Biomedical subjects

Joseph Cuschieri

Publications and source records attributed to Joseph Cuschieri.

At least 19 recordsLinked to original sources

PubMed2015

Advanced age is associated with worsened outcomes and a unique genomic response in severely injured patients with hemorrhagic shock.

INTRODUCTION: We wished to characterize the relationship of advanced age to clinical outcomes and to transcriptomic responses after severe blunt traumatic injury with hemorrhagic shock. METHODS: We performed epidemiological, cytokine, and transcriptomic analyses on a prospective, multi-center cohort of 1,928 severely injured patients. RESULTS: We found that there was no difference in injury severity between the aged (age ≥55, n = 533) and young (age <55, n = 1395) cohorts. However, aged patients had more comorbidities. Advanced age was associated with more severe organ failure, infectious complications, ventilator days, and intensive care unit length of stay, as well as, an increased likelihood of being discharged to skilled nursing or long-term care facilities. Additionally, advanced age was an independent predictor of a complicated recovery and 28-day mortality. Acutely after trauma, blood neutrophil genome-wide expression analysis revealed an attenuated transcriptomic response as compared to the young; this attenuated response was supported by the patients' plasma cytokine and chemokine concentrations. Later, these patients demonstrated gene expression changes consistent with simultaneous, persistent pro-inflammatory and immunosuppressive states. CONCLUSIONS: We concluded that advanced age is one of the strongest non-injury related risk factors for poor outcomes after severe trauma with hemorrhagic shock and is associated with an altered and unique peripheral leukocyte genomic response. As the general population's age increases, it will be important to individualize prediction models and therapeutic targets to this high risk cohort.

Adult↗

PubMed2014

A better understanding of why murine models of trauma do not recapitulate the human syndrome.

OBJECTIVE: Genomic analyses from blood leukocytes have concluded that mouse injury poorly reflects human trauma at the leukocyte transcriptome. Concerns have focused on the modest severity of murine injury models, differences in murine compared with human age, dissimilar circulating leukocyte populations between species, and whether similar signaling pathways are involved. We sought to examine whether the transcriptomic response to severe trauma in mice could be explained by these extrinsic factors, by utilizing an increasing severity of murine trauma and shock in young and aged mice over time, and by examining the response in isolated neutrophil populations. DESIGN: Preclinical controlled in vivo laboratory study and retrospective cohort study. SETTING: Laboratory of Inflammation Biology and Surgical Science and multi-institution level 1 trauma centers. SUBJECTS: Six- to 10-week-old and 20- to 24-month-old C57BL/6 (B6) mice and two cohorts of 167 and 244 severely traumatized (Injury Severity Score > 15) adult (> 18 yr) patients. INTERVENTIONS: Mice underwent one of two severity polytrauma models of injury. Total blood leukocyte and neutrophil samples were collected. MEASUREMENTS AND MAIN RESULTS: Fold expression changes in leukocyte and neutrophil genome-wide expression analyses between healthy and injured mice (p < 0.001) were compared with human total and enriched blood leukocyte expression analyses of severe trauma patients at 0.5, 1, 4, 7, 14, and 28 days after injury (Glue Grant trauma-related database). We found that increasing the severity of the murine trauma model only modestly improved the correlation in the transcriptomic response with humans, whereas the age of the mice did not. In addition, the genome-wide response to blood neutrophils (rather than total WBC) was also not well correlated between humans and mice. However, the expression of many individual gene families was much more strongly correlated after injury in mice and humans. CONCLUSIONS: Although overall transcriptomic association remained weak even after adjusting for the severity of injury, age of the animals, timing, and individual leukocyte populations, there were individual signaling pathways and ontogenies that were strongly correlated between mice and humans. These genes are involved in early inflammation and innate/adaptive immunity.

Adult↗

PubMed2007

Vitamin E inhibits endotoxin-mediated transport of phosphatases to lipid rafts.

