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Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is a global health care emergency. Anti-SARS-CoV-2 serological profiling of critically ill COVID-19 patients was performed to determine their humoral response. Blood was collected from critically ill ICU patients, either COVID-19 positive (+) or COVID-19 negative (−), to measure anti-SARS-CoV-2 immunoglobulins: IgM, IgA, IgG, and Total Ig (combined IgM/IgA/IgG). Cohorts were similar, with the exception that COVID-19+ patients had a greater body mass indexes, developed bilateral pneumonias more frequently and suffered increased hypoxia when compared to COVID-19- patients (p &lt, 0.05). The mortality rate for COVID-19+ patients was 50%. COVID-19 status could be determined by anti-SARS-CoV-2 serological responses with excellent classification accuracies on ICU day 1 (89%), ICU day 3 (96%), and ICU days 7 and 10 (100%). The importance of each Ig isotype for determining COVID-19 status on combined ICU days 1 and 3 was: Total Ig, 43%, IgM, 27%, IgA, 24% and IgG, 6%. Peak serological responses for each Ig isotype occurred on different ICU days (IgM day 13 &gt, IgA day 17 &gt, IgG persistently increased), with the Total Ig peaking at approximately ICU day 18. Those COVID-19+ patients who died had earlier or similar peaks in IgA and Total Ig in their ICU stay when compared to patients who survived (p &lt, 0.005). Critically ill COVID-19 patients exhibit anti-SARS-CoV-2 serological responses, including those COVID-19 patients who ultimately died, suggesting that blunted serological responses did not contribute to mortality. Serological profiling of critically ill COVID-19 patients may aid disease surveillance, patient cohorting and help guide antibody therapies such as convalescent plasma.

Objectives:. Coronavirus disease 2019 continues to spread worldwide with high levels of morbidity and mortality. We performed anticoronavirus immunoglobulin G profiling of critically ill coronavirus disease 2019 patients to better define their underlying humoral response. Design:. Blood was collected at predetermined ICU days to measure immunoglobulin G with a research multiplex assay against four severe acute respiratory syndrome coronavirus 2 proteins/subunits and against all six additionally known human coronaviruses. Setting:. Tertiary care ICU and academic laboratory. Subjects:. ICU patients suspected of being infected with severe acute respiratory syndrome coronavirus 2 had blood collected until either polymerase chain reaction testing was confirmed negative on ICU day 3 (coronavirus disease 2019 negative) or until death or discharge if the patient tested polymerase chain reaction positive (coronavirus disease 2019 positive). Interventions:. None MEASUREMENTS AND MAIN RESULTS:. Age- and sex-matched healthy controls and ICU patients who were either coronavirus disease 2019 positive or coronavirus disease 2019 negative were enrolled. Cohorts were well-balanced with the exception that coronavirus disease 2019 positive patients had greater body mass indexes, presented with bilateral pneumonias more frequently, and suffered lower Pao2:Fio2 ratios, when compared with coronavirus disease 2019 negative patients (p < 0.05). Mortality rate for coronavirus disease 2019 positive patients was 50%. On ICU days 1–3, anti–severe acute respiratory syndrome coronavirus 2 immunoglobulin G was significantly elevated in coronavirus disease 2019 positive patients, as compared to both healthy control subjects and coronavirus disease 2019 negative patients (p < 0.001). Weak severe acute respiratory syndrome coronavirus immunoglobulin G serologic responses were also detected, but not other coronavirus subtypes. The four anti–severe acute respiratory syndrome coronavirus 2 immunoglobulin G were maximal by ICU day 3, with all four anti–severe acute respiratory syndrome coronavirus 2 immunoglobulin G providing excellent diagnostic potential (severe acute respiratory syndrome coronavirus 2 Spike 1 protein immunoglobulin G, area under the curve 1.0, p < 0.0005; severe acute respiratory syndrome coronavirus receptor binding domain immunoglobulin G, area under the curve, 0.93–1.0; p ≤ 0.0001; severe acute respiratory syndrome coronavirus 2 Spike proteins immunoglobulin G, area under the curve, 1.0; p < 0.0001; severe acute respiratory syndrome coronavirus 2 Nucleocapsid protein immunoglobulin G area under the curve, 0.90–0.95; p ≤ 0.0003). Anti–severe acute respiratory syndrome coronavirus 2 immunoglobulin G increased and/or plateaued over 10 ICU days. Conclusions:. Critically ill coronavirus disease 2019 patients exhibited anti–severe acute respiratory syndrome coronavirus 2 immunoglobulin G, whereas serologic responses to non–severe acute respiratory syndrome coronavirus 2 antigens were weak or absent. Detection of human coronavirus immunoglobulin G against the different immunogenic structural proteins/subunits with multiplex assays may be useful for pathogen identification, patient cohorting, and guiding convalescent plasma therapy.

Supplemental Digital Content is available in the text., Objectives: Coronavirus disease 2019 is caused by the novel severe acute respiratory syndrome coronavirus 2 virus. Patients admitted to the ICU suffer from microvascular thrombosis, which may contribute to mortality. Our aim was to profile plasma thrombotic factors and endothelial injury markers in critically ill coronavirus disease 2019 ICU patients to help understand their thrombotic mechanisms. Design: Daily blood coagulation and thrombotic factor profiling with immunoassays and in vitro experiments on human pulmonary microvascular endothelial cells. Setting: Tertiary care ICU and academic laboratory. Subjects: All patients admitted to the ICU suspected of being infected with severe acute respiratory syndrome coronavirus 2, using standardized hospital screening methodologies, had daily blood samples collected until testing was confirmed coronavirus disease 2019 negative on either ICU day 3 or ICU day 7 if the patient was coronavirus disease 2019 positive. Interventions: None. Measurement and Main Results: Age- and sex-matched healthy control subjects and ICU patients that were either coronavirus disease 2019 positive or coronavirus disease 2019 negative were enrolled. Cohorts were well balanced with the exception that coronavirus disease 2019 positive patients were more likely than coronavirus disease 2019 negative patients to suffer bilateral pneumonia. Mortality rate for coronavirus disease 2019 positive ICU patients was 40%. Compared with healthy control subjects, coronavirus disease 2019 positive patients had higher plasma von Willebrand factor (p < 0.001) and glycocalyx-degradation products (chondroitin sulfate and syndecan-1; p < 0.01). When compared with coronavirus disease 2019 negative patients, coronavirus disease 2019 positive patients had persistently higher soluble P-selectin, hyaluronic acid, and syndecan-1 (p < 0.05), particularly on ICU day 3 and thereafter. Thrombosis profiling on ICU days 1–3 predicted coronavirus disease 2019 status with 85% accuracy and patient mortality with 86% accuracy. Surface hyaluronic acid removal from human pulmonary microvascular endothelial cells with hyaluronidase treatment resulted in depressed nitric oxide, an instigating mechanism for platelet adhesion to the microvascular endothelium. Conclusions: Thrombosis profiling identified endothelial activation and glycocalyx degradation in coronavirus disease 2019 positive patients. Our data suggest that medications to protect and/or restore the endothelial glycocalyx, as well as platelet inhibitors, should be considered for further study.

Background COVID19 is caused by the SARS-CoV-2 virus and has been associated with severe inflammation leading to organ dysfunction and mortality. Our aim was to profile the transcriptome in leukocytes from critically ill patients positive for COVID19 compared to those negative for COVID19 to better understand the COVID19-associated host response. For these studies, all patients admitted to our tertiary care intensive care unit (ICU) suspected of being infected with SARS-CoV-2, using standardized hospital screening methodologies, had blood samples collected at the time of admission to the ICU. Transcriptome profiling of leukocytes via ribonucleic acid sequencing (RNAseq) was then performed and differentially expressed genes as well as significantly enriched gene sets were identified. Results We enrolled seven COVID19 + (PCR positive, 2 SARS-CoV-2 genes) and seven age- and sex-matched COVID19- (PCR negative) control ICU patients. Cohorts were well-balanced with the exception that COVID19− patients had significantly higher total white blood cell counts and circulating neutrophils and COVID19 + patients were more likely to suffer bilateral pneumonia. The mortality rate for this cohort of COVID19 + ICU patients was 29%. As indicated by both single-gene based and gene set (GSEA) approaches, the major disease-specific transcriptional responses of leukocytes in critically ill COVID19 + ICU patients were: (i) a robust overrepresentation of interferon-related gene expression; (ii) a marked decrease in the transcriptional level of genes contributing to general protein synthesis and bioenergy metabolism; and (iii) the dysregulated expression of genes associated with coagulation, platelet function, complement activation, and tumour necrosis factor/interleukin 6 signalling. Conclusions Our findings demonstrate that critically ill COVID19 + patients on day 1 of admission to the ICU display a unique leukocyte transcriptional profile that distinguishes them from COVID19− patients, providing guidance for future targeted studies exploring novel prognostic and therapeutic aspects of COVID19.

