Neutrophil Chemotactic Activity Research Articles

Increased of exhaled breath condensate neutrophil chemotaxis in acute exacerbation of COPD

BackgroundNeutrophils have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Underlying mechanisms of neutrophil accumulation in the airways of stable and exacerbated COPD patients are poorly understood. The aim of this study was to assess exhaled breath condensate (EBC) neutrophil chemotactic activity, the level of two chemoattractants for neutrophils (GRO-α and LTB4) during the course of an acute exacerbation of COPD (AECOPD).Methods50 ex smoking COPD patients (33 with acute exacerbation and 17 in stable disease) and 20 matched ex smoking healthy controls were compared. EBC was collected by using a commercially available condenser (EcoScreen®). EBC neutrophil chemotactic activity (NCA) was assessed by using Boyden microchambers. Chemotactic index (CI) was used to evaluate cell migration. LTB4 and GROα levels were measured by a specific enzyme immunoassay in EBC.ResultsStable COPD and outpatients with AECOPD, but not hospitalized with AECOPD, had raised EBC NCA compared to healthy subjects (p < 0.05 and p < 0.01 respectively). In outpatients with AECOPD EBC NCA significantly decreased 6 weeks after the exacerbation. Overall EBC NCA was weakly correlated with sputum neutrophil counts (r = 0.26, p < 0.05).EBC LTB4 levels were increased in all groups of COPD compared to healthy subjects while GRO-α was only raised in patients with AECOPD. Furthermore, EBC LTB4 and GRO-α significantly decreased after recovery of the acute exacerbation. Increasing concentrations (0.1 to 10 μg/mL) of anti- human GRO-α monoclonal antibody had no effect on EBC neutrophil chemotactic activity of 10 exacerbated COPD patients.ConclusionsEBC NCA rose during acute exacerbation of COPD in ambulatory patients and decreased at recovery. While LTB4 seems to play a role both in stable and in exacerbated phase of the disease, the role of GRO-α as a chemotactic factor during AECOPD is not clearly established and needs further investigation.

Synergic production of neutrophil chemotactic activity by colonic epithelial cells and eosinophils

The presence of eosinophils in the lumen and mucosa of the intestine is characteristic of both ulcerative colitis (UC) and Crohn's disease (CD). There is evidence of eosinophil activation in the intestine during acute inflammatory episodes of these diseases; these episodes are also characterized by an influx of neutrophils, which have the potential to cause extensive tissue damage. We undertook a study to determine whether eosinophils in contact with colonic epithelial cells produce factors that may attract neutrophils in response to immunological stimulation. Neutrophil chemotactic activity (NCA) and concentrations of three neutrophil-attracting CXC chemokines – CXCL1 (Groα), CXCL5 (Ena78) and CXCL8 (IL8) – were measured in supernatants of T84 colonic epithelial cells and blood eosinophils or eosinophil-like myeloid leukaemia cells (AML14.3D10), alone or in combination. Cells were stimulated with serum-opsonized zymosan (OZ) particles. NCA (P<0.005) and CXCL5 levels (P<0.05) in the supernatants of OZ-stimulated epithelial/eosinophil co-cultures were significantly higher than in the supernatants of either cell type alone. Release of CXCL1 (P<0.05) and CXCL8 (P<0.01) from OZ-stimulated co-culture supernatants was significantly higher than from OZ-stimulated eosinophils but not higher than from OZ-stimulated epithelial cells. Eosinophils and colonic epithelial cells exhibit synergy in production of neutrophil chemoattractants in response to immunological stimulation. This may represent a mechanism for exaggerated recruitment of neutrophils to the intestine in response to acute infection in conditions that are characterized by the presence of eosinophils in the bowel.

