion, but not respiratory burst, was specifically enhanced in co-cultures of A549 cells and neutrophils. Moreover, interference with COX-2 activity by indomethacin or the specific COX-2 inhibitor NS-398 also blunted the increased A549 proliferation in the presence of neutrophils. In parallel, a massive amplification of COX-2-dependent prostaglandin E2 synthesis was detected in A549-neutrophil co-cultures. These findings suggest that direct cell-cell interactions between neutrophils and tumor cells cause release of inflammatory mediators which, in turn, may enhance tumor growth in NSCLC.

Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation.

Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E(2) were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE(2) release and proliferation in response to LPS. Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.

Mechanism of transfusion-related acute lung injury induced by HLA class II antibodies.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality in the United States and other countries. In most TRALI cases, human leukocyte antigen (HLA) class II antibodies are detected in implicated donors. However, the corresponding antigens are not present on the cellular key players in TRALI: neutrophils and endothelium. In this study, we identify monocytes as a primary target in HLA class II-induced TRALI. Monocytes become activated when incubated with matched HLA class II antibodies and are capable of activating neutrophils, which, in turn, can induce disturbance of an endothelial barrier. In an ex vivo rodent model, HLA class II antibody-dependent monocyte activation leads to severe pulmonary edema in a relevant period of time, whenever neutrophils are present and the endothelium is preactivated. Our data suggest that in most TRALI cases, monocytes are cellular key players, because HLA class II antibodies induce TRALI by a reaction cascade initiated by monocyte activation. Furthermore, our data support the previous assumption that TRALI pathogenesis follows a threshold model. Having identified the biologic mechanism of HLA class II antibody-induced TRALI, strategies to avoid plasma from immunized donors, such as women with a history of pregnancy, appear to be justified preventive measures.

Amplification of lipopolysaccharide-induced cytokine synthesis in non-small cell lung cancer/neutrophil cocultures.

Proinflammatory cytokines are centrally involved in tumor progression and survival in non-small cell lung cancer, and both the presence of infiltrating neutrophils and bacterial infection in the lung may indicate a poor prognosis. Against this background, we investigated the effect of the bacterial cell wall component lipopolysaccharide (LPS) on interleukin (IL)-6 and IL-8 synthesis in the non-small cell lung cancer line A549 and in A549-neutrophil cocultures. The LPS induced a dose-dependent and time-dependent release of IL-8 from A549 cells, whereas IL-6 could not be detected. Interestingly, in A549-neutrophil cocultures, IL-8 synthesis was massively amplified and IL-6 was also released, compared with the respective monocultures. The A549 cells were identified as the primary cellular source of these cytokines, as enhanced cytokine mRNA transcription was detected in this cell type, although not in neutrophils in the coculture system. Experiments done in transwells indicated that direct cell-cell contact was a prerequisite for the increased cytokine generation. Inhibition of tumor necrosis factor-alpha bioactivity by neutralizing antibodies and blocking cyclooxygenase-2 activity blunted the enhanced cytokine generation in the coculture system. Amplification of LPS-induced cytokine secretion could be reproduced when the small cell lung cancer cell line H69 was cocultured with neutrophils. When the Gram-positive cell wall component lipoteichoic acid was used instead of LPS, cytokine synthesis was also amplified in A549-neutrophil cocultures, to a similar extent to that observed with LPS. These data indicate that interaction between bacterial pathogens, neutrophils, and tumor cells might amplify the release of proinflammatory cytokines which may promote tumor growth in vivo.

Staphylococcus aureus alpha-toxin and Escherichia coli hemolysin impair cardiac regional perfusion and contractile function by activating myocardial eicosanoid metabolism in isolated rat hearts.

