Neutrophil Chemotactic Activity Research Articles

Angiopoietin‐1 but not angiopoietin‐2 induces IL‐8 synthesis and release from human neutrophils

Angiopoietins (Ang1 and Ang2) can promote neutrophil chemotactic activities by activating Tie2 receptor. Moreover, pretreatment with Ang1 or Ang2 enhances the chemotactic effect of interleukin‐8 (IL‐8). We wanted to assess if angiopoietins can promote IL‐8 protein synthesis and release from human neutrophils. Only a treatment with Ang1 at 10−8 M induced a significant increase of IL‐8 protein synthesis (3.6‐fold) and release (5.5‐fold), whereas the combination of Ang1 and Ang2 induced the same effect as Ang1 alone. Moreover, IL‐8 mRNA production was also increased (4.7‐fold) as compared to PBS. Cycloheximide, a protein synthesis inhibitor, reduced Ang1‐mediated IL‐8 protein synthesis and release by up to 96 and 92% respectively, whereas IL‐8 mRNA was increased by up to 18‐fold compared to PBS‐treated neutrophils. Actinomycin D, an mRNA synthesis inhibitor, reduced Ang1‐mediated IL‐8 mRNA and protein synthesis only within the first hour of treatment by 54 and 52% respectively. Using specific kinase inhibitors, we observed that Ang1‐driven IL‐8 mRNA and protein increase is p42/44 MAPK dependent and independent from p38 MAPK and PI3K activity. In summary, Ang1 (10−8 M) induces IL‐8 mRNA synthesis, as well as IL‐8 protein synthesis and release, through the activation of p42/44 MAPK. This work was supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Quebec.

Cilomilast counteracts the effects of cigarette smoke in airway epithelial cells

Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Cilomilast, a phosphodiesterase-4 inhibitor, inhibits cigarette smoke-induced neutrophilia. This study was aimed to explore whether cilomilast, in a human bronchial epithelial cell line (16-HBE), counteracted CSE effects. In particular, TLR4 expression, IP-10 and IL-8 release, lymphocyte and neutrophil chemotactic activity and ERK and IkBa phosphorylation in CSE and LPS-stimulated 16-HBE were assessed. CSE increased TLR4 expression, reduced IP-10 release and lymphocyte chemotactic activity and increased IL-8 release and neutrophil chemotactic activity. Cilomilast reduced TLR4 expression, IL-8 release and neutrophil chemotactic activity as well as it increased IP-10 release and lymphocyte chemotactic activity. All these cilomilast mediated effects were associated with a reduced ERK1/2 and with an increased IkBa phosphorylation. In conclusion, the present study provides compelling evidences that cilomilast may be considered a possible valid therapeutic option in controlling inflammatory processes present in smokers.

Effect of Jungle honey on the chemotactic activity of neutrophils

Effect of Jungle honey on the chemotactic activity of neutrophils

TLR4 upregulation underpins airway neutrophilia in smokers with chronic obstructive pulmonary disease and acute respiratory failure

Activation of Toll-like receptors (TLR) seems to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Upon TLR activation the release of defensins, including human beta defensin 2 (HBD-2), may occur. In this study, we explored the innate responses in patients with respiratory failure, with and without COPD, requiring intubation and mechanical ventilation. Mini–bronchoalveolar lavage (mini-BAL) samples were collected from nonsmoker subjects without COPD ( n = 10), smokers without COPD ( n = 6), and smokers with COPD ( n = 15). TLR4, TLR2, and HBD-2 expression was evaluated by immunocytochemistry; interleukin (IL)–8, IP-10, and HBD-2 concentrations were evaluated by enzyme-linked immunosorbent assay; chemotactic activity toward neutrophils and lymphocytes; and cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] and by flow cytometry with anti-TLR4 and with HBD-2 depleted and not depleted mini-BAL). COPD mini-BAL showed increased neutrophil numbers, reduced neutrophil apoptosis, increased TLR4 and HBD-2 expression, increased neutrophil chemotactic activity, reduced IP-10 concentrations, and reduced lymphocyte chemotactic activity compared with those in nonsmoker subjects without COPD. In the smokers without COPD the mini-BAL showed reduced TLR4 and HBD-2 expression, higher IP-10 concentrations, and higher chemotactic activity than in patients with COPD. The blocking of TLR4 activation and HBD-2 depletion increased neutrophil apoptosis. No differences were observed for TLR2 expression and IL-8 concentrations. This study strengthens the contribution of TLR4 to promoting airway neutrophilia in COPD.

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Enhances Activation of p38 and ERK MAPKs, Increasing Interleukin-6 Synthesis in Airway Epithelial Cells Exposed to Pseudomonas aeruginosa

In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRDeltaF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRDeltaF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRDeltaF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRDeltaF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.