The Role of Bone Morphogenetic Protein 6 in Accumulation and Regulation of Neutrophils in the Human Ovary

Bone morphogenetic protein (BMP) cytokine is known to regulate ovulation, as BMP-6 null mice exhibit a decrease in the number of ovulatory follicles without effect on either the morphology or the dynamics of follicular development. In the present study, the role of BMP-6 in ovulatory process was investigated using human granulosa-lutein cells (GCs). Granulosa-lutein cells, obtained from in vitro fertilization patients, were cultured with BMP-6 followed by RNA extraction. The neutrophil-chemotactic activity of the supernatant of cultured GC was investigated. Bone morphogenetic protein 6 significantly increased growth-regulated oncogene α (GRO-α) messenger RNA (mRNA) and protein expression in GC. In the neutrophil-chemotaxis assay, the GC supernatant cultured with BMP-6 attracted more neutrophils than control samples, which was negated with anti-GRO-α neutralizing antibody. Bone morphogenetic protein 6 also suppressed the relative expression of the protease inhibitors, secretory leukocyte peptidase inhibitor, and whey acid protein 14 mRNA in GC. Bone morphogenetic protein 6 might play a role in ovulation by increasing the accumulation of neutrophils in the ovulatory follicle and suppressing the effect of protease inhibitors.

Long-acting muscarinic receptor antagonists for the treatment of chronic airway diseases.

Acetylcholine (neuronal and non-neuronal origin) regulates bronchoconstriction, and mucus secretion. It has an inflammatory effect by inducing attraction, survival and cytokine release from inflammatory cells. Muscarinic receptors throughout the bronchial tree are mainly restricted to muscarinic M1, M2 and M3 receptors. Three long-acting muscarinic receptor antagonists (LAMAs) were approved for the treatment of chronic obstructive pulmonary disease (COPD) in Europe: once-daily tiotropium bromide; once-daily glycopyrronium bromide; and twice-daily aclidinium bromide. All have higher selectivity for M3 receptors than for M2 receptors, and dissociate more slowly from the M3 receptors than they do from the M2 receptors. Some LAMAs showed anti-inflammatory effects [inhibition of neutrophil chemotactic activity and migration of alveolar neutrophils, decrease of several cytokines in the bronchoalveolar lavage (BAL) including interleukin (IL)-6, tumor necrosis factor (TNF)-α and leukotriene (LT)B4] and antiremodeling effects (inhibition of mucus gland hypertrophy and decrease in MUC5AC-positive goblet cell number, decrease in MUC5AC overexpression). In the clinic, LAMAs showed a significant improvement of forced expiratory volume in 1 second (FEV1), quality of life, dyspnea and reduced the number of exacerbations in COPD and more recently in asthma. This review will focus on the three LAMAs approved in Europe in the treatment of chronic airway diseases.

Rhino-orbito-cerebral mucormycosis in a child with diabetic ketoacidosis.