Alveolar macrophages are key contributors to both the promotion and resolution of inflammation in the lung and are categorized into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. The change in M1/M2 balance has been reported in various pulmonary diseases and is a target for therapeutic intervention. The aim of this study was to assess the modulation of M1/M2 phenotype in alveolar macrophages by water-soluble carbon monoxide-releasing molecule-3 (CORM-3). Rat alveolar macrophages (AM) (NR8383) in culture were stimulated with LPS (5 ng/ml)/IFN-γ (10 U/ml) or IL-4 (10 ng/ml)/IL-13 (10 ng/ml) to induce M1 and M2 phenotypes, respectively. Expression of M1 phenotype markers, iNOS and TNF-α, and M2 phenotype markers, CD206 and Ym-1, was assessed by western blotting after 1, 3, 6, or 24 h in the absence or presence of CORM-3 (0.15 mM) treatment. Inactive CORM-3 (iCORM-3) was used as a control. Treatment of naïve (unstimulated) AM with CORM-3 promoted progression of the M2 phenotype as evidenced by the increased expression of CD206 (at 1 h; 1.8-fold) and Ym-1 (at 3 h; 1.9-fold), respectively. Surprisingly, CORM-3 treatment also upregulated the expression of iNOS protein as assessed 6 h following stimulation of AM with CORM-3 (2.6-fold). On the contrary, CORM-3 effectively reduced LPS/IFN-γ-induced expression of iNOS protein (0.6-fold); however, it had no effect on TNF-α expression. Finally, CORM-3 acutely (1-3 h) upregulated CD206 (1.4-fold) and Ym-1 (1.6-fold) levels in IL-4-/IL-13-treated (M2-stimulus) macrophages. These findings indicate that CORM-3 modulates macrophage M1 and M2 phenotypes in vitro with respect to continuous suppression of iNOS expression in M1-polarized macrophages and transient (early-phase) upregulation of CD206 and Ym-1 proteins in M2-polarized macrophages.

OBJECTIVE: Diabetic ketoacidosis in children is associated with vasogenic cerebral edema, possibly due to the release of destructive polymorphonuclear neutrophil azurophilic enzymes. Our objectives were to measure plasma azurophilic enzyme levels in children with diabetic ketoacidosis, to correlate plasma azurophilic enzyme levels with diabetic ketoacidosis severity, and to determine whether azurophilic enzymes disrupt the blood-brain barrier in vitro. DESIGN: Prospective clinical and laboratory study. SETTING: The Children's Hospital, London Health Sciences Centre. SUBJECTS: Pediatric type 1 diabetes patients; acute diabetic ketoacidosis or age-/sex-matched insulin-controlled. MEASUREMENTS AND MAIN RESULTS: Acute diabetic ketoacidosis in children was associated with elevated polymorphonuclear neutrophils. Plasma azurophilic enzymes were elevated in diabetic ketoacidosis patients, including human leukocyte elastase (p < 0.001), proteinase-3 (p < 0.01), and myeloperoxidase (p < 0.001). A leukocyte origin of human leukocyte elastase and proteinase-3 in diabetic ketoacidosis was confirmed with buffy coat quantitative real-time polymerase chain reaction (p < 0.01). Of the three azurophilic enzymes elevated, only proteinase-3 levels correlated with diabetic ketoacidosis severity (p = 0.002). Recombinant proteinase-3 applied to human brain microvascular endothelial cells degraded both the tight junction protein occludin (p < 0.05) and the adherens junction protein VE-cadherin (p < 0.05). Permeability of human brain microvascular endothelial cell monolayers was increased by recombinant proteinase-3 application (p = 0.010). CONCLUSIONS: Our results indicate that diabetic ketoacidosis is associated with systemic polymorphonuclear neutrophil activation and degranulation. Of all the polymorphonuclear neutrophil azurophilic enzymes examined, only proteinase-3 correlated with diabetic ketoacidosis severity and potently degraded the blood-brain barrier in vitro. Proteinase-3 might mediate vasogenic edema during diabetic ketoacidosis, and selective proteinase-3 antagonists may offer future vascular- and neuroprotection.

Polymorphonuclear leukocyte (PMN)-derived myeloperoxidase (MPO) contributes to the pathophysiology of numerous systemic inflammatory disorders through: (1) direct peroxidation of targets and (2) production of strong oxidizing compounds, e.g., hypohalous acids, particularly hypochlorous acid, which furthers oxidant damage and contributes to the propagation of inflammation and tissue injury/dysfunction. Carbon monoxide-releasing molecules (CORMs) offer potent anti-inflammatory effects; however, the mechanism(s) of action is not fully understood. This study assessed the potential of MPO activity inhibition by a water-soluble CORM, CORM-3. To this end, we used in vitro assays to study CORM-3-dependent modulation of MPO activity with respect to: (1) the inhibition of MPO's catalytic activity generally and (2) the specific inhibition of MPO's peroxidation and halogenation (i.e., production of hypochlorous acid) reactions. Further, we employed primary human umbilical vein endothelial cells (HUVECs) to investigate MPO-dependent cellular activation and dysfunction by measuring intracellular oxidant stress (DHR-123 oxidation) and HUVEC permeability (flux of Texas red-dextran), respectively. The results indicate that CORM-3 significantly inhibits MPO activity as well as MPO's peroxidation and hypohalous acid cycles specifically (p

Diabetic ketoacidosis (DKA) in children is associated with intracranial vascular complications, possibly due to leukocyte-endothelial interactions. Our aim was to determine whether DKA-induced inflammation promoted leukocyte adhesion to activated human cerebrovascular endothelium. Plasma was obtained from children with type 1 diabetes either in acute DKA or in an insulin-controlled state (CON). Plasma concentrations of 21 inflammatory analytes were compared between groups. DKA was associated with altered circulating levels of ↑CXCL1 (GROα), ↑CXCL8 (IL-8), ↑IL-6, ↑IFNα2, and ↓CXCL10 (IP-10) compared with CON. These plasma analyte measurements were then used to create physiologically relevant cytokine mixtures (CM). Human cerebral microvascular endothelial cells (hCMEC/D3) were stimulated with either plasma (DKA-P or CON-P) or CM (DKA-CM or CON-CM) and assessed for polymorphonuclear leukocyte (PMN) adhesion. Stimulation of hCMEC/D3 with DKA-P or DKA-CM increased PMN adhesion to hCMEC/D3 under “flow” conditions. PMN adhesion to hCMEC/D3 was suppressed with neutralizing antibodies to CXCL1/CXCL8 or their hCMEC/D3 receptors CXCR1/CXCR2. DKA-P, but not DKA-CM, initiated oxidative stress in hCMEC/D3. Expression of ICAM-1, VCAM-1, and E-selectin were unaltered on hCMEC/D3 by either DKA-P or DKA-CM. In summary, DKA elicits inflammation in children associated with changes in circulating cytokines/chemokines. Increased CXCL1/CXCL8 instigated PMN adhesion to hCMEC/D3, possibly contributing to DKA-associated intracranial vascular complications.