The production and release of inflammatory mediators is regulated by the coordinated activity of kinases and phosphatases. These proteins are known to regulate one another through an unknown mechanism. Previously, we have demonstrated that autocrine release of oxidants regulates macrophage activation in a similar fashion. The purpose of this study is to determine if attenuated oxidant activity by antioxidant exposure can regulate endotoxin-mediated kinase and phosphatase activity. Human promonocytic THP-1 cells were stimulated with lipopolysaccharide. Selected cells were pretreated with alpha-tocopherol succinate, LY294002, or an AKT inhibitor (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate). Lipid raft and cellular protein were analyzed for lipid raft toll-like receptor 4 (TLR4) receptor formation and mitogen-activated protein kinase (MAPK) activation. Harvested supernatants were analyzed for tumor necrosis factor (TNF)-alpha production. Lipopolysaccharide stimulation led to the lipid raft mobilization of TLR4 and heat shock protein 70. This was followed by lipid raft mobilization of SH related complex homology 2 domain-containing inositol-5-phosphate (SHIP), activation of the MAPK, and production of TNF-alpha. Pretreatment with alpha-tocopherol succinate did not affect mobilization of TLR4 or heat shock protein 70, but did result in attenuated mobilization of SHIP, activation of the MAPK, and production of TNF-alpha. In addition, alpha-tocopherol succinate was associated with increased activation of the counter-regulatory kinase protein kinase B. Pretreatment with LY294002 or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate reversed the effects of alpha-tocopherol succinate. Thus, it seems that endotoxin-mediated activation requires the coordinated activity of kinases and phosphatases. Antioxidant exposure in the form of vitamin E seems to attenuate endotoxin-mediated SHIP activation resulting in increased AKT activity, and attenuated MAPK activation and TNF-alpha production.

Cell Differentiation↗

PubMed2006

Endotoxin tolerance attenuates LPS-induced TLR4 mobilization to lipid rafts: a condition reversed by PKC activation.

Endotoxin tolerance is characterized by attenuated macrophage activation to subsequent LPS challenge and can be reversed through nonspecific protein kinase C (PKC) activation, and activation by LPS within naïve cells requires the activation of the cell surface receptors CD14 and TLR4 on lipid rafts. The effect of PKC activation and endotoxin tolerance on lipid raft receptor complex assembly is unknown and the focus of this study. Tolerance was induced in THP-1 cells through LPS pre-exposure. Naïve and tolerant cells were stimulated with LPS, with or without PMA pretreatment to activate PKC. TLR4 surface expression and LPS binding were determined by flow cytometry and immunohistochemistry. Cellular and lipid raft protein was analyzed for the presence and activation of the TLR4 complex components. Harvested supernatants were examined for TNF-alpha production. Total TLR4 surface expression and LPS binding were not affected by tolerance induction. LPS stimulation of naïve cells resulted in TLR4 and heat shock protein (HSP)70 lipid raft mobilization, MAPK activation, and TNF-alpha production. LPS stimulation of tolerant cells was associated with attenuation of all of these cellular events. Although PKC activation by PMA had no effect on naïve cells, it did result in reversal in tolerance-induced suppression of TLR4 and HSP70 lipid raft mobilization, MAPK activation, and TNF-alpha production. In addition, the effects associated with PMA were reversed with exposure to a myristoylated PKC-zeta pseudosubstrate. Thus, endotoxin tolerance appears to be induced through attenuated TLR4 formation following LPS stimulation. This complex formation appears to be PKC-dependent, and restoration of PKC activity reverses tolerance.

Cell Line↗

PubMed2006

Phosphatidylcholine-specific phospholipase C (PC-PLC) is required for LPS-mediated macrophage activation through CD14.