Sir, Rhino-orbito-cerebral mucormycosis is an unusual complication of diabetic ketoacidosis (DKA). Most of them have been fatal. High index of suspicion is critical for diagnosis and early treatment. A 9-year-old, female child presented with fever, abdominal pain, and vomiting for 2 days. Examination revealed acidotic breathing and multiple furuncles over the face. She had hyperglycemia of 485 mg/dl, ketonuria, wide anion gap metabolic acidosis (pH 6.8) and polymorphonuclear leucocytosis. She was diagnosed as Type 1 diabetes with DKA and treated according to the DKA management protocol of the unit. In view of persistent altered sensorium anti-edema measures were initiated. At 48 h of admission, she had a dark colored bloody nasal discharge. Injection amphotericin B was started empirically for suspected mucormycosis. At 72 h, the child developed ophthalmoplegia of the right eye. A black necrotic palatal lesion was noted at 96 h [Figure 1]. Computed tomography (CT) brain was normal. CT paranasal sinuses showed right maxillary sinusitis. Nasal endoscopy revealed black necrotic eschar over the right middle and inferior turbinates, floor and lateral wall of nasal cavity. Despite surgical debridement child worsened with right facial palsy and left hemiparesis. Repeat CT brain revealed massive infarct involving right frontal, temporal and parietal regions. The child expired on day 9. Figure 1 Black necrotic eschar in palate Histopathology of the necrotic lesion revealed broad, aseptate hyphae, branching at right angles with vascular invasion. Culture on saboraud dextrose agar medium grew black colonies typical of Rhizopus. On lactophenol cotton blue mount presence of sporangium and rhizoids typical of Rhizopus was seen. Postmortem cerebral spinal fluid was unusually orange-red colored with no cells, with 1250 mg% proteins and 35 mg% sugar. Hemoglobin A1c was high (12.6%). Mucormycosis is an acute, frequently fatal, fungal disease in immunocompromised children due to Rhizopus[1] followed by Mucor. Risk factors include DKA, prolonged neutropenia, malignancy, chronic corticosteroid therapy and deferoxamine therapy. Rhinocerebral (most common in DKA),[2] pulmonary, cutaneous, gastrointestinal, and disseminated involvement can be seen. In DKA, the chemotactic activity of neutrophils and their ability to produce reactive oxygen species is diminished. Acidosis disrupts the transferrin binding to iron,[3] releasing free iron favoring Rhizopus growth. As the advanced glycosylation end products increases the transendothelial migration of neutrophils decreases. Following inhalation, the disease begins with facial pain and numbness followed by sinusitis and dark bloody nasal discharge. In this child, nasal discharge was the first sign. The infected tissue appears normal initially then become erythematous, violaceous and finally turns black due to thrombosis and tissue infarction.[4] Rapid progression of the disease occurs in hours to days. Direct microscopic examination of the specimen and culture are diagnostic. Management includes reversal of the risk factor, surgical debridement along with antifungal therapy. Amphotericin B (polyene) is the drug of choice given over few weeks. Amphotericin B liposomal complex, echinocandins with polyenes, posaconazole and deferasirox can also be used. Clinical suspicion warrants treatment without awaiting confirmation. Mortality is more than 40% with cerebral involvement. It is less than 10% without cerebral involvement.[2] Poor prognostic factors are delayed diagnosis, inadequate surgical debridement, spread beyond sinonasal cavity, and cerebral involvement.

Tissue Inhibitor of Metalloproteinases–3 Moderates the Proinflammatory Status of Macrophages

Tissue inhibitor of metalloproteinases-3 (TIMP-3) has emerged as a key mediator of inflammation. Recently, we reported that the resolution of inflammation is impaired in Timp3(-/-) mice after bleomycin-induced lung injury. Here, we demonstrate that after LPS instillation (another model of acute lung injury), Timp3(-/-) mice demonstrate enhanced and persistent neutrophilia, increased numbers of infiltrated macrophages, and delayed weight gain, compared with wild-type (WT) mice. Because macrophages possess broad immune functions and can differentiate into cells that either stimulate inflammation (M1 macrophages) or are immunosuppressive (M2 macrophages), we examined whether TIMP-3 influences macrophage polarization. Comparisons of the global gene expression of unstimulated or LPS-stimulated bone marrow-derived macrophages (BMDMs) from WT and Timp3(-/-) mice revealed that Timp3(-/-) BMDMs exhibited an increased expression of genes associated with proinflammatory (M1) macrophages, including Il6, Il12, Nos2, and Ccl2. Microarray analyses also revealed a baseline difference in gene expression between WT and Timp3(-/-) BMDMs, suggesting altered macrophage differentiation. Furthermore, the treatment of Timp3(-/-) BMDMs with recombinant TIMP-3 rescued this altered gene expression. We also examined macrophage function, and found that Timp3(-/-) M1 cells exhibit significantly more neutrophil chemotactic activity and significantly less soluble Fas ligand-induced caspase-3/7 activity, a marker of apoptosis, compared with WT M1 cells. Macrophage differentiation into immunosuppressive M2 cells is mediated by exposure to IL-4/IL-13, and we found that Timp3(-/-) M2 macrophages demonstrated a lower expression of genes associated with an anti-inflammatory phenotype, compared with WT M2 cells. Collectively, these findings indicate that TIMP-3 functions to moderate the differentiation of macrophages into proinflammatory (M1) cells.

