Immunomodulatory gene expression analysis in LPS-stimulated human polymorphonuclear leukocytes treated with antibiotics commonly used for multidrug-resistant strains.

Conventional antibiotics used for the treatment of severe infections such as sepsis and septic shock confer immunomodulatory benefits. However, the growing problem of multidrug resistant infections has led to an increase in the administration of non-conventional last-resort antibiotics, including quinolones, aminoglycosides, and polypeptides, and the effects of these drugs on immunomodulatory gene expression in activated human polymorphonuclear leukocytes (PMNs) have not been reported. In this study, lipopolysaccharide-stimulated PMNs were incubated with piperacillin, rifampicin, fosfomycin (FOM), levofloxacin (LVFX), minocycline (MINO), colistin, tigecycline, or amikacin, and the mRNA expression levels of pattern recognition receptors (TLR2, TLR4, and CD14), inflammatory cytokines (TNFα and IL6), and chemokine receptors (IL8Rs and ITGAM) in these cells were quantitated using real-time qPCR. Many of the tested antibiotics altered the expression of the investigated cytokines. Notably, FOM, LVFX, and MINO significantly downregulated the expression of IL6, which is associated with pro- and anti-inflammatory defense mechanisms. Treatment of FOM and LVFX reduced IL-6 production as well as observed for IL6 gene expression. These findings indicated transcription and translation cooperation under the used experimental conditions. Therefore, our findings suggest that administration of these antibiotics suppresses the host anti-inflammatory response.

Lipopolysaccharide-Deficient Acinetobacter baumannii Due to Colistin Resistance Is Killed by Neutrophil-Produced Lysozyme.

Acinetobacter baumannii causes nosocomial infections due to its multidrug resistance and high environmental adaptability. Colistin is a polypeptide antibacterial agent that targets lipopolysaccharide (LPS) and is currently used to control serious multidrug-resistant Gram-negative bacterial infections, including those caused by A. baumannii. However, A. baumannii may acquire colistin resistance by losing their LPS. In mouse models, LPS-deficient A. baumannii have attenuated virulence. Nevertheless, the mechanism through which the pathogen is cleared by host immune cells is unknown. Here, we established colistin-resistant A. baumannii strains and analyzed possible mechanisms through which they are cleared by neutrophils. Colistin-resistant, LPS-deficient strains harbor mutations or insertion sequence (IS) in lpx genes, and introduction of intact lpx genes restored LPS deficiency. Analysis of interactions between these strains and neutrophils revealed that compared with wild type, LPS-deficient A. baumannii only weakly stimulated neutrophils, with consequent reduced levels of reactive oxygen species (ROS) and inflammatory cytokine production. Nonetheless, neutrophils preferentially killed LPS-deficient A. baumannii compared to wild-type strains. Moreover, LPS-deficient A. baumannii strains presented with increased sensitivities to antibacterial lysozyme and lactoferrin. We revealed that neutrophil-secreted lysozyme was the antimicrobial factor during clearance of LPS-deficient A. baumannii strains. These findings may inform the development of targeted therapeutics aimed to treat multidrug-resistant infections in immunocompromised patients who are unable to mount an appropriate cell-mediated immune response.

Multidrug-resistant Acinetobacter baumannii resists reactive oxygen species and survives in macrophages.

We investigated the intracellular survival of multidrug-resistant Acinetobacter baumannii (MDRAB) clinical isolates in macrophages, after phagocytosis, to determine their virulence characteristics. After ATCC 19606 and 5 clinical isolates of MDRAB were phagocytosed by mouse and human macrophages, the bacterial count of MDRAB strains, R4 and R5, increased in the mouse macrophages, 24 hours after phagocytosis. Bacterial count of the strains, R1 and R2, was almost equal 4 and 24 hours after phagocytosis. Intracellular reactive oxygen species was detected in the macrophages after phagocytosis of these bacteria. Further, the strains R1, R2, R4, and R5 showed higher catalase activity than ATCC 19606. Additionally, strains R1, R4, and R5 grew more efficiently than ATCC 19606 in the presence of H2O2, whereas growth of strains R2 and R3 was marginally more than that of ATCC 19606 in the presence of H2O2. The MDRAB clinical isolates altered the expression of TNF-α, IL-1ß, IL-6, and MIP-2 mRNA induced in J774A.1 cells, 24 hours after phagocytosis. These results provide insights into the renewed virulence characteristics of MDRAB clinical isolates. Finally, tigecycline killed MDRAB phagocytosed by the macrophages more effectively than colistin, although colistin and tigecycline are both considered effective antibiotics for the treatment of MDRAB.