OBJECTIVE: In sepsis, cardiac function is frequently depressed. Microcirculatory disturbances as evidenced in most organs may extend to the coronary circulation and may play a role in the occurrence of cardiac dysfunction. Staphylococcal alpha-toxin and Escherichia coli hemolysin (ECH), pore-forming exotoxins of clinically relevant bacteria, have recently been demonstrated to evoke cardiac dysfunction in isolated rat hearts by activating myocardial eicosanoid metabolism. alpha-Toxin activates synthesis of thromboxane (Tx) A2, ECH of cysteinyl-leukotrienes (Sibelius U, Grandel U, Buerke M, et al: Leukotriene-mediated coronary vasoconstriction and loss in myocardial contractility evoked by low doses of Escherichia coli hemolysin in perfused reat hearts. Crit Care Med 2003; 3:683-688, Sibelius U, Grandel U, Buerke M, et al: Staphylococcal alpha-toxin provokes coronary vasoconstriction and loss in myocardial contractility in perfused rat hearts-Role of Tx formation. Circulation 2000; 101:78-85). We now investigated whether cardiac dysfunction in response to alpha-toxin and ECH is caused by disturbances of regional cardiac perfusion. DESIGN: A prospective, experimental study. SETTING: A research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Changes of regional perfusion were investigated by using colored microspheres in isolated rat hearts perfused with alpha-toxin or ECH either at constant coronary perfusion pressure or constant coronary flow rate. Significance of toxin-activated eicosanoid generation was evaluated by pharmacologic interventions. MEASUREMENTS AND MAIN RESULTS: By eliciting eicosanoid formation, both toxins caused an increase in coronary vascular resistance and a loss in contractile function. In ECH-perfused hearts, reduction of regional perfusion predominantly occurred in subendocardial sections in either perfusion mode (coronary perfusion pressure or coronary flow rate). When synthesis of cysteinyl-leukotrienes was blocked by the 5-lipooxygenase inhibitor MK-886, disturbances of regional perfusion and the associated cardiac dysfunction were largely prevented. Coronary perfusion of alpha-toxin caused a decrease of regional perfusion that was more pronounced in subepicardial layers. Inhibiting the release of TxA2 by blocking the cyclooxygenase with indomethacin attenuated the perfusion abnormalities and the cardiodepression in response to alpha-toxin. CONCLUSIONS: Bacterial exotoxins of clinically relevant bacteria may impair cardiac function by eliciting distinct coronary perfusion abnormalities via release of vasoactive eicosanoids.

Lipoteichoic acid (LTA) from Staphylococcus aureus stimulates human neutrophil cytokine release by a CD14-dependent, Toll-like-receptor-independent mechanism: Autocrine role of tumor necrosis factor-[alpha] in mediating LTA-induced interleukin-8 generation.

OBJECTIVE: In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils. SUBJECTS: Isolated human neutrophils from healthy volunteers. INTERVENTIONS: Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies. MEASUREMENTS: Measurement of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction. CONCLUSIONS: The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-alpha and IL-1beta and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-alpha, as it was largely abrogated by neutralizing anti-TNF-alpha antibodies. In contrast, IL1-beta was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-alpha generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-alpha generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.

Antibody-induced neutrophil activation as a trigger for transfusion-related acute lung injury in an ex vivo rat lung model.

Transfusion-related acute lung injury (TRALI) is a hazardous complication of transfusion and has become the leading cause of transfusion-related death in the United States and United Kingdom. Although leukoagglutinating antibodies have been frequently shown to be associated with the syndrome, the mechanism by which they induce TRALI is poorly understood. Therefore, we reproduced TRALI in an ex vivo rat lung model. Our data demonstrate that TRALI induction by antileukocyte antibodies is dependent on the density of the cognate antigen but does not necessarily require leukoagglutinating properties of the antibody or the presence of complement proteins. Rather, antibody-mediated activation of neutrophils seems to initiate TRALI, a process that could be triggered by neutrophil stimulation with fMLP. Antibody-mediated neutrophil activation and subsequent release of reactive oxygen species may thus represent key events in the pathophysiologic cascade that leads to immune TRALI.

Free arachidonic versus eicosapentaenoic acid differentially influences the potency of bacterial exotoxins to provoke myocardial depression in isolated rat hearts.