Lipid rafts, composed of sphingolipids, are critical to Toll-like receptor 4 (TLR4) assembly during lipopolysaccharide (LPS) exposure, as a result of protein kinase C (PKC)-zeta activation. However, the mechanism responsible for this remains unknown. The purpose of this study is to determine if LPS-induced TLR4 assembly and activation are dependent on the sphingolipid metabolite ceramide produced by phosphatidylcholine-specific phospholipase C (PC-PLC) or CD14. To study this, THP-1 cells were stimulated with LPS. Selected cells were pretreated with the PC-PLC inhibitor D609, exogenous C2 ceramide, CD14 neutralizing antibody, or TLR4 neutralizing antibody. LPS led to production of ceramide, phosphorylation of PKC-zeta, and assembly of the TLR4 within lipid rafts. This was followed by activation of the mitogen-activated protein kinase family and the liberation of cytokines. Pretreatment with D609 or CD14 blockade was associated with attenuated LPS-induced ceramide production, TLR4 assembly on lipid rafts, and cytokine production. Pretreatment with TLR4 blockade did not affect LPS-induced ceramide production but was associated with significant attenuation in cytokine production. Treatment with C2 ceramide prior to LPS reversed the inhibitory effects induced by D609 but not of CD14 or TLR4 blockade. C2 ceramide alone induced the activation of PKC-zeta and the assembly of TLR4 but was not associated with cytokine liberation. This study demonstrates that TLR4 assembly and activation following LPS exposure require the production of ceramide by PC-PLC, which appears to be CD14-dependent.

Bridged-Ring Compounds↗

PubMed2006

Surgical management of acute necrotizing lung infections.

BACKGROUND: Surgical resection for acute necrotizing lung infections is not widely accepted due to unclear indications and high risk. OBJECTIVE: To review results of resection in the setting of acute necrotizing lung infections. METHODS: A retrospective review of patients who underwent parenchymal resection between January 1, 2000, and January 1, 2006, for management of necrotizing pneumonia or lung gangrene. RESULTS: Thirty-five patients underwent resection for lung necrosis. At the time of consultation, all patients presented with pulmonary sepsis, and also had the following: empyema (n = 17), hemoptysis (n = 5), air leak (n = 7), septic shock requiring pressors (n = 8) and inability to oxygenate adequately (n = 7). Twenty-four patients were ventilated preoperatively. Eleven patients had frank lobar gangrene, and the other patients had combinations of necrotizing pneumonia and abscesses. In 10 patients, preresection procedures were performed, including percutaneous drainage of an abscess (n = 4), thoracoscopic decortication (n = 4) and open decortication (n = 2). Procedures included pneumonectomy (n = 4), lobectomy (n = 18), segmentectomy (n = 2), wedge resection (n = 4) and debridement (n = 7). There were three (8.5%) postoperative deaths--two due to multiple organ failure and one due to anoxic brain injury. All patients not ventilated preoperatively were weaned from ventilatory support within three days. Of those ventilated preoperatively, three died, while four remained chronically ventilator dependent. CONCLUSIONS: Surgical resection for necrotizing lung infections is a reasonable option in patients with persistent sepsis who are failing medical therapy. Ventilated patients have a worse prognosis but can still be candidates for resection. Patients who are hemodynamically unstable appear to have better outcomes if they can be stabilized before resection.

Acute Disease↗

PubMed2005

Central venous-arterial carbon dioxide difference as an indicator of cardiac index.

OBJECTIVE: The mixed venous-arterial (v-a) pCO(2) difference has been shown to be inversely correlated with the cardiac index (CI). A central venous pCO(2), which is easier to obtain, may provide similar information. The purpose of this study was to examine the correlation between the central venous-arterial pCO(2) difference and CI. DESIGN: Prospective, cohort study. SETTING: Intensive care unit of an urban tertiary care hospital. PATIENTS AND PARTICIPANTS: Eighty-three consecutive intensive care unit patients. MEASUREMENTS: Simultaneous blood gases from the arterial, pulmonary artery (PA), and central venous (CV) catheters were obtained. At the same time point, cardiac indices were measured by the thermodilution technique (an average of three measurements). The cardiac indices obtained by the venous-arterial differences were compared with those determined by thermodilution. RESULTS: The correlation (R(2)) between the mixed venous-arterial pCO(2) difference and cardiac index was 0.903 (p <0.0001), and the correlation between the central venous-arterial pCO(2) difference and cardiac index was 0.892 (p <0.0001). The regression equations for these relationships were natural log (CI)=1.837-0.159 (v-a) CO(2) for the PA and natural log (CI)=1.787-0.151 (v-a) CO(2) for the CV (p <0.0001 for both). The root-mean-squared error for the PA and CV regression equations were 0.095 and 0.101, respectively. CONCLUSION: Venous-arterial pCO(2) differences obtained from both the PA and CV circulations inversely correlate with the cardiac index. Substitution of a central for a mixed venous-arterial pCO(2) difference provides an accurate alternative method for calculation of cardiac output.