EGF-Induced Bronchial Epithelial Cells Drive Neutrophil Chemotactic and Anti-Apoptotic Activity in Asthma

Chronic damage and repair of the bronchial epithelium are features of asthma. We have previously reported that ex vivo stimulation of normal bronchial epithelial cells with epidermal growth factor (EGF), a key factor of epithelial repair, enhances the mechanisms of neutrophil accumulation, thereby promoting neutrophil defences during acute injury but potentially enhancing inflammation in chronic airway diseases. We have now sought to (i) determine whether this EGF-dependent pro-neutrophil activity is increased in asthma, where EGF and its epithelial receptor are over-expressed, and (ii) elucidate some of the mechanisms underlying this asthmatic epithelial-neutrophil interaction. Primary bronchial epithelial cells (PBEC) from healthy subjects, mild asthmatics and moderate-to-severe asthmatics (Mod/Sev) were stimulated with EGF, a model that mimics a repairing epithelium. Conditioned culture media (EGF-CM) were assessed for neutrophil chemotactic and anti-apoptotic activities and inflammatory mediator production. EGF induced the epithelium to produce soluble mediators with neutrophil chemotactic (p<0.001) and pro-survival (p = 0.021) activities which were related to the clinical severity of asthma (trend p = 0.010 and p = 0.009, respectively). This was associated with enhanced IL-6, IL-8, GM-CSF and TNF-α release, and cytokine-neutralising experiments using EGF-CM from Mod/Sev asthmatics demonstrated a role for GM-CSF in neutrophil survival (p<0.001). Pre-treatment of neutrophils with specific inhibitors of the myeloid-restricted class I phosphatidylinositol-3-OH kinase (PI(3)K) isoforms showed that the EGF-CM from Mod/Sev asthmatics depended on the γ (p<0.021) but not δ isoforms, while neutrophil survival required multiple class I PI(3)Ks. The EGF-induced chemotactic, but not pro-survival activity, involved RhoA signaling in neutrophils (p = 0.012). EGF whose activity is upregulated in asthma induces ex vivo the epithelium from asthmatic patients to produce pro-neutrophil activities; these are related to asthma severity and, in moderate-to-severe asthmatics, involves class IB PI(3)Kγ signaling, providing a potential therapeutic target for neutrophilic forms of asthma.

Cyclic Stretch Augments Production of Neutrophil Chemokines and Matrix Metalloproteinases‐1 (MMP‐1) from Human Decidual Cells, and the Production was Reduced by Progesterone

The purpose of this study was to evaluate the impact of mechanical stretch caused by uterine contraction and progesterone (P₄) on decidual cells (DC), neutrophil chemokines, and MMP-1 expression. DC were cultured, and cyclic stretch was applied using a computer-operated cell stretch system. Interleukin (IL)-8, growth-regulated oncogene (GRO) α, matrix metalloproteinase (MMP)-1, and mRNA and/or protein expression/activity was measured using RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated using migration assays. The effect of P₄ was also studied. Cyclic mechanical stretch increased IL-8, GROα, mRNA and protein, and MMP-1 production and activity level. Supernatant from stretched cells induced neutrophil chemotactic activity significantly. P₄ suppressed the effect of stretch. The current study demonstrates that cyclic mechanical stretch stimulates the production of neutrophil chemokines and MMP-1 from human decidual cells, and the production was reduced by progesterone. These findings suggest that decidual cells are responding to mechanical and endocrine signals and induce biochemical factors and thereby contribute to the regulation of human labor.

Effect of Hot Water Extract from Agaricus Blazei Murill on Chemotaxis of Neutrophils

Hot water extract from the edible Brazilian mushroom, Agaricus Blazei Murill (ABM), is used for both traditional and alternative medicine. ABM is reported to stimulate anti-tumor, anti-infection, and immune activity. However, there are few reports of how ABM affects neutrophils. Therefore, in this study, we examined the effect of hot water ABM extract on neutrophil migration, phagocytosis, and reactive oxygen species production using neutrophils from guinea pig. Migratory direction and velocity as indicators of chemotactic activity of neutrophils were significantly (p 0.001) increased at concentration of 50 and 100 mg/ml in ABM extract compared with control. Phagocytic activity of neutrophil was significantly (p 0.01) increased at concentration of 5 mg/ml in ABM extract compared with control. Production of reactive oxygen species (ROS: H2O2 or ) by neutrophils was significantly (p 0.01) increased at concentration of 5 mg/ml in ABM extract compared with control. These results suggest that enhancement in neutrophil chemotactic activity, phagocytic activity and ROS production are mechanisms by which ABM extract inhibits bacterial infection in the skin and dermatitis.