Detection of Pediatric Pulmonary Arterial Hypertension by School Electrocardiography Mass Screening.

Rationale: To detect pulmonary arterial hypertension (PAH) at any early stage is a promising approach to optimize the outcome. Objectives: To investigate the impact of school ECG-based screening on detecting idiopathic or heritable (I/H)-PAH in the general pediatric population. Methods: This was a nationwide survey of patients with I/H-PAH newly diagnosed at 3 months to 18 years of age in Japan during 2005-2012. Measurements and Main Results: Eighty-seven eligible patients (age range, 1-16 yr) were recruited. Among 68 (78%) patients diagnosed at greater than or equal to 6 years of age (the age of the first ECG-based screening), 28 (41%) were detected by the ECG-based screening (screening group) and 40 (59%) were recognized by their symptoms (n = 37) or coincidental occasions (n = 3; nonscreening group). In the screening group, the proportion of patients in World Health Organization functional class I/II at diagnosis was higher (96% vs. 60%; P < 0.001), plasma brain natriuretic peptide level was lower (149 ± 290 vs. 398 ± 559 pg/ml; P = 0.045), and 6-minute-walk distance was longer (420 ± 109 vs. 327 ± 104 m; P < 0.001) than the nonscreening group, despite similar values in mean pulmonary artery pressure (58 ± 17 vs. 61 ± 17 mm Hg; P = 0.42) and pulmonary vascular resistance index (18 ± 8 vs. 21 ± 11 Wood units ⋅ m2; P = 0.24) between groups. The proportion of patients on intravenous epoprostenol at the final visit was lower in the screening group than the nonscreening group (14% vs. 50; P = 0.004). Conclusions: These findings suggest that the ECG-based screening detects a unique subpopulation of pediatric patients with I/H-PAH that is associated with already established pulmonary hypertension but without obvious right heart failure and warrants investigating the prognostic significance of this system.

Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion.

Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

Acinetobacter baumannii Lipopolysaccharide Influences Adipokine Expression in 3T3-L1 Adipocytes.

Acinetobacter baumannii is one of the most important nosocomial opportunistic pathogen worldwide. In addition, obesity has been associated with an increased risk of nosocomial infection, suggesting that there may be an association between A. baumannii and white adipose tissue. However, the effects of A. baumannii on adipocytes have not been well studied at the molecular level. Here, we investigated the potential role of A. baumannii-derived lipopolysaccharides (LPS) as signaling molecules that affect adipocyte functionality. We tested the effect of increasing concentrations of A. baumannii-derived LPS (10, 100, or 1000 ng/mL) on the 3T3-L1 adipocyte cell line. Exposure to LPS was found to increase the expression of several adipokines (e.g., MIP-2, MCP-1, TNF-α, IL-6, lipocalin-2, and FABP4) in 3T3-L1 adipocytes and significantly reduced the expression of leptin and adiponectin. The effects of A. baumannii-derived LPS on MIP-2 expression were similar in comparison with that of LPS prepared from Pseudomonas aeruginosa and Escherichia coli in our cell culture-based system. This study suggests that A. baumannii-derived LPS functions as a signaling molecule that impacts the inflammatory function of white adipose tissue on the level of gene expression.

The TNF-α of mast cells induces pro-inflammatory responses during infection with Acinetobacter baumannii.

Mast cells serve important roles as sentinels against bacterial infection by secreting mediators stored in granules. Much of their effectiveness depends upon recruiting and/or modulating other immune cells. The location of mast cells implies that they recognize pathogens invading tissues or mucosal tissues. Acinetobacter baumannii is a gram-negative bacterium that is considered an emerging nosocomial pathogen and causes a wide range of infections associated with high morbidity and mortality. To date, the interaction of A. baumannii with mast cells remains unclear. In this study, we demonstrated an interaction between human LAD2 mast cells and A. baumannii in vitro. When LAD2 cells were co-cultured with live A. baumannii or Pseudomonas aeruginosa PAO1 in vitro for 4h, TNF-α and IL-8 were produced in the culture supernatant. These inflammatory cytokines were not detected in the supernatant after the cells were treated with live bacteria without serum. Gene expression analysis showed that TNF-α and IL-8 mRNA expression increased in A. baumannii- and P. aeruginosa-infected LAD2 cells. Scanning electron microscopy showed that A. baumannii was tightly attached to the surface of LAD2 cells and suggested that A. baumannii may bind to FcγRII (CD32) on LAD2 cells. TNF-α in the culture supernatant from A. baumannii-infected LAD2 cells, showed that PMN activation and migration increased in Boyden chamber assays. These results suggest that mast cells recognize and initiate immune responses toward A. baumannii by releasing the preformed mediator TNF-α to activate effector neutrophils.