Adult↗

PubMed2005

Oxidative-induced calcium mobilization is dependent on annexin VI release from lipid rafts.

BACKGROUND: Oxidative stress results in macrophage reprogramming through the formation of focal adhesion-like complexes on lipid rafts. Although the cellular mechanisms responsible for this reprogramming remain unknown, oxidative stress is known to result in a transient increase in intracellular calcium. This transient flux is thought to occur through the membrane dissociation of the calcium-bound protein annexin VI. The purpose of this study is to clarify the source of the calcium, and determine if it is responsible for the formation of focal adhesion-like complexes during oxidative stress through the activation of calcium/calmodulin dependent protein kinase II (CaMK II). METHODS: THP-1 cells were stimulated with hydrogen peroxide. Selected cells were pretreated with methyl beta-cyclodextrin (MbetaCD), a cholesterol-depleting agent; 1,2-bis aminophenoxy ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator; or autocamtide 2-related inhibitory peptide, a CaMK II inhibitor. Intracellular calcium flux was determined by a Fluo-3 technique. Lipid raft and cellular protein were extracted and analyzed for active CaMK II, annexin VI, and components of focal adhesion-like complexes. RESULTS: Hydrogen peroxide exposure led to mobilization of annexin VI from lipid rafts to the cytosol, which was followed by an increase in cytosolic calcium, phosphorylation of CaMK II, and formation of focal adhesion-like complexes. Cholesterol depletion from lipid rafts attenuated all of these effects. 1,2-bis Aminophenoxy ethane-N,N,N',N'-tetraacetic acid and autocamtide 2-related inhibitory peptide pretreatment attenuated CaMK II phopshorylation and formation of focal ahdesionlike complexes. CONCLUSIONS: Macrophage reprogramming during oxidative stress occurs through the cytosolic mobilization of annexin VI from lipid rafts. As a result, bound calcium dissociates, resulting in the activation of CaMK II and the formation of focal adhesion-like complexes.

Annexin A6↗

PubMed2005

Mitogen-activated protein kinase (MAPK).

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Critical Illness↗

PubMed2005

Calcium/calmodulin-dependent kinase II is required for platelet-activating factor priming.

Platelet-activating factor (PAF) primes the macrophage proinflammatory response to inflammatory stimuli, such as lipopolysaccharide (LPS). The cellular events responsible for this priming or reprogramming remain unresolved, but may occur through an increase in cytosolic calcium, inducing calcium/calmodulin-dependent kinase (CaMK) activation. To study this, differentiated THP-1 cells were used to study the effect of CaMK II and IV inhibition on PAF-induced reprogramming of TLR4-mediated events. LPS induced p38, ERK 1/2, and JNK/SAPK phosphorylation, NF-kappaB and AP-1 activation, and TNF-alpha and IL-10 production. PAF pretreatment selectively increased LPS-induced ERK 1/2, JNK/SAPK, NF-kappaB and AP-1 activation, and TNF-alpha production. Inhibition of CaMK II prevented PAF-induced priming of these events. Inhibition of CaMK IV prevented LPS-induced ERK 1/2, JNK/SAPK, NF-kappaB and AP-1 activation, and TNF-alpha production, but increased IL-10 production with or without PAF pretreatment. Neither CaMK II nor IV inhibition had any affect on p38 activity. These data suggest that the function of CaMK II is essential for PAF-induced macrophage priming. This priming event is mediated in part by modulation of ERK 1/2, JNK/SAPK, NF-kappaB, and AP-1 activation. CaMK IV, on the other hand, is not specific for priming by PAF and appears to have a direct link in TLR4-mediated events.

Blood Platelets↗

PubMed2005

The role of repeat angiography in the management of pelvic fractures.