The NLRP3 inflammasome is an intracellular multiple-protein complex that controls the maturation and release of interleukin (IL)-1β and IL-18. Endogenous carbon monoxide (CO) is anti-inflammatory. The aim of this study was to assess the effects/mechanisms of CO-releasing molecule-3 (CORM-3)-dependent modulation of the NLRP3 inflammasome in cardiac fibroblasts (CF) and its effect on myocardial function in sepsis. CF were treated with CORM-3 or inactive CORM-3 (iCORM-3) before NLRP3 inflammasome priming with lipopolysaccharides (LPS) or following activation with adenosine triphosphate (ATP). In parallel, cardiomyocytes (CM) were challenged with supernatants of LPS/ATP-stimulated CF or a cytokine mixture (Cyto-mix) containing IL-1β, IL-18, and HMGB1. In vivo, mice were treated with CORM-3 before or after LPS to induce sepsis (endotoxemia). Pretreatment of CF with CORM-3 prevented an LPS-induced increase in NLRP3 and pro-IL-1β expression. Treatment of CF with CORM-3 before ATP prevented ATP-induced activation of the NLRP3 inflammasome. Challenging CF with LPS/ATP promoted NLRP3 interactions with adaptor ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), which was prevented by CORM-3. Challenging CM with supernatants of CF with LPS/ATP or Cyto-mix (IL-1β, IL-18, and HMGB1) resulted in CM apoptosis, which was attenuated with either a CORM-3 or IL-1 receptor antagonist. Finally, myocardial NLRP3 inflammasome activation and myocardial dysfunction in septic mice were abolished by CORM-3. In NLRP3-deficient mice with sepsis, CORM-3 did not show additional benefits in improving myocardial function. Our results indicate that CORM-3 suppresses NLRP3 inflammasome activation by blocking NLRP3 interactions with the adaptor protein ASC and attenuates myocardial dysfunction in mice with sepsis.

A compelling strategy for treatment of spinal cord injury is the blockade of integrin-mediated leukocyte extravasation using a monoclonal antibody (mAb) against the alpha4 subunit of the alpha4beta1-integrin. However, little is known with respect to neutrophil function following anti-alpha4 mAb treatment. This study assessed the effects of anti-alpha4 mAb binding on neutrophil activation [reactive oxygen species (ROS) production], function (phagocytic activity) and anti-alpha4-mAb/alpha4beta1-integrin-complex internalization.Resting, primed or stimulated rat neutrophils were incubated ex vivo with anti-alpha4 mAb or isotype-control antibody. ROS production, phagocytic activity, and anti-alpha4-mAb/alpha4beta1-integrin-complex internalization were determined by flow cytometry using dihydrorhodamine (DHR1,2,3), fluorescent microspheres, and indirect immunolabeling, respectively.Brief (0.5 h) incubation of resting, primed or activated neutrophils with anti-alpha4 mAb had no effect on ROS production and did not change neutrophil phagocytic activity. However, prolonged incubation (2 h), assessed only in resting neutrophils, increased ROS production. The anti-alpha4-mAb/alpha4beta1-integrin-complex was internalized after 1 h of anti-alpha4 mAb treatment and remained internalized up to 6 h.Neutrophil ROS production and phagocytic function remain unaltered after brief anti-alpha4 mAb exposure, demonstrating that use of this mAb as a treatment should not adversely affect important beneficial roles of these cells.

Systemic inflammatory response syndrome, as a consequence of ischemia/reperfusion (I/R), negatively influences the function of the affected organs. The objective of this study was to assess the role of nitric oxide (NO) in remote intestinal inflammatory response elicited by hindlimb I/R. To this end, C57BL/6 (wild type; WT) and inducible nitric-oxide synthase (iNOS)-deficient mice were subjected to bilateral hindlimb ischemia (1 h) followed by 6 h of reperfusion. Some WT mice were injected with iNOS inhibitor N-[3-(aminomethyl)benzyl] acetamidine (1400W) (5 mg/kg s.c.) immediately before reperfusion, and proinflammatory response was assessed 6 h later. Hindlimb I/R resulted in dysfunction of the small intestine as assessed by the increase in permeability [blood-to-lumen clearance of Texas Red-dextran (molecular mass 3 kDa)] and an increase in the luminal levels of tumor necrosis factor (TNF)-alpha protein and nitrate/nitrite (NO(2)(-)/NO(3)(-)). The above-mentioned changes were accompanied by up-regulation of the proinflammatory phenotype in the mucosa of small intestine with respect to 1) an increase in TNF-alpha and iNOS protein expression, 2) leukocyte accumulation, 3) formation of edema, 4) an increase in leukocyte rolling/adhesion in the submucosal microvasculature, and 5) activation of transcription factor nuclear factor-kappaB and up-regulation of adhesion molecule expression. Interestingly, the most profound changes with respect to intestinal dysfunction were found in jejunum and ileum, whereas duodenum was affected the least. Interfering with iNOS activity (1400W and iNOS-deficient mice) significantly attenuated hindlimb I/R-induced inflammatory response and dysfunction of the small intestine with respect to the above-mentioned markers of inflammation. The obtained results indicate that hindlimb I/R induces remote inflammatory response in the small intestine through an iNOS-derived NO-dependent mechanism.

Sepsis-associated encephalopathy is an early manifestation of sepsis, resulting in a diffuse dysfunction of the brain. Recently, nitric oxide (NO) has been proposed to be one of the key molecules involved in the modulation of inflammatory responses in the brain. The aim of this study was to assess the role of NO in cerebrovascular endothelial cell activation/dysfunction during the early onsets of sepsis. To this end, we employed an in vitro model of sepsis in which cultured mouse cerebrovascular endothelial cells (MCVEC) were challenged with blood plasma (20% vol/vol) obtained from sham or septic (feces-induced peritonitis, FIP; 6 h) mice. Exposing MCVEC to FIP plasma for 1 h resulted in increased production of reactive oxygen species and NO as assessed by intracellular oxidation of oxidant-sensitive fluorochrome, dihydrorhodamine 123 (DHR 123), and nitrosation of NO-specific probe, DAF-FM, respectively. The latter events were accompanied by dissociation of tight junction protein, occludin, from MCVEC cytoskeletal framework and a subsequent increase in FITC-dextran (3-kDa mol mass) flux across MCVEC grown on the permeable cell culture supports, whereas Evans blue-BSA (65-kDa mol mass) or FITC-dextran (10-kDa mol mass) flux were not affected. FIP plasma-induced oxidant stress, occludin rearrangement, and MCVEC permeability were effectively attenuated by antioxidant, 1-pyrrolidinecarbodithioic acid (PDTC; 0.5 mM), or interfering with nitric oxide synthase (NOS) activity [0.1 mM nitro-l-arginine methyl ester (l-NAME) or endothelial NOS (eNOS)-deficient MCVEC]. However, treatment of MCVEC with PDTC failed to interfere with NO production, suggesting that septic plasma-induced oxidant stress in MCVEC is primarily a NO-dependent event. Taken together, these data indicate that during early sepsis, eNOS-derived NO exhibits proinflammatory characteristics and contributes to the activation and dysfunction of cerebrovascular endothelial cells.

Objective: Both superoxide dismutase (SOD) and nitric oxide synthase (NOS) have been implicated in delayed preconditioning (DP) to ischemia/reperfusion (I/R) in the heart. We used isolated cardiac myocytes to test the hypothesis that SOD and NOS may interact in the development of DP. Methods: Mouse neonatal cardiac myocytes were challenged with anoxia/reoxygenation (A/R; an in vitro counterpart to I/R) and normoxia/normoxia (N/N) served as the control. Two indices of inflammation were measured: oxidant stress (DHR oxidation) and polymorphonuclear leukocyte (PMN) transendothelial migration (cell culture inserts). The role of SOD was assessed using an antisense approach and the role of NOS was assessed using iNOS and eNOS deficient myocytes. Results: Cardiac myocytes exposed to A/R (1) produced more oxidants (intracellular fluorescence emission from 2.0±0.1 for N/N to 3.0±0.3 for A/R; P

During the systemic inflammatory response, circulating cytokines interact with the vascular endothelium, resulting in activation and nuclear accumulation of the nuclear transcription factor, nuclear factor kappa B (NFκB). In turn, NFκB transactivates relevant proinflammatory genes, resulting in an amplification of the inflammatory response. Because this scenario is potentially detrimental to the host, mechanisms exist to limit this amplification. Using an in vitro system that mimics the vascular–interstitial interface during inflammation (cell culture inserts), we provide evidence for the existence of a novel negative feedback mechanism on NFκB activity. We show that the interleukin 1β–induced accumulation of nuclear NFκB in human umbilical vein endothelial cell monolayers is dramatically reduced when polymorphonuclear leukocytes (PMN) are allowed to migrate across these monolayers. This effect does not appear to be due to PMN-derived elastase or nitric oxide. Fixed PMN (adhere but do not migrate) did not affect nuclear NFκB. Furthermore, cross-linking of platelet-endothelial cell adhesion molecule-1 (PECAM-1), but not intercellular adhesion molecule-1, reduces human umbilical vein endothelial cell nuclear NFκB induced by interleukin 1β. Finally, interaction of PMN with PECAM-1–deficient endothelial cells does not reduce nuclear NFκB. These observations indicate that engagement of PECAM-1 by emigrating PMN is a pivotal event in this negative feedback on NFκB activity.