Cyclic Stretch Augments Production of Neutrophil Chemokines and Matrix Metalloproteinase‐1 in Human Uterine Smooth Muscle Cells

The aim of this study was to investigate the impact of uterine contraction on the immune environment within the uterus during parturition. Uterine smooth muscle cells (USMC) were isolated from uterine myometrial tissues and cultured. The effects of cyclic stretch on mRNA and/or protein expression of IL-8, Groα, and pro-MMP-1 by USMC were measured using RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated using migration assays. To evaluate the effect of progesterone (P(4) ), USMC were pretreated with P(4) for 24hr. Cyclic stretch increased IL-8 and Groα mRNA and protein and pro-MMP-1 production significantly. Supernatants from stretched cells induced neutrophil chemotactic activity significantly; these effects were abrogated by anti-IL-8 or Groα neutralizing antibodies. Stretch effects were reduced by P(4) . These results suggest that uterine contraction may induce neutrophil infiltration and MMP-1 production, which may contribute to cervical ripening and rupture of membrane. The inhibitory effects of P(4) may explain the mechanism by which progestin prevents preterm labor.

Hepoxilin A3 Facilitates Neutrophilic Breach of Lipoxygenase-Expressing Airway Epithelial Barriers

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.

Lipocalin‐2 ameliorates granulocyte functionality

Attraction of neutrophils to sites of infection or tissue injury is an essential prerequisite for an efficient innate immune response. Herein, we provide novel evidence that the antimicrobial protein, neutrophil gelatinase associated lipocalin (24p3 or lipocalin-2, Lcn2) is a central regulator of this process. Lcn2 is produced by several cell types but high amounts are released by neutrophils. Using human and murine neutrophils, we found that the addition of recombinant Lcn2 significantly stimulated their migration, which was independent of IL-8/keratinocyte chemokine formation. Mechanistically, this could be traced back to Lcn2-mediated changes of Erk1/2 signaling. Accordingly, the i.p. injection of Lcn2 into C57BL/6 mice stimulated the mobilization of neutrophils while we found a significantly reduced neutrophil chemotactic activity of cells obtained from Lcn2 KO mice. This observation transmitted to a reduced accumulation of neutrophils in intra-dermal lesions infected with Salmonella typhimurium in Lcn2 KO mice as compared to WT mice. This was not only due to a reduced chemotaxis but also to an impaired cellular adhesion of neutrophils in the absence of Lcn2. We herein describe a novel role of Lcn2 as an important paracrine chemoattractant and an indispensable factor for neutrophil function in inflammation.

Mitochondrial dysfunction increases inflammatory responsiveness to cytokines in normal human chondrocytes

Alterations in mitochondria play a key role in the pathogenesis of osteoarthritis (OA). The role of inflammation in the progression of OA has also acquired important new dimensions. This study was undertaken to evaluate the potential role of mitochondrial dysfunction in increasing the inflammatory response of normal human chondrocytes to cytokines. Mitochondrial dysfunction was induced by commonly used inhibitors. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were used as inflammatory mediators. IL-8 and cyclooxygenase 2 (COX-2) protein and messenger RNA (mRNA) expression and prostaglandin E(2) (PGE(2) ) levels were assessed. The chemotactic activity of neutrophils was assayed. Additionally, inhibitors of reactive oxygen species (ROS) and NF-κB were used to identify possible inflammatory response pathways induced by mitochondrial dysfunction, and the effects of the natural antioxidant resveratrol were tested. Pretreatment with antimycin A or oligomycin (inhibitors of mitochondrial respiratory chain complexes III and V, respectively) triggered a strong potentiation of IL-1β-induced IL-8 mRNA and protein expression (mean ± SEM at 18 hours 5,932 ± 1,995 pg/50,000 cells for IL-1β alone versus 16,241 ± 5,843 pg/50,000 cells for antimycin A plus IL-1β and 20,087 ± 5,407 pg/50,000 cells for oligomycin plus IL-1β; P < 0.05). Similar results were observed with TNFα or when expression of the inflammatory mediator COX-2 or PGE(2) production was assessed. Mitochondrial dysfunction increased the chemotactic activity induced by cytokines, and ROS and NF-κB inhibitors decreased the production of IL-8. Resveratrol significantly reduced the inflammatory response. Our findings indicate that mitochondrial dysfunction could amplify the responsiveness to cytokine-induced chondrocyte inflammation through ROS production and NF-κB activation. This pathway might lead to the impairment of cartilage and joint function in OA.