BACKGROUND: Angiographic embolization has emerged as the treatment modality of choice for bleeding pelvic fractures. The purpose of this study is to identify potential indicators for ongoing pelvic hemorrhage despite initial therapeutic or non-diagnostic angiography. METHODS: The trauma registry of a Level I trauma center was used to identify patients with pelvic fractures between January 2000 and June 2002. Records were reviewed for demographics, severity of injury, hemodynamic status, initial and subsequent base deficit, blood and fluid requirements, length of stay, and mortality. Statistical analysis was performed using Student's t test, and univariate and multivariate analysis, significance was assigned to p < or = 0.05. RESULTS: During the study period, 678 patients had pelvic fractures. Angiography was performed in 31 (4.6%) of these patients. Arterial hemorrhage was diagnosed initially on 16 (51.6%) patients requiring embolization. Three (18.8%) of these embolized patients required repeat angiography and embolization due to ongoing pelvic hemorrhage. Of the initial 15 patients with negative angiograms, five (33.3%) had repeat angiograms due to continued hypotension and acidosis. Four (80.0%) of these five patients were found to have arterial hemorrhage requiring embolization. Of the seven (22.6%) patients requiring repeat angiography for control of ongoing pelvic hemorrhage, three independent factors were predictive: continued or recurrent hypotension (SBP < 90), absence of intra-abdominal injury, and persistent base deficit of 10 for greater than 6 hours. The presence of all three independent predictors was associated with a 97% probability of pelvic bleeding (p = 0.001). CONCLUSION: Angiographic embolization is highly effective in controlling arterial bleeding associated with pelvic fractures. However, repeat angiography should be performed in patients with pelvic fractures with ongoing evidence of hemorrhage demonstrated by persistent base deficit and hypotension once other potential sources of bleeding have been excluded.

Acidosis↗

PubMed2004

Implications of proteasome inhibition: an enhanced macrophage phenotype.

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.

Cell Line↗

PubMed2004

PKC-zeta is essential for endotoxin-induced macrophage activation.

BACKGROUND: A critical element in sepsis-induced tissue injury is the release of pro-inflammatory mediators from LPS-activated macrophages. The cellular mechanisms involved in this process remain incompletely understood. The aim of the current study was to further clarify the mechanism of LPS activation through the TLR4 receptor complex by examining the roles of the various isoforms of PKC. MATERIALS AND METHODS: Differentiated THP-1 cells were subjected to LPS stimulation. Selected cells were pretreated with various concentrations of Gö6983 to inhibit conventional, novel, and atypical PKC isoforms. Lipid raft, cellular, and nuclear proteins were then extracted and analyzed by Western blot and EMSA for components of the TLR4 pathway. Supernatants harvested under the various conditions were analyzed by ELISA for the production of TNF-alpha. RESULTS: LPS stimulation led to the mobilization of TLR4 to lipid rafts followed by phosphorylation and activation of IRAK, ERK 1/2, p38, and JNK/SAPK. Subsequently, LPS induced the activation of NF-kappaB and AP-1. Activation of these TLR4-signaling components resulted in the production of TNF-alpha. Inhibition of conventional and novel PKC isoforms had no significant effect on macrophage activation. Inhibition of the atypical PKC, PKC-zeta, was associated with significant attenuation in the mobilization of TLR4 to lipid rafts, the activation of all TLR4-signaling components, and the production of TNF-alpha. CONCLUSION: This study demonstrates that the atypical PKC isoform, PKC-zeta, is critical to regulation of LPS-induced TLR4 lipid raft mobilization within macrophages, TLR4-signaling, and TNF-alpha production. Although the mechanism of its activation remains unresolved, it appears that modulation of PKC-zeta activity during Gram-negative infections may limit associated inflammatory-induced morbidity.

Enzyme Activation↗

PubMed2004

Implications of lipid raft disintegration: enhanced anti-inflammatory macrophage phenotype.