OBJECTIVE: Staphylococcal alpha-toxin and Escherichia coli hemolysin (ECH) evoke cardiac dysfunction in isolated rat hearts by provoking myocardial synthesis of arachidonic acid-derived thromboxane A2 or the cysteinyl-leukotrienes, LTC4, LTD4, and LTE4, respectively. We investigated whether low doses of either toxin, which fail to induce cardiac depression by themselves, induce cardiac dysfunction when combined with free arachidonic acid. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Hearts were perfused with low doses of ECH or alpha-toxin in the absence or presence of arachidonic acid or the alternative eicosanoid precursor eicosapentaenoic acid (EPA). MEASUREMENTS AND MAIN RESULTS: Application of low-dose ECH with arachidonic acid increased coronary perfusion pressure, depressed left ventricular contractile function, provoked electrical instability, and induced a release of creatine kinase concomitant with the liberation of LTC4, LTD4, and LTE4 into the perfusate. All events were abolished when formation of cysteinyl-leukotrienes was blocked by the 5-lipoxygenase activity inhibitor MK-886, targeting 5-lipoxygenase activating protein. In the presence of arachidonic acid, low doses of alpha-toxin caused an increase in cerebral perfusion pressure and a decline of contractile performance, attributable to the release of thromboxane A2, as both events were mitigated by the cyclooxygenase-inhibitor indomethacin. High doses of ECH caused cardiac dysfunction even in the absence of arachidonic acid. However, in the presence of EPA, the cardiodepressant effect of ECH was blunted. Release of EPA-derived LTE5 at the expense of arachidonic acid-derived LTC4, LTD4, and LTE4 was noted in these hearts. CONCLUSIONS: The potency of the bacterial exotoxins ECH and alpha-toxin to cause coronary vasoconstriction and myocardial depression is dependent on the availability of free arachidonic acid and may be influenced by supplying omega-3 fatty acids as alternative lipid precursors.

Mechanisms of cardiac depression caused by lipoteichoic acids from Staphylococcus aureus in isolated rat hearts.

BACKGROUND: Lipoteichoic acid (LTA) represents a major virulence factor in gram-positive sepsis. METHODS AND RESULTS: In the present study we perfused isolated rat hearts for 180 minutes with highly purified LTA from Staphylococcus aureus. A progressive decline of left ventricular contractile function paralleled by the expression of myocardial tumor necrosis factor-alpha (TNF-alpha) mRNA and protein as well as the release of TNF-alpha into the perfusate was observed in LTA-perfused hearts. Employment of an anti-TNF-alpha antibody completely prevented the loss in contractile function. When CD14, a prominent pathogen recognition receptor, was blocked by a specific antibody, induction of TNF-alpha mRNA and protein release as well as the associated cardiodepression was diminished in response to LTA. Synthesis of TNF-alpha protein was located to interstitial cells of LTA-challenged hearts as detected by immunohistochemistry. Besides progressive cardiodepression, coronary perfusion pressure (CPP) was moderately increased in LTA-perfused hearts. This was accompanied by the release of thromboxane A2 (TXA2) into the perfusate and the induction of cyclooxygenase (Cox)-2 mRNA and protein in the myocardium. Blocking of TXA2 by the nonspecific Cox inhibitor indomethacin, the thromboxane receptor antagonist daltroban, or the selective Cox-2 inhibitor NS-398 prevented the increase in CPP. CONCLUSIONS: LTA causes cardiac depression by activating myocardial TNF-alpha synthesis via CD14 and induces coronary vascular disturbances by activating Cox-2-dependent TXA2 synthesis. These phenomena may contribute to cardiac depression in gram-positive sepsis.

Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation.

In Wegener's granulomatosis (WG), a pathogenetic role has been proposed for circulating anti-neutrophil-cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3). Disease activation in WG appears to be triggered by bacterial infections. In the present study, we characterized the effect of anti-PR3 antibodies on in vitro activation of isolated monocytes and neutrophils by the bacterial cell-wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Although sole incubation of monocytes and neutrophils with monoclonal anti-PR3 antibodies induced the release of minor quantities of the chemokine interleukin-8 (IL-8), preincubation with anti-PR3 antibodies, but not with isotype-matched control immunogloblin G (IgG), resulted in a markedly enhanced IL-8 liberation upon LPS challenge. The priming response was evident after 2 h of preincubation with anti-PR3 and peaked after 6 h. The anti-PR3-related priming was also observed for tumor necrosis factor alpha (TNF-alpha) and IL-6 synthesis. Comparable priming occurred when leukocytes were preincubated with ANCA-IgG derived from WG serum but not with normal IgG. The priming effect of the anti-PR3 antibody pretreatment was reproduced for LTA challenge of monocytes and neutrophils but not for leukocyte stimulation with TNF-alpha. Flow cytometric analysis revealed an increase in monocyte and neutrophil membrane CD14 expression during the anti-PR3 priming. We conclude that cytoplasmic ANCA specifically prime CD14-dependent monocytes and neutrophils for activation. The resulting enhanced responsiveness to bacterial pathogens may contribute to the development and maintenance of inflammatory lesions during active WG.