In vitro studies have indicated that polymorphonuclear leukocytes (PMNs) traverse endothelial cell monolayers via the paracellular pathway (i.e., through endothelial cell-cell junctions. Herein, we assessed whether the adherens junctions (AJs) are disrupted during PMN transendothelial cell migration.Human umbilical vein endothelial cells (HUVECs) were grown to confluence on porous membranes and activated with interleukin-1beta, and PMN transendothelial migration was facilitated by formyl-methionyl-leucyl-phenylalanine. Using dual immunofluorescence staining and laser scanning confocal microscopy, we assessed the effects of PMN-endothelial cell adhesive interactions (i.e., adhesion to and emigration across monolayers) on the AJ components vascular endothelial (VE)-cadherin, beta-catenin, alpha-catenin, and gamma-catenin.In the AJ immediately adjacent to the adherent PMN, there was a loss of staining for some of the AJ components. AJ components further away from HUVEC-PMN adhesive interactions were unaffected. An iterative approach indicated that the four components were sequentially lost from the AJ. beta-catenin was lost first, followed by VE-cadherin, alpha-catenin, and, finally, gamma-catenin. In the absence of PMNs, the cross-linking of VE-cadherin, but not platelet endothelial cell adhesion molecule-1 or intercellular adhesion molecule-1, increased the cytoplasmic accumulation of beta-catenin. During PMN transendothelial migration, all of the junctional components under study were lost at the immediate site of monolayer penetration. Again, at regions removed from the actual site of PMN penetration of the monolayers, the AJ components were unaffected. PMN-induced disorganization of the AJs was partially prevented by an elastase inhibitor.These findings suggest that adherent PMNs induce a localized, sequential disassembly of AJs, which is partially mediated by PMN-derived elastase and involves the initial loss of an intracellular component of AJs (i.e., beta-catenin).

During sepsis, the production of both nitric oxide and superoxide are increased. Furthermore, NO and O(2)(-) may interact to produce peroxynitrite. The major aim of the present study was to assess the relative roles of NO, O(2)(-), and ONOO- in the regulation of nuclear factor kappaB (NF-kappaB) activity and subsequent E-selectin expression during the early stages of sepsis.Mice were administered 5 microg lipopolysaccharide (LPS) intraperitoneally, and NF-kappaB activity and E-selectin expression in the small intestine were assessed 3 hours later.In wild-type mice, increased levels of NF-kappaB in nuclear extracts were noted. By contrast, in both inducible nitric oxide synthase-deficient and transgenic Cu/Zn superoxide dismutase-overexpressing mice, NF-kappaB mobilization to the nucleus was diminished. Pretreatment with a ONOO- decomposition catalyst (5,10,15,20-tetrakis[4-sulfonatophenyl]porphyrinato iron [III] [FeTPPS]) resulted in a diminished impact of LPS on NF-kappaB activation. LPS increased vascular E-selectin expression in wild-type mice. E-selectin expression was diminished in inducible nitric oxide synthase-deficient mice after LPS challenge, but E-selectin expression remained elevated in both Cu/Zn superoxide dismutase transgenic mice and wild-type mice pretreated with FeTPPS.Our observations suggest that NO, O(2)(-), and ONOO- production are all important mediators in the induction of NF-kappaB activity during endotoxemia and that, in vivo, E-selectin expression on endothelium may not always be associated with whole-organ NF-kappaB activation.

Objective Exogenously administered CO interferes with PMN recruitment to the inflamed organs. The mechanisms of CO-dependent modulation of vascular proadhesive phenotype, a key step in PMN recruitment, are unclear. Methods We assessed the effects/mechanisms of CO liberated from a water-soluble CORM-3 on modulation of the proadhesive phenotype in hCMEC/D3 in an in vitro model of endotoxemia. To this end, hCMEC/D3 were stimulated with LPS (1 μg/mL) for six hours. In some experiments hCMEC/D3 were pretreated with CORM-3 (200 μmol/L) before LPS-stimulation. PMN rolling/adhesion to hCMEC/D3 were assessed under conditions of laminar shear stress (0.7 dyn/cm2). In parallel, expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 (qPCR), activation of transcription factors, NF-κB and AP-1 (ELISA), and MAPK-signaling (expression/phosphorylation of p38, ERK1/2, and JNK1/2; western blot) were assessed. Results The obtained results indicate that CORM-3 pretreatment reduces PMN rolling/adhesion to LPS-stimulated hCMEC/D3 (p

Aims The present study was to investigate the role of calpain in reactive oxygen species (ROS) production in endothelial cells and endothelium-dependent vascular dysfunction under experimental conditions of diabetes. Methods and results Exposure to high glucose activated calpain, induced apoptosis and reduced nitric oxide (NO) production without changing eNOS protein expression, its phosphorylation and dimers formation in primary human umbilical vein endothelial cells (HUVECs). These effects of high glucose correlated with intracellular ROS production and mitochondrial superoxide generation. Selectively scavenging mitochondrial superoxide increased NO production in high glucose-stimulated HUVECs. Inhibition of calpain using over-expression of calpastatin or pharmacological calpain inhibitor prevented high glucose-induced ROS production, mitochondrial superoxide generation and apoptosis, which were concurrent with an elevation of NO production in HUVECs. In mouse models of streptozotocin-induced type-1 diabetes and OVE26 type-1 diabetic mice, calpain activation correlated with an increase in ROS production and peroxynitrite formation in aortas. Transgenic over-expression of calpastatin reduced ROS production and peroxynitrite formation in diabetic mice. In parallel, diabetes-induced reduction of endothelium-dependent relaxation in aortic ring was reversed by over-expression of calpastatin in mouse models of diabetes. However, the protective effect of calpastatin on endothelium-dependent relaxation was abrogated by eNOS deletion in diabetic mice. Conclusions This study suggests that calpain may play a role in vascular endothelial cell ROS production and endothelium-dependent dysfunction in diabetes. Thus, calpain may be an important therapeutic target to overcome diabetes-induced vascular dysfunction.

Previous work has demonstrated that carbon monoxide releasing molecules (CORMs) suppress inflammation, however, the mechanisms are not well understood. Neutrophil (PMN)-derived myeloperoxidase (MPO...

Objective: Endothelial cells (ECs) in septic skeletal muscle may be exposed to large amounts of NO and superoxide generated by the skeletal muscle cells. We tested the hypothesis that inducible nitric oxide synthase (iNOS) induction in ECs (i.e., one of the steps in the septic process) is modulated by extravascularly generated nitric oxide (NO) and superoxide. Methods: To model sepsis in vitro, monolayers of microvascular ECs derived from rat skeletal muscle were incubated with a mixture of lipopolysaccharide (LPS) (25 ng/mL) and interferong (IFNg) (100 U/mL) for up to 24 hours. Next, a long-term release NO donor (DETA NONOate), a superoxide-generating mixture (xanthine oxidase/xanthine; XO/X), or DETA + XO/X were added to the LPS + IFNg mixture. The iNOS protein and activity, as well as intracellular oxidative stress, were measured at intervals up to 24 hours, whereas the activation of AP-1, IRF-1, and NFkB transcription factors was determined at 2 and 24 hours. Results: LPS + IFNg caused time-dependent increases in iNOS protein and activity. Increasing concentrations of DETA (up to 500 mM) decreased, whereas XO/X (10 mU per mL/0.1 mM, respectively) markedly enhanced, iNOS expression and activity. DETA attenuated the enhancement by XO/X. Although intracellular oxidative stress was not altered by LPS + IFNg, modulations of iNOS expression by DETA, XO/X, and DETA + XO/X correlated with changes in oxidative stress. Among the three transcription factors, only IRF-1 and NFkB seemed to be involved in iNOS induction and its modulation by DETA and XO/X. Conclusions: LPS + IFNg can induce iNOS expression in microvascular ECs from rat skeletal muscle, whereas NO and superoxide modulate this expression. On the basis of these observations we suggest that NO and superoxide from the extravascular tissue may play a key role in the inflammatory response of septic ECs. Microcirculation (2001) 8, 415‐425.