Functional Capacity of Neutrophils From Class III Obese Patients

Class III obesity is associated with chronic inflammation and a variety of changes in immune function. Yet surprisingly little was known about the status of neutrophils that represent the first line of immune defense. The aim of this study was to assess key functions of neutrophils from class III obese patients, namely phagocytosis, superoxide production, chemotaxis, and response to endotoxin challenge, and compare their responses with lean controls. Thirty obese patients (BMI 48.8 ± 6.6 kg/m(2)) with comorbidities such as diabetes, hyperlipidemia, high blood pressure, etc. and nine lean (BMI between 20 and 25) subjects were enrolled in the study. Neutrophils from class III obese patients phagocytosed Escherichia coli (E. coli) at similar rates and with adequate numbers of bacteria taken up per cell compared with cells from lean subjects. Neutrophil production of superoxide, which is key to rapid killing of pathogens, showed modest diminution in the class III obese, which increased among patients with BMI >50. Chemotactic activity of neutrophils from class III obese patients was not altered. However, neutrophils from obese subjects showed an increased response to low-dose endotoxin, with concomitant reduced apoptosis and extension of their half-life compared with lean subjects, which suggests possible hyperresponsiveness of these neutrophils. Overall, neutrophil activity was not significantly altered by age, gender, diabetic status, or hyperlipidemia. Collectively, these results suggest that class III obese patients, even with comorbidities, have normal or nearly normal phagocytic, chemotactic, and superoxide generating capacity.

LPS challenge in healthy subjects: An investigation of neutrophil chemotaxis mechanisms involving CXCR1 and CXCR2

LPS inhalation was used to investigate whether sputum supernatant post-LPS challenge increases neutrophil chemotactic activity and to elucidate the role of CXCR1/CXCR2 signalling in this process.14 healthy non-smoking subjects inhaled 30μg of LPS. Sputum was induced at baseline, 6 and 24h post-LPS challenge. Differential cell counts were determined and supernatants CXCL8, CXCL1, IL-6 and CCL2 levels measured. Peripheral blood neutrophils obtained from healthy volunteers were used for chemotaxis experiments using sputum supernatant. To delineate signalling mechanisms, the effects of a CXCR2/CXCR1 (dual) antagonist (Sch527123) and a CXCR2 specific antagonist (SB656933) were tested.LPS inhalation significantly increased sputum neutrophil counts from 45.3% to 76.7% and 69.3% at 6 and 24h respectively. LPS increased CXCL8, IL-6 and CCL2 levels but not CXCL1. Neutrophil chemotaxis significantly increased (2.7 fold) at 24h compared to baseline. Chemotaxis was inhibited by 79.0% with Sch527123 and 52.0% with SB656933.We conclude that LPS challenge increases sputum supernatant CXCL8 levels, which is associated with increased chemotactic activity which is dependent on both CXCR1 and CXCR2.

TLR2 ligand engagement upregulates airway smooth muscle TNFα-induced cytokine production