BACKGROUND: Lipid rafts are membrane microdomains characterized by an enriched cholesterol environment and appear to serve as a platform for signaling. Their role within the macrophage during endotoxin exposure is unknown. METHODS: THP-1 cells were subjected to lipopolysaccharide stimulation with or without methyl-beta-cyclodextrin (MbetaCD) pretreatment, a cholesterol depleting agent. Cell surface expression of toll-like receptor-4 (TLR4) and platelet-activating factor receptor (PAFr) was determined by flow cytometry. Membrane receptor components and activation of the mitogen-activated protein kinases (MAPK) was determined from lipid raft and cellular protein by immunoblot. Inflammatory mediator production was determined from harvested supernatants by enzyme-linked immunosorbent assay. RESULTS: Surface expression of TLR4 and PAFr was not affected by MbetaCD. Lipopolysaccharide stimulation led to TLR4 mobilization to lipid rafts, MAPK activation, and inflammatory mediator production. Pretreatment with MbetaCD did not affect TLR4 mobilization to lipid rafts, but did result in lost lipid raft expression of the PAFr coupled G-protein, Galpha1. MbetaCD treatment led to selective attenuation of MAPK activation through ERK 1/2. This dysregulated signaling was associated with attenuated production of tumor necrosis factor-alpha, but increased production of interleukin-10. CONCLUSION: Lipid raft disintegration results in lost expression of Galpha1, dysregulated MAPK signaling, and selective anti-inflammatory mediator production. Therefore, modulation of lipid raft cholesterol content may represent a potential mechanism for regulation of macrophage phenotypic differentiation.

Cell Line↗

PubMed2004

Modulation of macrophage responsiveness to lipopolysaccharide by IRAK-1 manipulation.

Local activation of the macrophage by endotoxin is essential for the eradication of invasive gram-negative infections. Circulating endotoxin at lower concentrations results in immune cell activation at distant sites leading to tissue injury. Although the cellular mechanisms involved in these potentially dissimilar events are incomplete, it appears that the proximal kinase IRAK-1 plays a role. Thus, sense and antisense IRAK-1 oligonucleotides were used to determine the role IRAK-1 plays in macrophage activation by systemic (1-100 ng/mL) and local (1000 ng/mL) concentration of lipopolysaccharide (LPS) within THP-1 cells. Within the sense group, 1-1000 ng/mL of LPS within the sense group resulted in cellular activation of ERK-1/2, p38, and JNK/SAPK and the nuclear activation of NF-kappaB and AP-1. This activation was associated with proinflammatory cytokine production and cellular spreading. Systemic concentrations of LPS within the antisense group were associated with significant attenuation of intracellular signaling, cytokine production, and cellular spreading compared with the sense group. Local concentrations of LPS within the antisense group, however, were associated only with a delay in intracellular signaling, with no effect on cytokine production or cell spreading compared with the sense group. Based on these results, it appears that IRAK-1 is essential to macrophage activation at systemic, but not local, concentrations of LPS. These data suggest that redundant pathways exist that are functional at higher concentrations of LPS. Therefore, IRAK-1 appears to be the central kinase involved in the activation of the macrophage at distant sites during septic shock but is not necessary for activation in areas of local infection.

Blotting, Western↗

PubMed2003

NADPH oxidase activation in fibronectin adherent human neutrophils: A potential role for beta1 integrin ligation.

BACKGROUND: Hydrogen peroxide production by human neutrophils is tightly coupled to integrin ligation. This provides a means for spatial localization of oxidant production. However, integrin specificity and consequent signaling mechanisms for this process remain undefined. In the present study we demonstrate that otherwise unstimulated neutrophils adherent to fibronectin, a beta(1) ligand, but not fibrinogen, a beta(2) ligand, produce hydrogen peroxide in a time-dependent fashion. We hypothesized that signaling proceeded through focal, adhesionlike structures. METHODS: Triton-X insoluble (actin cytoskeleton) fractions from suspension phase cells and cells adherent to the beta(1) integrin ligand fibronectin were assessed for the presence of the integrin-associated kinase Pyk2, the scaffolding protein paxillin, and the downstream Src family kinase Lyn. Lysates were subjected to immunoprecipitation with anti-phosphotyrosine and probed for Pyk2, paxillin, and Lyn. Associations between focal adhesion kinase (FAK) and paxillin were determined by immunoprecipitation with anti-FAK and probing with anti-paxillin antibody. Activation of NADPH oxidase was determined by demonstration of redistribution of p47(phox) in Triton-X insoluble fractions. RESULTS: NADPH oxidase activation, as judged by H(2)O(2) production, occurred with fibronectin-, but not in suspension or fibrinogen-adherent cells. Cells adherent to fibronectin for 20 minutes demonstrated marked increases in Pyk2, paxillin, and Lyn activation in comparison to fibronectin-adherent and suspension phase cells. CONCLUSIONS: This study demonstrates that fibronectin adherence is a significant initiating factor for NADPH oxidase assembly in human neutrophils. This appears to be mediated by beta(1) integrins. We demonstrate formation of focal adhesions containing Pyk2/FAK, paxillin, and Lyn, and translocation of p47(phox).