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)−/− mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS−/− and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS−/− mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS−/− mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS−/− mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS−/− mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.

To investigate the effects of endotoxin on adhesion of human red blood cells to human vascular endothelial cells under conditions of flow.Prospective, randomized, controlled in vitro study.University-affiliated cell biology laboratory.Human erythrocytes and human vascular endothelial cells.Fresh human erythrocytes and human vascular endothelial cells grown as monolayers were incubated with either saline or endotoxin. After incubation, endothelial monolayers were superfused with erythrocytes, and the number of erythrocytes adhering to the endothelial monolayer was quantified.Adhesion of erythrocytes to vascular endothelium was measured under conditions of continuous flow in different settings: a) exposure of both endothelial cells and erythrocytes to saline; b) incubation of both erythrocytes and endothelial cells with endotoxin; c) exposure of erythrocytes only to endotoxin; d) incubation of endothelial cells only to endotoxin; and e) both the endothelial cells and erythrocytes incubated with different concentrations of endotoxin. Erythrocyte adhesion in the saline control group was 71 +/- 8 cells/mm2. Incubation of both components with endotoxin increased the number of adhesive erythrocytes to 172 +/- 9 cells/mm2 (p.05). When only the endothelial cells were treated with endotoxin, 142 +/- 8 cells/mm2 adhered to the endothelial monolayer, whereas the incubation of the erythrocytes only to endotoxin resulted in adhesion of 102 +/- 3 cells/mm2. Decreasing concentrations of endotoxin reduced adhesion from 172 +/- 9 cells/mm2 (endotoxin, 75 microg/mL) to 165 +/- 9 cells/mm2 (endotoxin, 25 microg/mL), 153 +/- 4 cells/mm2 (endotoxin, 1 microg/mL), and 146 +/- 6.1 cells/mm2 (endotoxin, 5 ng/mL).Exposure of human erythrocytes and human venous vascular endothelial cells to an inflammatory stimulus such as endotoxin promotes a dose-dependent adhesion of erythrocytes to endothelium in a dynamic environment. These adhesive erythrocyte-endothelium interactions can be produced by exposure of either red blood cells or endothelial cells to endotoxin, with a higher degree of adhesion after activation of the endothelial cell component.

Cytokine release from inflammatory (CD14+) cells is reduced after repeated stimulation with lipopolysaccharide (LPS; LPS tolerance). However, it is not known whether LPS tolerance can be induced in CD14−cells. The aim of the present study was to determine whether endothelial cells [human umbilical vein endothelial cells (HUVEC)] could be rendered tolerant to LPS with respect to LPS-induced polymorphonuclear neutrophil (PMN) adhesion. LPS stimulation (0.5 μg/ml; 4 h) of naive HUVEC increased PMN adhesion. Pretreatment of HUVEC with LPS (0.5 μg/ml) for 24 h resulted in a reduction in the proadhesive effects of a subsequent LPS challenge. The initial LPS stimulation increased 1) mobilization of the nuclear transcription factor NF-κB to the nucleus and 2) surface levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and E-selectin. In LPS-tolerant HUVEC, a second LPS challenge resulted in 1) less accumulation of NF-κB in the nucleus, 2) a reduction in E-selectin expression, and 3) unchanged ICAM-1 expression. LPS-tolerant cells were still capable of mobilizing NF-κB in response to stimulation with either interleukin-1β or tumor necrosis factor-α, resulting in elevated E-selectin levels and increased PMN adhesion. These studies show for the first time that LPS tolerance can be induced in endothelial cells with respect to PMN adhesion. This tolerance is specific for LPS and is associated with an inability of LPS to mobilize NF-κB, resulting in less E-selectin expression.

Abstract —Exposing human umbilical vein endothelial cells (HUVECs) to anoxia/reoxygenation (A/R) results in an increase in polymorphonuclear leukocyte (PMN) adhesion to HUVECs. This A/R-induced hyperadhesion is completely prevented by a previous (24 hours earlier) exposure of HUVECs to A/R. This phenomenon has been termed “A/R tolerance.” Exposing HUVECs to A/R induces an increase in nuclear factor κB (NF-κB) in HUVEC nuclei within 4 hours. Interfering with either NF-κB activation (proteasome inhibitor) or translocation (double-stranded oligonucleotides containing NF-κB binding sequence) prevents the development of A/R tolerance (ie, the increase in A/R-induced PMN adhesion to HUVECs is the same after the first and second A/R challenges). NO production by HUVECs is increased after the second A/R challenge, but not after the first A/R challenge. Inhibition of NO synthase (NOS) during the second A/R challenge prevents the development of A/R tolerance with respect to PMN adhesion. However, while HUVECs contained endothelial NOS protein, no inducible NOS was detected in either tolerant or nontolerant cells. Further studies indicated that inhibition of GTP-cyclohydrolase I (an enzyme involved in de novo synthesis of an important cofactor for NOS activity, tetrahydrobiopterin) prevented the generation of NO in A/R-tolerant cells. Extracellular generation of NO (NO donor) did not effect the hyperadhesion response induced by the initial A/R challenge. A/R also induced an oxidant stress in naive HUVECs, but not in A/R-tolerant HUVECs. Inhibition of NOS during the second A/R insult results in the generation of an oxidant stress similar to that observed after the first A/R challenge. Taken together, the findings of the present study are consistent with a role for NF-κB in the development of A/R tolerance (with respect to PMN adhesion), perhaps by transcriptional regulation of GTP-cyclohydrolase. The increased NO production during the second A/R insult reduces PMN adhesion most likely by reducing the intracellular oxidant stress induced by A/R.

In vivo, mucosal epithelial cells are capable of rapidly resealing an injured epithelial lining by migrating into the denuded area (restitution). Since mucosal injury is associated with epithelial cell destruction and mast cell degranulation, we used an in vitro model of restitution to assess the effects of epithelial and mast cell products on the rate of epithelial cell migration. Confluent monolayers of IEC-18 cells were wounded by scratching with a razor blade and the number of migrating cells entering the denuded area were quantitated. Sonicates of IEC cells stimulated epithelial cell migration. An antibody against epidermal growth factor (EGF) inhibited the promigratory effects of the IEC sonicates. By contrast, RBL (mast cells) sonicates inhibited epithelial cell migration. Protamine sulfate (an heparin antagonist) and heparinase I (a heparin degrading enzyme) reversed the inhibitory effects of the RBL sonicates. Our findings suggest that epithelial cells destroyed during mucosal injury release substances which promote epithelial restitution, most likely, EGF or EGF-like peptides. By contrast, degranulation of mast cells during mucosal injury inhibits epithelial cell migration, an effect most likely attributable to mast cell-derived heparin.

Recent findings indicate that carbon monoxide (CO) in non-toxic doses exerts a beneficial anti-inflammatory action in various experimental models. However, the precise anti-inflammatory mechanism of CO in the intestine remains unclear. Here, we assessed the effects of a novel water-soluble CO-releasing molecule, CORM-3, on trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice.To induce colitis, C57BL/6 male mice received an enema of TNBS. CORM-3 or its inactive compound, iCORM-3, were administered intraperitoneally, once immediately before, and twice daily after receiving an enema of TNBS. Three days after TNBS administration, the distal colon was removed, assessed for colonic damage and histological scores, polymorphonuclear leukocyte recruitment (tissue-associated myeloperoxidase, MPO activity), and TNF-α, IFN-γ and IL-17A expression (mRNA and protein levels in the colon mucosa). CD4(+) T cells isolated from murine spleens were stimulated with anti-CD3/CD28, in the presence or absence of CORM-3/iCORM-3. The cell supernatants were assessed for TNF-α and IFN-γ expression, 24 h following stimulation.Colonic damage and histological scores were significantly increased in TNBS-induced mice compared to sham-operated mice. Tissue-associated MPO activity and expression of TNF-α, IFN-γ, and IL-17A in the colonic mucosa were higher in TNBS-induced colitis mice. The above changes were attenuated in CORM-3-treated mice. Further, CORM-3 was effective in reducing TNF-α and IFN-γ production in anti-CD3/CD28-stimulated CD4(+) T cells.These findings indicate that CO released from CORM-3 ameliorates inflammatory responses in the colon of TNBS-challenged mice at least in part through a mechanism that involves the suppression of inflammatory cell recruitment/activation.