Inhibition of Neutrophil Adhesion and Antimicrobial Activity by Diluted Hydrosol Prepared from Rosa damascena.

Hydrosol prepared from the flowers of Rosa damascena (rose water) has been traditionally used for various health-related issues, including skin troubles such as erythema, itchiness, swelling. For the care of these skin troubles caused by microbial infection, both antimicrobial and antiinflammatory effects are required. Here, we investigated the effects of rose water on the growth of Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA), which cause skin infections, and on the function of neutrophils, which play a major role in the regulation of inflammatory reactions. To assess its modulatory effects on neutrophils, the effects of rose water against neutrophil adhesion response were evaluated. Rose water inhibited mycelial growth of C. albicans at a concentration of ca. 2.2%, and reduced viability of MRSA within 1 h. Rose water suppressed neutrophil activation induced by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), and N-formyl-Met-Leu-Phe (fMLP) at 5-15%. It also reduced the LPS- and TNF-α-induced cell surface expression of the adhesion-related molecule, cluster of differentiation (CD) 11b, but did not affect the migratory capacity of neutrophils with or without chemoattractant. These results suggest that rose water may reduce the pathogenicity of microbes, and attenuate neutrophil stimulation, which is involved in inflammatory responses. These findings suggest that rose water has a potential effect to inhibit skin inflammation caused by microbes.

Virulence characteristics of Acinetobacter baumannii clinical isolates vary with the expression levels of omps.

PURPOSE: We investigated the expression levels of virulence factors (ompA, omp33-36 and carO) in five clinical isolates and in a standard ATCC 19606 strain of Acinetobacter baumannii to determine their effect on the virulence characteristics of the isolates. METHODOLOGY: The mRNA levels of omps and proinflammatory cytokines were analyzed by quantitative real-time PCR. For adherence assay, after human lung epithelial cells (A549) were co-cultured with A. baumannii at 37 °C for 2 h, the cell-adherent bacteria was counted. Pearson correlation analysis was used to compare the omps mRNA levels, the proinflammatory cytokines and the number of adherent bacteria. RESULTS: The mRNA levels of ompA in the clinical isolates were higher and similar compared with those in ATCC 19606, whereas the mRNA levels of omp33-36 in the clinical isolates were lower and similar compared with those in ATCC 19606. The mRNA levels of carO in the clinical isolates were significantly higher than those in ATCC 19606. The number of cell-adherent clinical isolates was higher than that of cell-adherent ATCC 19606. Furthermore, the number of cell-adherent clinical isolates was positively and significantly correlated with ompA mRNA level. The mRNA levels of TNF-α, IL-6 and IL-8 in A549 cells co-cultured with the clinical isolates were lower than those in A549 cells co-cultured with ATCC 19606. Moreover, the mRNA levels of TNF-α, IL-6 and IL-8 were negatively and significantly correlated with those of carO in the isolates. CONCLUSION: These results provide insights into the renewed virulence characteristics of A. baumannii clinical isolates that depend on cell adherence capacity and the expression level of omp mRNAs.

Clinical Features of Acute and Fulminant Myocarditis in Children　- 2nd Nationwide Survey by Japanese Society of Pediatric Cardiology and Cardiac Surgery.

BACKGROUND: The 1st nationwide survey by the Japanese Society of Pediatric Cardiology and Cardiac Surgery of acute or fulminant myocarditis (AMC/FMC) in children revealed that the survival rate of FMC was only 51.6%. The 2nd nationwide survey was performed to evaluate the recent outcomes of pediatric myocarditis.Methods and Results:Questionnaires regarding patients aged ≤18 years with AMC/FMC during the period from January 2006 to December 2011 were mailed. A total of 221 cases (age 6.5±5.3 years, 116 boys and 105 girls) were reported. There were 145 (65.6%) and 74 cases (33.5%) of AMC/FMC, respectively; the type of myocarditis was not reported in the remaining 2 cases (0.9%). Viruses were identified in 56 cases (25.3%), including coxsackie B in 9 and influenza A in 8. Histopathology by either endomyocardial biopsy or autopsy was obtained in 38 cases (19.2%). Intravenous immunoglobulin was effective in 49 (34.3%) of 143 cases. Steroid therapy was effective in 20 (32.8%) of 61 cases. Mechanical circulatory support was given in 54 cases (24.4%) and 94.2% of them were patients with FMC. The survival rates for the whole study population, acute myocarditis, and FMC were 75.6%, 91.0%, and 48.6%, respectively. CONCLUSIONS: The survival rate of children with myocarditis was almost identical to that of 10 years ago. (Circ J 2016; 80: 2362-2368).

Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide.

We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accelerating disulfide-exchange reactions for incorrectdisulfides during folding in E. coli. Furthermore, the persulfides enhanced the expression level, likely due to upregulation of the enzymatic activity of T7 RNA polymerase. The recombinant protein was purified via affinity chromatography followed by cleavage with sodium fluoride, resulting in complete proteolytic removal of the N-terminal tag. The endotoxin-free fusion protein from ClearColi(®) BL21(DE3) could bind to the resin in the standard protocol using sodium phosphate (pH 7.2). Furthermore, purified rhSCF enhanced the proliferation and maturation of the human mast cell line LAD2. Thus, we conclude that use of the protein expression system employing E. coli by disulfide shuffling with persulfide addition could be a very useful method for efficient protein production.

Current trends in stenting for aortic coarctation in Japan: Subanalysis of Japanese Society of Pediatric Interventional Cardiology (JPIC) stent survey.

BACKGROUND: Stenting for aortic coarctation (CoA) has been accepted as an alternative to surgery for adolescents and adults, but only a few case have been reported in Japan. The purpose of this study was to provide a detailed review of Japanese national data on stenting of CoA. METHODS: In a subanalysis of the data of the Japanese Society of Pediatric Interventional Cardiology (JPIC), we identified 35 patients with CoA who underwent stenting. We analyzed procedural characteristics including factors that may have contributed to hemodynamic effectiveness, and we compared these parameters between the patients under and over 15 years of age. RESULTS: The mean ratio of balloon diameter/minimum lumen diameter (MLD) before stenting was 1.7 (range, 1.2-4.0), and the mean difference between the balloon diameter and the reference vessel diameter was -0.7 mm (range, -5.0 to +3.0 mm). %MLD/balloon diameter, which was defined as [(balloon diameter - MLD after dilation)/balloon diameter] × 100 predicted achievement of <10 mmHg pressure gradient after stenting. The sensitivity and the specificity of its cut-off of 7% were 93% and 47% (AUC, 0.7), respectively. There was no statistical difference between the two age groups under and over 15 years of age, in terms of selection criteria of stent size, balloon type used for deployment and immediate angiographic and hemodynamic result. CONCLUSIONS: Stenting for CoA was clinically effective with few complications in Japan, even in patients not fully grown.

Expression of bioactive soluble human stem cell factor (SCF) from recombinant Escherichia coli by coproduction of thioredoxin and efficient purification using arginine in affinity chromatography.

Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.

Priming effects of tumor necrosis factor-α on production of reactive oxygen species during Toxoplasma gondii stimulation and receptor gene expression in differentiated HL-60 cells.

Neutrophils are among the principal effector cells that protect against infectious agents, in part by producing reactive oxygen species (ROS) via the actions of tumor necrosis factor-α (TNF-α). In this study, we investigated whether HL-60 cells that had been differentiated into neutrophil-like cells by all-trans retinoic acid could be primed with TNF-α similar to human neutrophils. Our results showed that when differentiated HL-60 (dHL-60) cells were primed with TNF-α for 10 min, ROS production induced by zymosan A or phorbol myristate acetate (PMA) was enhanced in a TNF-α-dose-dependent manner. In addition, when dHL-60 cells were stimulated with live tachyzoites of Toxoplasma gondii after TNF-α priming, ROS production was also enhanced. Thus, dHL-60, similar to neutrophils, produced ROS after PMA, zymosan A, or T. gondii stimulation. Furthermore, we examined gene expression in dHL-60 cells after TNF-α treatment. The pro-inflammatory cytokine IL-6 was up-regulated more than 1.6-fold by 0.1 ng/mL TNF-α. Endogenous TNF-α was down-regulated by priming. IL-8 receptors genes were not affected by priming with 0.1 ng/mL or 1 ng/mL TNF-α. Complement receptor (CR) 1 and CR3 gene expression was not affected by TNF-α priming for 10 min. However, when the priming period was extended to 1 h, CR1 and CR3 genes were up-regulated 1.3 and 1.4-fold, respectively. Expression of the cell-surface CR3 (CD11b) was not significantly affected by TNF-α for 15 min but was slightly enhanced after priming for 2 h. These results suggest that dHL-60 cells may be used as a substitute for neutrophils when evaluating the effects of cytokines or immunomodulator agents.

Effects of erythromycin and rifampicin on immunomodulatory gene expression and cellular function in human polymorphonuclear leukocytes.