Increased neutrophil mediator release in patients with pulmonary hypertension--suppression by inhaled iloprost.

Polymorphonuclear neutrophils (PMN) have been implicated in various vascular inflammatory processes. We isolated PMN from venous blood samples of 10 patients with severe primary pulmonary arterial hypertension (PPH), 7 patients with pulmonary hypertension secondary to chronic thromboembolism (CTEPH), and 12 healthy controls. When stimulated with the calcium-ionophore A23187, platelet activating factor (PAF) or the microbial agent n-formyl-Methionyl-Leucyl-Phenylalanine (fMLP), significantly increased release of elastase and superoxide anion was noted in both groups with pulmonary hypertension. Moreover, the neutrophils of CTEPH patients responded with an enhanced liberation of leukotriene (LT) B(4) and 5-hydroxyeicosatetraenoic acid (5-HETE). Inhalation of aerosolized iloprost (5 microg) caused a rapid decline in pulmonary vascular resistance, in both PPH and CTEPH. This hemodynamic response was paralleled by a significant suppression of ionophore- and ligand-induced elastase and superoxide release, as well as LTB(4) and 5-HETE formation. The neutrophil inhibitory effect of the inhalation maneuver was fully reproduced by in vitro incubation of neutrophils with 1-10 pg/ml iloprost for 2 hours. This is the first study to demonstrate that circulating neutrophils from patients with PPH and CTEPH possess an enhanced readiness to respond with inflammatory mediator generation to different stimulatory agents ex-vivo, and that PMN respiratory burst, elastase secretion and leukotriene generation are promptly reduced by an iloprost inhalation maneuver. Neutrophils might participate in the inflammatory processes in pulmonary arterial hypertension.

Omega-3 vs. omega-6 lipid emulsions exert differential influence on neutrophils in septic shock patients: impact on plasma fatty acids and lipid mediator generation.

OBJECTIVE: To compare the effects of a conventional omega-6 lipid infusion and a fish oil based (omega-3) lipid infusion for parenteral nutrition on neutrophil function, lipid mediators, and plasma free fatty acids. DESIGN AND SETTING: Open-label, randomized, pilot study in a university hospital medical intensive care unit and experimental laboratory. PATIENTS AND PARTICIPANTS: Ten patients with septic shock and eight healthy controls. INTERVENTIONS: Patients (five per group) requiring parenteral nutrition received intravenously either a omega-3 or a omega-6 lipid emulsion for a 10-day period. MEASUREMENTS AND RESULTS: At baseline levels of plasma free fatty acids were elevated several-fold, including high concentrations of the omega-6 lipid precursor arachidonic acid (AA). Neutrophils isolated from septic patients displayed markedly reduced responsiveness to ex vivo stimulation, including lipid mediator generation [leukotrienes (LT), PAF], respiratory burst, and phosphoinositide hydrolysis signaling. Under the omega-6 lipid infusion regimen abnormalities in plasma free fatty acids and impairment of neutrophil functions persisted or worsened. In contrast, a rapid switch in the plasma free fatty acid fraction to predominance of the omega-3 acids eicosapentaenoic acid and docosahexaenoic acid over AA occurred in response to omega-3 lipid infusion. LTB(5), in addition to LTB(4), appeared upon neutrophil stimulation originating from these patients, and neutrophil function was significantly improved in the omega-3 lipid group. CONCLUSIONS: omega-3 vs. omega-6 lipid emulsions differentially influence the plasma free fatty acid profile with impact on neutrophil functions. Lipid-based parenteral nutrition in septic patients may thus exert profound influence on sequelae and status of immunocompetence and inflammation.

Parenteral nutrition with fish oil modulates cytokine response in patients with sepsis.