BACKGROUND & AIMS: Previous studies indicate that a water extract of Helicobacter pylori promotes leukocyte adhesion and emigration as well as endothelial barrier disruption (increased vascular protein leakage) in rat mesenteric venules. The aims of this study were to assess whether H. pylori extract-activated neutrophils disrupt endothelial cell monolayers and to identify the mechanisms involved in this process. METHODS: Human neutrophils were incubated with monolayers of human umbilical vein endothelial cells (HUVECs) in the presence or absence of H. pylori extract. RESULTS: H. pylori extract-activated human neutrophils produced endothelial cell detachment from HUVEC monolayers, the severity of which was dependent on the duration of exposure. Endothelial cell detachment was prevented by a monoclonal antibody directed against CD11/CD18 on neutrophils or a monoclonal antibody against intercellular adhesion molecule 1 on endothelial cells. HUVEC monolayer disruption was also prevented by superoxide dismutase, catalase, and a monoclonal antibody against elastase. Further studies indicated that H. pylori extract was capable of inhibiting human neutrophil elastase. The antielastase activity was not diminished by oxidants. CONCLUSIONS: These studies indicate that H. pylori extract-activated human neutrophils can disrupt HUVEC monolayers only when human neutrophils are allowed to adhere to HUVECs and may provide an explanation for the H. pylori extract-induced, neutrophil- dependent vascular protein leakage observed in vivo. The possibility that H. pylori releases antiproteases may explain, in part, why this bacterium is so virulent. (Gastroenterology 1996 Jan;110(1):21-9)

Objective To determine if the DKA-induced inflammation in juvenile mice provokes activation and dysfunction of CVECs. Methods DKA in juvenile mice was induced with administration of STZ and ALX. Blood from DKA mice was assessed for cytokines and soluble cell adhesion proteins, and either DKA plasma or exogenous compounds were applied to immortalized bEND3. Results DKA increased circulating levels of IL-6, IL-8(KC), MCP-1, IL-10, sE-selectin, sICAM-1, and sVCAM-1. Stimulation of bEND3 with DKA plasma caused cellular activation (increased ROS and activation of NF-κΒ), upregulation of a proadhesive phenotype (E-selectin, ICAM-1, and VCAM-1), and increased leukocyte-bEND3 interaction (leukocyte rolling/adhesion). TEER, a measure of bEND3 monolayer integrity, was decreased by DKA plasma. Activation and dysfunction of bEND3 with DKA plasma were suppressed by plasma heat treatment (56°C, 1 hour) and replicated with the application of DKA recombinant cytomix (IL-6, IL-8[KC], MCP-1, and IL-10), implicating circulating inflammatory protein(s) as mediators. Treatment of bEND3 with β-OH-butyrate, the main ketone elevated in DKA, failed to mimic the DKA plasma–induced activation and dysfunction of bEND3. Conclusions DKA elicits systemic inflammation associated with CVEC activation and dysfunction, possibly contributing to DKA-associated intracranial microvascular complications.

In stable organ systems, such as the heart and kidneys, an oxidant stress induces an increase in endogenous antioxidant systems resulting in an increased resistance of the tissue to a subsequent oxidant challenge. The development of this oxidant tolerance requires 1.5-6 d. The aim of the present study was to determine whether oxidant tolerance can be induced in the small intestinal mucosa, a labile system whose epithelium turns over every 2-3 d. Ischemia/reperfusion-induced epithelial barrier dysfunction of the small intestinal mucosa was monitored in Sprague-Dawley rats whose intestines had been exposed to an ischemic insult 1, 24, or 72 h previously. At 24 h, but not 1 or 72 h after the initial ischemic insult, the mucosa was more resistant to ischemia/reperfusion-induced barrier dysfunction. The antioxidant status of the mucosa was enhanced at 24 h, but not at 1 or 72 h after the initial ischemic insult. This adaptation appears to be specific for oxidants, since an initial ischemic insult imposed 24 h earlier also protected against H2O2-induced, but not acid- or ethanol-induced, barrier dysfunction. Further studies indicated that the increase in antioxidant status of the mucosa observed 24 h after the initial ischemic insult was a result of adaptational changes in the lamina propria, rather than the epithelium. In vitro studies with isolated epithelial cells also indicated that epithelial cells do not develop oxidant tolerance. We conclude that the development of oxidant tolerance in the small intestinal mucosa does not involve an active participation of the epithelial lining.

The role of CO in the modulation of antioxidant enzyme function has not been investigated, yet. In this study we assessed the effects and potential mechanisms of the ruthenium-based water-soluble CO-releasing molecule CORM-3 in the modulation of superoxide dismutase (SOD) activity/binding in vascular endothelial cells (HUVECs). To this end, HUVECs were treated with CORM-3 (100 μM) and assessed for total SOD activity in cell lysates (cell-associated SOD activity) and cell culture supernatants (soluble SOD). In parallel, release/binding of extracellular SOD (SOD-3) in the absence or presence of heparin (1-10 IU/ml), a key factor regulating SOD-3 cell-surface binding, was investigated. In addition, the effects of CORM-3 on the modulation of purified SOD-1 and SOD-2 activity in a cell-free system were also assessed. The results obtained indicate that CORM-3 effectively suppresses the activity of both purified SOD-1 and SOD-2. These findings were accompanied by CORM-3-dependent attenuation of total cell-associated SOD activity (without affecting SOD-1/SOD-2 protein expression) and a subsequent increase in ROS production (DHR123 oxidation) in HUVECs. In parallel, a concomitant increase in soluble-SOD activity (due to increased SOD-3 release from the cell surface) was observed in the cell culture supernatants. However, in the presence of heparin, total cell-associated SOD activity was significantly increased by CORM-3, because of increased binding of SOD-3 to HUVECs. Taken together these findings indicate for the first time that CORM-3 modulates both the activity of intracellular SOD (i.e., SOD-1 and SOD-2) and the binding of extracellular SOD (SOD-3) to the cell surface.

AIM: To determine whether Carbon (CO) liberated from CO-releasing molecules attenuates leukocyte infiltration in the small intestine of thermally injured mice. METHODS: Thirty-six mice were assigned to four groups. Mice in the sham group (n = 9) were underwent to sham thermal injury; mice in the burn group (n = 9) received 15% total body surface area full-thickness thermal injury; mice in the burn + CORM-2 group (n = 9) were underwent to the same thermal injury with immediate administration of tricarbonyldichlororuthenium (II) dimer CORM-2 (8 mg/kg, i.v.); and mice in the burn+DMSO group (n = 9) were underwent to the same thermal injury with immediate administration of 160 μL bolus injection of 0.5% DMSO/saline. Histological alterations and granulocyte infiltration of the small intestine were assessed. Polymorphonuclear neutrophil (PMN) accumulation (myeloperoxidase assay) was assessed in mice mid-ileum. Activation of nuclear factor (NF)-κΒ, expression levels of intercellular adhesion molecule-1 (ICAM-1) and inducible heme oxygenase in mid-ileum were assessed. RESULTS: Treatment of thermally injured mice with CORM-2 attenuated PMN accumulation and prevented activation of NF-κΒ in the small intestine. This was accompanied by a decrease in the expression of ICAM-1. In parallel, burn-induced granulocyte infiltration in mid-ileum was markedly decreased in the burn mice treated with CORM-2. CONCLUSION: CORM-released CO attenuates leukocyte infiltration in the small intestine of thermally injured mice by interfering with NF-κΒ activation and protein expression of ICAM-1, and therefore suppressing the pro-adhesive phenotype of endothelial cells.