BACKGROUND: We investigated the effects of two antibiotics, erythromycin and rifampicin, on the immunomodulatory gene expression and cellular function of human polymorphonuclear leukocytes (PMNs). METHODS: We used real-time quantitative PCR to examine the expression of immunomodulatory genes. The production of reactive oxygen species (ROS) was determined by fluorescence-activated cell sorting. PMN chemotaxis was analyzed using a KK chemotaxis chamber. RESULTS: Stimulation of PMNs with lipopolysaccharide (LPS) resulted in increases in the mRNA levels of immunomodulatory genes. Rifampicin significantly inhibited the overexpression of TLR2, TLR4, CD14 and IL8Rs. However, erythromycin suppressed only the upregulation of TLR2 and TNFA. Neither antibiotic had an effect on the production of ROS. Rifampicin significantly inhibited PMN chemotaxis, but erythromycin had no effect. CONCLUSIONS: Erythromycin and rifampicin may play anti-inflammatory roles by affecting the expression levels of immunomodulatory genes or the chemotaxis of PMNs.

Enhancement of interleukin-8-induced chemotactic response and reactive oxygen species production in HL-60 cells expressing CXCR1.

Neutrophils play a pivotal role in immunity against infection by ingesting and killing invading microbes. Neutrophils isolated from human peripheral blood have been used for a number of studies conducted for evaluation of immunomodulating drugs, cytokines, and microbe products. Human promyelocytic leukemia cells, HL-60, have been extensively studied because they can differentiate into neutrophil-like cells by addition of all-trans retinoic acid or dimethyl sulfoxide. For a system that would always allow experimental use of granulocytic cells in a uniformly activated state, we have established HL-60 cell lines with increased migratory activity by transducing the CXC chemokine receptor 1 (CXCR1) gene. When these cell lines were primed with CXC chemokine ligand 8 (IL-8), a slight increase in reactive oxygen species production induced by phorbol myristate acetate (PMA) or zymosan A stimuli was observed. A significance increase in migratory activity was noticed when the HL-60 cells transduced CXCR1 were stimulated with IL-8 in the Boyden chamber method. The gene-transduced HL-60 cell lines may be used as a substitute for neutrophils in screening the effects of various immunomodulating drugs on the migratory activity induced by IL-8.

Etanercept for the treatment of intractable hemophagocytic syndrome with systemic lupus erythematosus.

A 41-year-old woman presented with continuous fever, and her laboratory data suggested the recrudescence of systemic lupus erythematosus. She was treated with 60 mg/day prednisolone. With a dose reduction of prednisolone, high fever and pancytopenia were observed again. A bone marrow biopsy revealed hemophagocytosis. The effects of steroid pulse therapy, high-dose intravenous immunoglobulin, cyclosporine A, and methotrexate were insufficient. However, after four injections of etanercept (25 mg, twice a week) subcutaneously, her symptoms had completely resolved. In such cases, therapy with etanercept may be effective.

Apoptotic signaling in endothelial cells with neutrophil activation.

As is the case for tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), degranulated substances (DS) released from polymorphonuclear leukocytes (PMN) and H(2)O(2) cause endothelial cell apoptosis through the phosphorylation of members of the mitogen-activated protein kinase (MAPK) family. Stimulation of human umbilical vein endothelial cells (HUVEC) with IL-1ß or TNF-α/cycloheximide (CHX) was found to enhance the phosphorylation of p38 and Jun-N-terminal kinase (JNK) in a time-dependent fashion, but did not affect the time-dependent phosphorylation of extracellular signal-regulated kinase. In addition, IL-1ß and TNF-α/CHX induced the phosphorylation of activating transcription factor-2 (ATF-2), but not c-Jun. Moreover, the p38 in HUVEC was phosphorylated by DS released from PMN and also by H(2)O(2), but not by •O(2) (-) induced by myeloperoxidase (MPO) of PMN. On the other hand, caspase 8 in HUVEC was activated by DS, but not by H(2)O(2) and/or •O(2) (-). In addition, caspases 3 and 7 were cleaved by the treatment of DS and turned into active forms. DS was concentrated, analyzed by electrophoresis, and revealed to contain precursor and subunits of MPO (90, 60, and 14 kDa) and another peptide with a molecular weight of about 28 kDa. Because SB203580 that was an inhibitor of p38 MAPK did not repress phosphorylation of ATF-2 in HUVEC, it was suggested that JNK was more important than p38 in a series of signaling courses. These results suggest the possibility that not only TNF-α/CHX and IL-1ß but also DS released from PMN and the cell-permeable reactive oxygen species H(2)O(2) induce blood vessel injury through endothelial apoptosis.