Airway inflammation and respiratory infections are important factors contributing to disease exacerbation in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle (ASM) cells express Toll-like receptors (TLRs) and may be involved in the amplification of airway inflammatory responses during infectious exacerbations. We determined whether infectious stimuli (mimicked using Pam3CSK4, a synthetic bacterial lipopeptide that binds to TLR2/TLR1) further enhance ASM cell inflammatory responses to TNFα in vitro and the signaling pathways involved. Human ASM cells were pretreated for 1 h with Pam3CSK4 (1 μg/ml) in the absence or presence of TNFα (10 ng/ml), and IL-6 and IL-8 release was measured after 24 h. As expected, stimulation with Pam3CSK4 or TNFα alone induced significant IL-6 and IL-8 release. Furthermore, Pam3CSK4 significantly increased TNFα-induced IL-6 and IL-8 mRNA expression and protein release and neutrophil chemotactic activity. The potentiating effect of Pam3CSK4 on TNFα-induced inflammatory responses was not due to enhanced TLR2 expression nor did it involve augmentation of NF-κB or MAPK signaling pathways. Rather, Pam3CSK4 induced cAMP response element (CRE) binding protein phosphorylation and induced CRE-mediated transcriptional regulation, suggesting that Pam3CSK4 and TNFα are acting in concert to enhance ASM cytokine secretion via parallel transcriptional pathways. Our findings suggest that ASM cells may be involved in the amplification of airway inflammatory responses during infectious exacerbations in chronic airway disease.

Respiratory epithelial cell lines exposed to anoxia produced inflammatory mediator

Human epithelial cell lines were utilized to examine the effects of anoxia on cellular growth and metabolism. Three normal human epithelial cells lines (A549, NHBE, and BEAS-2B) as well as a cystic fibrosis cell line (IB3-1) and its mutation corrected cell line (C38) were grown in the presence and absence of oxygen for varying periods of time. Interleukin-8 (IL-8) levels were measured by enzyme-linked immunosorbent assay technique. Cellular metabolism and proliferation were assayed by determining mitochondrial oxidative burst activity by tetrazolium compound reduction. The viability of cells was indirectly measured by lactate dehydrogenase release. A549, NHBE, and BEAS-2B cells cultured in the absence of oxygen showed a progressive decrease in metabolic activity and cell proliferation after one to three days. There was a concomitant increase in IL-8 production. Cell lines from cystic fibrosis (CF) patients did not show a similar detrimental effect of anoxia. However, the IL-8 level was significantly increased only in IB3-1 cells exposed to anoxia after two days. Anoxia appears to affect certain airway epithelial cell lines uniquely with decreased cellular proliferation and a concomitant increased production of a cytokine with neutrophilic chemotactic activity. The increased ability of the CF cell line to respond to anoxia with increased secretion of inflammatory cytokines may contribute to the inflammatory damage seen in CF bronchial airway. This study indicates the need to use different cell lines in in vitro studies investigating the role of epithelial cells in airway inflammation and the effects of environmental influences.

Inhibitory effects of bortezomib on restenosis of rat experimental vein graft

Objective The anti-inflammatory and vaso-protective effects of bortezomib were analyzed in rat model to test whether it inhibits neointima formation in transplant-induced vasculopathy.Methods Seventy-six rats were subjected to autologous vein grafting surgery.Thereafter,all post-surgical rats were randomly divided into two groups and dosed intravenously with 0.15 mg/kg bortezomib or vehicle on the day 0,3,7,and 10.After 24 and 72 h,rats were humanely killed and vein grafts were processed for real-time reverse transcription-polymerase chain reaction (RT-PCR) to test the gene expression of cytokineinduced neutrophil chemoattractant-2β (CINC-2β),monocyte chemoattractant protein ( MCP)-1,interleukin (IL)-1,IL-6 and tumor necrosis factor (TNF)-α.Meanwhile,24-h vein grafts were processed for enzyme linked immunosorbent assay ( ELISA ) or neutrophil chemotaxis assay.Subsequently,rats were euthanized at 4th week after grafting and samples were processed for morphometric analysis.Results The expression of mRNA for chemokines ( CINC-2β,MCP-1 ) and cytokines ( IL-1,IL-6 and TNF-α) were markedly increased in the injured vessels at the first day after surgery and declined during the following three days,and protein levels of them were markedly increased at the same time [ CINC-2β (415.23 ±56.50) pg/mg; MCP-1 (9894.58 ±2795.62) pg/mg; IL-1 (3087.51 ±609.19) pg/mg; IL-6 (2225.27±206.06) pg/mg; TNF-α (402.31 ± 134.13) pg/mg] (P ＜0.05) accompanied with a marked increase in neutrophil migration toward homogenates of injured vessels as chemotactic index (CI) was (241.52 ±33.43 ) ％ ( P ＜ 0.05 ).Morphometric analysis revealed that the intima of grafts was thickened markedly as (70.93 ± 11.25) μm at 4th week after surgery (P ＜0.05).It was important that bortezomib significantly attenuated gene expression and protein levels in most of the inflammatory mediators except IL-1 [ CINC-2β(231.48 ±36.32) pg/mg; MCP-1 (5846.10 ± 1044.33) pg/mg; IL-6 (1023.22 ± 109.84) pg/mg;TNF-α (218.40 ± 38.06) pg/mg] (P ＜ 0.05 ),and simultaneously inhibited neutrophil chemotactic activity of the vessel homogenates as CI was ( 167.81 ± 30.00) ％ (P ＜0.05 ).And notably,bortezomib resulted in significant inhibition of intimal hyperplasia as (35.23 ±5.21 ) μm at 4th week compared with untreated controls (P ＜ 0.05).Conclusion Bortezomib could inhibit neointima formation by attenuating the inflammatory response in transplant-induced vasculopathy. Key words: Proteasome inhibitor; Bypass graft; Intimal hyperplasia; Restenosis