Human pulmonary microvascular endothelial cell (HPMVEC) injury is central to the pathophysiology of human lung injury. However, septic HPMVEC barrier dysfunction and the contribution of neutrophils have not been directly addressed in vitro. Instead, human EC responses are often extrapolated from studies of human umbilical vein EC (HUVEC). We hypothesized that HUVEC was not a good model for investigating HPMVEC barrier function under septic conditions. HPMVEC was isolated from lung tissue resected from lung cancer patients using magnetic bead-bound anti-PECAM-1 antibody. In confluent monolayers in 3-mum cell-culture inserts, we assessed trans-EC Evans-Blue (EB)-conjugated albumin leak under basal, unstimulated conditions and following stimulation with either lipopolysaccharide or a mixture of equal concentrations of TNF-alpha, IL-1beta and IFN-gamma (cytomix). Basal EB-albumin leak was significantly lower across HPMVEC than HUVEC (0.64 +/- 0.06% vs. 1.13 +/- 0.10%, respectively, P < 0.001). Lipopolysaccharide and cytomix increased leak across both HPMVEC and HUVEC in a dose-dependent manner, with a similar increase relative to basal leak in both cell types. The presence of neutrophils markedly and dose-dependently enhanced cytomix-induced EB-albumin leak across HPMVEC (P < 0.01), but had no effect on EB-albumin leak across HUVEC. Both cytomix and lipopolysaccharide-induced albumin leak was not associated with a loss of cell viability. In conclusion, HPMVEC barrier dysfunction under septic conditions is dramatically enhanced by neutrophil presence, and HUVEC is not a suitable model for studying HPMVEC septic barrier responses. The direct study of HPMVEC septic responses will lead to a better understanding of human lung injury.

Cardiac myocytes isolated from rats with peritonitis (cecal ligation and perforation; CLP) promote PMN transendothelial migration. Herein, we assessed (1) the mechanisms involved in cardiac myocyte activation during peritonitis and (2) the means by which these activated myocytes promote PMN transendothelial migration. Plasma obtained from mice subjected to CLP (septic plasma) activated isolated cardiac myocytes as evidenced by (1) increased nuclear levels of nuclear factor-kappaB (NF-kappaB) and (2) their ability to promote PMN migration across endothelial cell monolayers. Pretreatment of septic plasma with an antibody against tumor necrosis factor-alpha (TNF-alpha), but not interleukin-1beta (IL-1beta), blunted the ability of septic plasma to activate the myocytes. However, septic plasma obtained from TNF-alpha-deficient mice could still activate the myocytes; an effect attenuated by an antibody against IL-1beta. If the myocytes were pretreated with a proteasome inhibitor (MG 132) to prevent NF-kappaB activation, the myocyte-induced PMN transendothelial migration was compromised. The activated myocytes released platelet-activating factor (PAF), and myocyte-induced PMN migration was abrogated by a PAF receptor antagonist (WEB 2086). These myocytes also released the CXC chemokines LIX and KC; an event prevented by MG 132. Antibodies against LIX and KC abrogated the myocyte-induced PMN migration. However, LIX and KC, but not PAF, could promote PMN migration when used at concentrations produced by activated myocytes. These observations indicate that TNF-alpha and IL-1beta are, in part, responsible for the ability of septic plasma to activate cardiac myocytes. The activated myocytes promote PMN transendothelial migration, an effect attributable to LIX and KC, and possibly, PAF.

Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), a counterpart of the human growth-regulated oncogene product (GRO), has been suggested to participate in neutrophil recruitment in an experimental model of gastritis in rat. However, the mechanism(s) involved in regulation of CINC-1 production by the gastric mucosa remains unclear. The aim of this study was to investigate the mechanism(s) of CINC-1 production by rat gastric mucosa in vitro. All experiments were performed using rat normal gastric mucosal cell line (RGM-1). RGM-1s were stimulated with tumor necrosis factor (TNF)-alpha, and CINC-1 mRNA levels (reverse transcription-polymerase chain reaction) and protein secretion (enzyme-linked immunosorbent assay) were assessed. The production of reactive oxygen species (ROS) and nuclear factor (NF)-kappaB activation (translocation to the nuclei) in response to TNF-alpha stimulation was evaluated using fluorescence microscopy in the presence or absence of the inhibitors of mitochondrial electron flow and NF-kappaB activation. Stimulation of RGM-1 cells with TNF-alpha resulted in an increase in intracellular oxidative stress, NF-kappaB translocation to the nuclei, and up-regulation of CINC-1 mRNA and protein, which was prevented by interfering with mitochondria-dependent ROS production and NF-kappaB activation. Taken together, these findings indicate that CINC-1, a counterpart of the human GRO, production by rat gastric epithelial cells in response to TNF-alpha stimulation is an oxidant stress-mediated and NF-kappaB-dependent event.

The goal of the present study was to assess whether cardiac myocytes exposed to anoxia-reoxygenation (A/R) could generate a chemotactic gradient for polymorphonuclear neutrophil (PMN) transendothelial migration. Exposure of neonatal mouse cardiac myocytes to A/R induced an oxidant stress in the myocytes. Supernatants obtained from A/R-conditioned myocytes promoted mouse PMN migration across mouse myocardial endothelial cell monolayers. This increase in PMN transendothelial migration could be prevented if catalase or a platelet-activating factor (PAF) antagonist was added to the supernatants before assay. Supernatants from A/R-conditioned myocytes activated endothelial cells by inducing an intracellular oxidant stress. The oxidant stress and PMN transendothelial migration induced by supernatants from A/R-conditioned myocytes were substantially reduced when endothelial cells derived from manganese superoxide dismutase overexpressing mice were used in the assays. Supernatants from A/R-conditioned myocytes also increased endothelial cell surface levels of E-selectin and intercellular adhesion molecule-1. Our results indicate that cardiac myocytes exposed to A/R can generate a chemotactic gradient, presumably due to production and release of stable oxidants and PAF. The ability of supernatants from A/R-conditioned myocytes to promote PMN transendothelial migration was largely dependent on induction of an oxidant stress in endothelial cells. In addition, these supernatants also induced a proadhesive phenotype in the endothelial cells.

Previous studies indicate that aspirin can promote neutrophil (PMN) adhesion to endothelial cells and neutrophil-mediated endothelial cell detachment. The objectives of the present study were to determine whether PMN adhesion is a prerequisite for aspirin-induced, PMN-mediated endothelial cell detachment and whether neutrophil-derived oxidants and/or proteases are responsible for the cell detachment. Human PMNs were added to confluent monolayers of human umbilical vein endothelial cells (HUVEC) and coincubated with or without aspirin at a clinically relevant concentration (300 micrograms/ml). Aspirin-activated PMNs induced endothelial cell detachment, but not cell lysis. Endothelial cell detachment was always preceded by retraction of endothelial cells within the monolayer. The aspirin-induced, neutrophil-mediated cell detachment was prevented by a monoclonal antibody directed against CD11/CD18 adhesion integrins on PMNs. Elastase inhibitors, but not superoxide dismutase or catalase, prevented both endothelial cell retraction and detachment. If aspirin-activated neutrophils were allowed to migrate across the monolayers, endothelial cell retraction or detachment did not occur. These studies indicate that aspirin-induced, PMN-mediated endothelial cell retraction and detachment requires PMN adhesion to the target cells and is due to neutrophil-derived elastase. Endothelial cell retraction, induced by activated neutrophils, may represent an exaggeration of a normal physiologic event, i.e., neutrophil emigration.

Epidermal growth factor (EGF) is present in biliary, pancreatic, and Brunner's gland secretions. The aim of the present study was to assess the effects of EGF on lipid-induced mucosal injury. The proximal jejunum of anesthetized rats was cannulated for perfusion of the lumen with emulsified oleic acid (40 mM oleic acid in 20 mM sodium taurocholate; pH 6.0). Mucosal epithelial integrity was monitored by measuring the blood-to-lumen clearance of 51Cr-labeled EDTA. Perfusion of the lumen with emulsified lipid increased EDTA clearance. Addition of EGF (0.5 ng/ml) to the lipid emulsion ameliorated the lipid-induced increase in EDTA clearance. Perfusion of the lumen with EGF alone stimulated mucus secretion from goblet cells. This effect of EGF was abolished by atropine. In addition, in atropinized animals there was 1) an exaggeration of the lipid-induced injury and 2) a loss of the protective effect of EGF. Our findings provide evidence supporting the hypothesis that EGF provides protection against lipid-induced mucosal injury, in part, by stimulating mucus production.