Cell Adhesion↗

PubMed2003

Modulation of endotoxin-induced endothelial activity by microtubule depolymerization.

BACKGROUND: Endotoxin not only activates the Toll-mediated signaling pathway within endothelial cells that leads to neutrophil migration but also causes the polymerization of microtubules. The potential role of this polymerization event, however, is unknown. METHODS: Human umbilical vein endothelial cells stimulated with endotoxin were pretreated with or without the microtubule depolymerizing agent colchicine. Toll-mediated signaling events and protein production were in turn investigated by Western blot, gel shift, and enzyme-linked immunosorbent assay. Finally, neutrophil adhesion was assayed fluorometrically under the various conditions. RESULTS: Endotoxin led to activation of the various Toll-mediated pathways, production of intercellular adhesion molecule-1 and interleukin-8, and subsequent neutrophil adhesion. Pretreatment with colchicine led to selective inhibition of anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1; selective enhancement of p38; and no effect on nuclear factor-kappaB. This selective modulation of intracellular signaling resulted in attenuated intercellular adhesion molecule-1, interleukin-8 and prostaglandin E2 production, but enhanced cyclooxygenase-2 expression. As a result, microtubule disruption led to a significant reduction in neutrophil adhesion. CONCLUSION: Microtubule formation is essential to optimal endotoxin-induced intracellular signaling through anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1. Failure of these signaling events is associated with a marked reduction in the formation of a proadhesive phenotype that may prove to be beneficial in modulating neutrophil recruitment during sepsis.

Adaptor Protein Complex 1↗

PubMed2003

Platelet-activating factor acetylhydrolase inhibits alveolar macrophage activation in vivo.

Platelet-activating factor (PAF) is an important proinflammatory mediator of septic shock. PAF is produced by activated macrophages and acts to perpetuate the response to endotoxin. PAF is metabolized by an endogenous PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of postinjury multiple organ failure. We have previously shown that PAF-AH significantly inhibits macrophage activation by lipopolysaccharide (LPS) in vitro. The purpose of these studies was to determine whether this effect would translate to an in vivo model of remote lung injury. Wistar rats were administered a single intravenous dose of PAF-AH (5 mg/kg) or its carrier solution simultaneous with the induction of zymosan peritonitis. After 24 h, alveolar macrophages were obtained by bronchoalveolar lavage and stimulated in vitro with LPS (1 microg/mL). Supernatants were collected at 18 h for cytokine production and cellular and nuclear protein extractions were performed at 30 and 60 min to assess the activation of p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases and the nuclear translocation of nuclear factor (NF)-kappaB. Administration of PAF-AH significantly inhibited LPS-induced tumor necrosis factor alpha and interleukin-1beta production by alveolar macrophages from zymosan-treated animals. This functional inhibition was associated with inhibition of ERK 1/2 kinase and NF-kappaB activation but not p38 kinase activation. Interleukin 6 production was depressed in the macrophages from zymosan-treated animals but no additional inhibition resulted from PAF-AH treatment. In conclusion, zymosan peritonitis leads to priming of alveolar macrophages such that their subsequent tumor necrosis factor alpha response to LPS is enhanced. In vivo administration of PAF-AH abrogates this response, suggesting that this priming may be PAF dependent. This effect of PAF-AH may be mediated by the inhibition of intracellular signaling via inhibition of ERK kinase and NF-kappaB activation.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