Up-regulation of human bradykinin B1 receptor by secreted components ofPseudomonas aeruginosavia a NF-κB pathway in epithelial cells

Pulmonary epithelial cells produce neutrophil chemotactic activity in response to pathogenic bacterial infections, resulting in neutrophil migration to infection sites. Elicited neutrophils in the inflamed tissues were found to be dependent on bradykinin B1 receptor (B1R), which shows high affinity for the active metabolites derived from bradykinin. Thus, the up-regulation of bradykinin and B1R expression represents an important host defense response against invading microbes such as Pseudomonas aeruginosa. However, the effect of P.aeruginosa on the expression of B1R remains unclear, while P.aeruginosa infection is known to stimulate the production of bradykinin. Here, we report that human B1R (hB1R) transcription is up-regulated in host cells co-cultured with P.aeruginosa. Components secreted from P.aeruginosa play a major role in the up-regulation, and the secretion of the components is not controlled by either type III secretion system or quorum sensing. Moreover, the B1R induction is mediated by a NF-κB signaling pathway in human lung epithelial cells. Taken together, this study demonstrates that P.aeruginosa is capable of up-regulating hB1R expression via the NF-κB signaling pathway.

A functional promoter polymorphism of the human IL18 gene is associated with aspirin-induced urticaria

Urticaria is the commonest cutaneous reaction caused by aspirin or other nonsteroidal anti-inflammatory drugs. The pathogenesis of aspirin-induced urticaria (AIU) is not fully understood, but appears to involve mast cell activation and neutrophil infiltration. To investigate the genetic contribution of interleukin (IL)-18, which can amplify acute inflammation by promoting mast cell activation, neutrophil migration and cytokine production, to the pathogenesis of AIU. A case-control association study was performed using 275 patients with AIU and 196 normal healthy controls in a Korean population. Two promoter polymorphisms of the IL18 gene (-607A/C and -137G/C) were genotyped using the primer extension method. The functional effect of the IL18 gene promoter polymorphism was investigated through in vitro studies including a luciferase reporter assay and electrophoretic mobility shift assays (EMSAs) and ex vivo studies involving neutrophil chemotaxis assays. A significant association was detected between both AIU in general and the aspirin-intolerant acute urticaria (AIAU) phenotype and the IL18 promoter polymorphism -607A/C. Patients with AIAU showed higher frequencies of the C(-607) G(-137) haplotype, ht1 [CG], compared with controls (P=0·02). Moreover, ht1 [CG] showed a high transcript haplotype by the luciferase activity assay, and EMSAs identified a -607C allele-specific DNA-binding protein as CREB2. Neutrophil chemotactic activity was highest in subjects with AIU exhibiting the high transcript haplotype, ht1 [CG] (P=0·019). The high transcript haplotype ht1 [CG] of the IL18 gene may contribute to the development of acute cutaneous inflammation sensitive to aspirin, leading to the clinical presentation of AIAU.