Effects of putrescine, spermidine, and spermine on lipid-induced injury to jejunal mucosa were assessed in anesthetized rats. Mucosal epithelial integrity was continuously monitored by measuring blood-to-lumen clearance of 51Cr-labeled EDTA. Perfusion of jejunal lumen with emulsified lipid (20 mM sodium taurocholate and 40 mM oleic acid) increased 51Cr-EDTA clearance. Addition of spermidine (0.5 mM), but not putrescine (2.0 mM) or spermine (0.25 mM), to the lipid perfusate reduced the increment in 51Cr-EDTA clearance. Histological evaluation of jejunal mucosa indicated that the epithelial lining of the villous tips was damaged by emulsified oleic acid and that this injury was ameliorated by spermidine. Pretreatment of jejunal mucosa with spermidine did not prevent disruption of mucosal integrity induced by a subsequent perfusion with emulsified lipids. Intravenous infusion of spermidine to achieve an extracellular concentration of 0.5 mM did not prevent the lipid-induced increase in 51Cr-EDTA. Spermidine also ameliorated lipid-induced disruption of Caco-2 cell monolayers in culture; this protective effect was dose dependent and was observed only when spermidine was applied to the apical aspect of the monolayers. These findings indicate that spermidine must be present on the apical portion of the epithelial cell during lipid insult. Substitution of lysine or arginine for spermidine did not reduce the extent of lipid-induced injury to jejunal mucosa, indicating that spermidine's protective effects cannot simply be attributed to its cationic nature. Spermidine did not alter the turbidity of a micellar oleic acid solution, indicating that spermidine was not removing oleic acid from the soluble phase.(ABSTRACT TRUNCATED AT 250 WORDS)

One of the cardinal signs of acute inflammation is neutrophil (PMN) emigration across the endothelium and into the affected tissue. We have previously shown that human PMN migration across human umbilical vein endothelial cell (HUVEC) monolayers is dependent on PMN-derived elastase. However, whether migrating PMN release elastase into the extracellular milieu or retain it on the cell surface is unclear. In the present study, we show that when PMN are activated by platelet activating factor (PAF), elastase was mobilized to and retained in the cell membrane; no elastase activity was detected in the supernatant. Neutroplasts (enucleated cells devoid of granules) prepared from PAF-activated PMN contained twice as much elastase as did neutroplasts prepared from unstimulated PMN. Neutroplasts from PAF-activated PMN migrated across HUVEC monolayers in response to a chemotactic gradient (PAF), while those prepared from unstimulated PMN did not. The neutroplast transendothelial migration was inhibited (80%) by a monoclonal antibody against elastase. Using confocal microscopy, we noted that the localization of elastase on the cell surface of PMN, which were adherent to HUVEC but not migrating, was largely confined to the apical aspect of the PMN. There was little or no elastase detectable on the basal aspect of the PMN membrane in contact with the endothelium. By contrast, in migrating PMN the membrane-bound elastase was primarily localized to the migrating front, i.e. pseudopodia penetrating the HUVEC monolayers. Taken together, our findings indicate that migrating PMN localize their membrane-bound elastase to the migrating front where it facilitates transendothelial migration.

Background/Aims: Dietary polyunsaturated fats are significant sources of luminal lipid hydroperoxides whose accumulation can be injurious to the intestinal epithelium. The current study examines the cytotoxicity of peroxidized fish oil to CaCo-2 cells. Methods: Chromate release from cells was used as an index of CaCo-2 injury, and day 1 and day 7 postconfluent monolayers were used to represent the immature and mature states, respectively. Results: Air oxidation of fish oil yielded equimolar quantities of hydroperoxyeicosapentaenoic (20:5) and docosahexaenoic (22:6) acids. Their cytotoxicity were time- and concentration-dependent and were related to the developmental stages. A 100-μmol/L dose of hydroperoxides caused a 40% and a 15% 51 Cr release from day 1 and day 7 cells, respectively. Cellular glutathione (GSH), GSH redox enzyme, and γ-glutamyl cysteine synthetase activities were significantly lower in day 1 than in day 7 cells, indicating that hydroperoxide metabolism in immature cells is rate limited by reductant supply. GSH supplementation increased cell GSH in day 7 cells (twofold) but not in day 1 cells, suggesting a limited ability of immature cells to use exogenous GSH. Conclusions: These results show that nondifferentiated cells are more sensitive to oxidant-induced injury than mature cells. This enhanced susceptibility is associated with a lower GSH-dependent detoxication capacity of the immature cells.

Background/Aims: Previous studies indicate that a water extract of Helicobacter pylori (HPE) can promote neutrophil-endothelial cell interactions in vivo and in vitro. The objectives of this study were to assess whether HPE alters the rate of albumin leakage in rat mesenteric venules and identify the factors that mediate the HPE-induced microvascular dysfunction. Methods: Intravital microscopy was used to continuously monitor leukocyte adherence and emigration and albumin leakage in rat mesenteric venules during superfusion with HPE. Results: HPE increased leukocyte adherence and emigration and microvascular albumin leakage. The enhanced albumin leak could be subdivided into two components: an early (within 10 minutes) and a later (within 30 minutes) phase. HPE also elicited perivenular mast cell degranulation and the formation of platelet-leukocyte aggregates within post-capillary venules. The HPE-induced early phase of albumin leakage was attenuated by pretreatment with a mast cell stabilizer. The HPE-induced late phase of albumin leakage was reduced by monoclonal antibodies directed against either CD11bCD18 or intercellular adhesion molecule 1. A monoclonal antibody against P-selectin also inhibited the HPE-induced platelet-leukocyte aggregation and reduced the later phase of albumin leak. Conclusions: HPE-induced microvascular dysfunction appears to be a consequence of interstitial and intravascular cell-cell interactions.

The aim of the present study was to characterize and compare the expression of intercellular adhesion molecule 1 (ICAM-1) on unstimulated and endotoxin-challenged endothelial cells in different tissues of the rat. ICAM-1 expression was measured using 125I-labeled anti-rat ICAM-1 monoclonal antibody (MAb) and an isotype-matched control MAb labeled with 131I (to correct for nonspecific accumulation of the binding MAb). Under baseline conditions, ICAM-1 MAb binding was observed in all organs. The binding of 125I-ICAM-1 MAb varied widely among organs, with the largest accumulation (per g tissue) in the lung, followed by heart (1/30th of lung activity), splanchnic organs (1/50th of lung activity), thymus (1/100th of lung activity), testes (1/300th of lung activity), and skeletal muscle (1/800th of lung activity). Endotoxin induced an increase in ICAM-1 MAb binding in all organs except the spleen. Endotoxin-induced upregulation of ICAM-1 was greatest in heart and skeletal muscle (5- to 10-fold), whereas the remaining organs exhibited a two- to fourfold increase in ICAM-1 expression. Maximal upregulation of ICAM-1 occurred at 9-12 h after endotoxin administration. A dose-dependent increase in ICAM-1 expression was elicited by 0.1-10 microgram/kg, with higher doses (up to 5 mg/kg) producing no further increment. Induction of ICAM-1 mRNA after endotoxin was observed in all tissues examined (lung, heart, intestine), peaked at 3 h, and then rapidly returned to control levels. These findings indicate that ICAM-1 is constitutively expressed on vascular endothelium in all organs of the rat and that there are significant regional differences in the magnitude and time course of endotoxin-induced ICAM-1 expression.

Gastric mucosal injury induced by strong irritants can be dramatically reduced by pretreating the mucosa with mild forms of the same irritant. This phenomenon has been termed "adaptive cytoprotection." The aim of the present study was to use in vivo and in vitro approaches to study adaptive cytoprotection in the small intestine using physiologically relevant concentrations of oleic acid. Anesthetized rats were instrumented for perfusion of the proximal jejunum with 10 or 40 mM oleic acid (in 20 mM sodium taurocholate). Mucosal epithelial integrity was continuously monitored by measuring the blood-to-lumen clearance of 51Cr-labeled EDTA. Perfusion of the lumen with 40 mM oleic acid produced a 10-fold increase in 51Cr-EDTA clearance, which was not affected by a previous perfusion with 10 mM oleic acid, i.e., no adaptive cytoprotection. In another series of experiments, oleic acid was placed in the lumen rather than perfused, and mucosal epithelial integrity was assessed histologically. Intraluminal placement of 10 mM oleic acid resulted in the generation of a mucus layer over the epithelium. Subsequent placement of 40 mM oleic acid did not produce significant epithelial cell injury, i.e., adaptive cytoprotection. In in vitro studies, mucin (1, 5, and 10 mg/ml) was layered over confluent monolayers of Caco-2 cells prior to addition of 2 mM oleic acid in 4 mM sodium taurocholate. The epithelial cell injury induced by oleic acid was inhibited by mucin in a dose-dependent manner. Further studies indicate that mucin does not prevent, but simply delays, the onset of cell injury.(ABSTRACT TRUNCATED AT 250 WORDS)