Infusion of fish oil-based (n-3) lipids may influence leukocyte function and plasma lipids in critical care patients. Twenty-one patients with sepsis requiring parenteral nutrition were randomized to receive an n-3 lipid emulsion rich in eicosapentaenoic acid and docosahexaenoic acid or a conventional (n-6) lipid emulsion (index fatty acid: arachidonic acid) for 5 days. The impact on plasma-free fatty acids, mononuclear leukocyte cytokine generation, and membrane fatty acid composition was examined. Cytokine synthesis by isolated mononuclear leukocyte was elicited by endotoxin. Before the onset of lipid infusion therapy, plasma-free fatty acid concentrations were greatly increased in septic patients, with arachidonic acid by far surpassing eicosapentaenoic acid and docosahexaenoic acid, a feature maintained during conventional lipid infusion. Within 2 days of fish oil infusion, free n-3 fatty acids increased, and the n-3/n-6 ratio was reversed, with rapid incorporation of n-3 fatty acids into mononuclear leukocyte membranes. Generation of proinflammatory cytokines by mononuclear leukocytes was markedly amplified during n-6 and was suppressed during n-3 lipid application. After termination of lipid administration, free n-3 fatty acid concentrations and mononuclear leukocyte cytokine synthesis returned to preinfusion values. Use of lipid infusions might allow us to combine intravenous alimentation with differential impact on inflammatory events and immunologic functions in patients with sepsis.

Distinct pathways of lipopolysaccharide priming of human neutrophil respiratory burst: role of lipid mediator synthesis and sensitivity to interleukin-10.

OBJECTIVE: Exposure of neutrophils to low doses of bacterial lipopolysaccharides enhances their readiness to respond with inflammatory mediator generation including oxygen radical formation to a subsequently applied inflammatory stimulus ("priming"). In the present study, we investigated the role of lipid mediator synthesis and the impact of the anti-inflammatory cytokine interleukin-10 on the lipopolysaccharide-dependent priming of human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated neutrophils from healthy volunteers. INTERVENTIONS: Incubation of isolated neutrophils with endotoxin. MEASUREMENTS AND MAIN RESULTS: Evidence for two distinct priming mechanisms was obtained. The first was strictly serum component dependent, proceeded via CD14, and was not inhibited by even high concentrations of interleukin-10. The second priming mechanism was serum component independent but nevertheless proceeded via CD14. It was linked with neutrophil synthesis of the platelet activating factor and resulted in the appearance of leukotrienes, in particular leukotriene B4, as far as exogenous arachidonic acid was provided. The employment of a platelet-activating factor receptor antagonist (WEB 2086) blocked leukotriene synthesis, and both WEB 2086 and a 5-lipoxygenase inhibitor (MK-886) suppressed the respiratory burst linked with this second priming pathway. This sequence of priming events was inhibited by interleukin-10, when this cytokine was coadministered with the priming agent lipopolysaccharide, whereas late interleukin-10 admixture was ineffective. CONCLUSIONS: We conclude that two mechanisms of lipopolysaccharide priming of human neutrophil respiratory burst can be differentiated. One displays serum component dependence, is independent of neutrophil lipid mediator generation, and is not affected by interleukin-10. The other is serum independent although being operated via CD14, employs autocrine loops of platelet-activating factor and leukotriene B4 synthesis, and is sensitive to the inhibitory capacity of interleukin-10. These features may be relevant when the goal is to pharmacologically modify neutrophil functions in septic events.

Wegener's granulomatosis: antiproteinase 3 antibodies induce monocyte cytokine and prostanoid release-role of autocrine cell activation.

Antineutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 [PR3; cytoplasmic ANCA (c-ANCA)], a leukocyte serine protease, are highly specific for Wegener's Granulomatosis (WG). A pathogenetic role for c-ANCA has been proposed as a result of their ability of activating neutrophils, whereas their interaction with monocytes is less well characterized. We investigated the influence of monoclonal anti-PR3 antibodies (anti-PR3) and c-ANCA from WG sera on monocyte cytokine and prostanoid release. We found that PR3 was expressed on the surface of isolated monocytes. Anti-PR3 challenge provoked a pronounced release of cytokines with early appearance of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-1beta and delayed release of IL-6, IL-8, and thromboxane A2 (TxA2). The secretory response was reproduced by c-ANCA but not by human and murine control IgG and anti-CD14 antibodies. Because F(ab)2 fragments of anti-PR3 were ineffective, coligation of Fc gamma receptors (FcgammaR) was apparently mandatory for monocyte activation. Using soluble receptors for TNF-alpha and IL-1beta and a Tx receptor antagonist, we noted that the "early" cytokines functioned as inducers of TxA2, which then activated IL-8 release. In contrast, IL-6 formation was an independent event. We concluded that anti-PR3 antibodies are potent inducers of monocyte cytokine and prostanoid release, and TNF-alpha, IL-1beta, and TxA2 function as facilitators of the secretory response. These mechanisms may contribute to inflammatory tissue injury in WG.