Surfactant Protein A Stimulates Release of Neutrophil Chemotactic Factors by Alveolar Type II Pneumocytes

Mucosal immunity is an important mechanism in the response to injury. Our hypothesis is that surfactant protein A (SP-A) is an autocrine factor that stimulates alveolar type II epithelial cell release of neutrophil chemotactic factors by binding to the SP-A receptor expressed by these cells. We examined (1) the effect of SP-A (20 μg/ml) or IL-1β (10 ng/ml) on release of neutrophil chemotactic factors by primary cultures of type II cells or alveolar macrophages, and (2) the effect of intratracheal instillation of the blocking antibody to the SP-A receptor on the response to oleic acid-induced lung injury in vivo. All media and cell culture supernates were assayed for neutrophil chemotactic activity, and bronchoalveolar lavage fluid from the in vivo experiments was analyzed for inflammatory cell counts. While SP-A and media used for the cell cultures has no intrinsic neutrophil chemotactic activity, supernates from primary cultures of type II cells incubated in either SP-A or IL-1β had twofold higher neutrophil chemotactic factor activity compared to supernates from controls. SP-A had no effect on release of neutrophil chemotactic factor by alveolar macrophages. Oleic acid-induced lung injury resulted in a marked influx of neutrophils into BAL, and this influx was reduced by 70% by pretreatment with the antibody to SP-A receptor. We conclude that SP-A stimulates the release of neutrophil chemotactic factor by alveolar type II cells, and this effect is mediated by the receptor for SP-A specifically expressed by these cells.

Skin Inflammation Induced by the Synergistic Action of IL-17A, IL-22, Oncostatin M, IL-1α, and TNF-α Recapitulates Some Features of Psoriasis

Keratinocytes play a crucial role in the regulation of skin inflammation, responding to environmental and immune cells stimuli. They produce soluble factors that can act in an autocrine or paracrine manner on immune cells or directly on aggressors. A screening of the activities of 36 cytokines on keratinocyte gene expression identified IL-17A, IL-22, oncostatin M, TNF-alpha, and IL-1alpha as potent cytokines in inducing cutaneous inflammation. These five proinflammatory cytokines synergistically increased production of CXCL8 and beta-defensin 2 (BD2). In addition, ex vivo studies on human skin explants demonstrated upregulation of BD2, S100A7, and CXCL8 expression in response to the same combination of cytokines. In vivo intradermal injection of these five cytokines in mouse increased CXCL1, CXCL2, CXCL3, S100A9, and BD3 expression, associated with neutrophil infiltration. We confirmed and extended this synergistic effect using quantitative real-time PCR analysis and observed increased expression of nine chemokines and 12 antimicrobial peptides. Production of CXCL, CXCL5, and CXCL8 by keratinocytes stimulated in the presence of this cytokine combination was associated with increased neutrophil chemotactic activity. Similarly, high production of BD2, BD3, and S100A7 was associated with an increased antimicrobial activity. Finally, the transcriptional profile observed in this in vitro model of inflammatory keratinocytes correlated with the one of lesional psoriatic skin. Our results demonstrate the important potentiating activities of IL-17A, IL-22, oncostatin M, TNF-alpha, and IL-1alpha on keratinocytes. This is particularly interesting in the context of psoriasis where these cytokines are overexpressed and could synergize to play an important role in upregulation of chemokines and antimicrobial peptides production.

Irradiation Stimulates Human Lung Fibroblasts to Release Inflammatory Cell Chemotactic Activity

Background:Radiation therapy plays an important role in the treatment of lung cancer.Howeverit also radiation therapy often induces serious complications such as radiation-induced pneumonitis and its underlying mechanism remains to be elucidated. Objectives:The present study examined whether radiation might stimulate human lung fibroblasts(HLFto release neutrophil and monocyte chemotactic activity(NCA and MCA. Methods:HLF were exposed to varying doses of radiation (3-12 Gyat varying incubation times(12-72 hr. The chemotaxis assays of NCA and MCA were performed by a 48-well microchemotaxis chamber method. The effect of polyclonal antibodies of various cytokines on NCA and MCA was evaluated.Furthermorethe protein and mRNA expression of these effective cytokines was assessed by ELISA and RT-PCR. Results:HLF released NCA and MCA in response to irradiation in a dose-and time-dependent manner.NCA was significantly inhibited by anti-interleukin (IL-8 antibody and MCA was significantly attenuated by anti- monocyte chemoattractant protein (MCP-1 antibody. The protein secretion of IL-8 and MCP-1 was significantly increased by irradiationand mRNA expression of IL-8 and MCP-1 was upregulated by irradia‑ tion. Conclusion:These findings suggest that HLF mayat least partlyparticipate in the development of radiation- induced pneumonitis.Shinshu Med J 58 : 57―68 2010 (Received for publication November 262009 ;accepted in revised form January 82010

C5 contents and neutrophil chemotactic activities in bronchiolar and alveolar regions

To clarify the localization and mechanism of neutrophil infiltration in the lower respiratory tract, we measured neutrophil number, neutrophil chemotactic factor (NCF) activity and content of C5 in bronchial lavage (BL) fluid and bronchoalveolar lavage (BAL) fluid. Numbers of neutrophils, NCF activity and C5 content were higher in the BL fluid from normal volunteers (NV) and control patients (CP) than those in the BAL fluid from the same subjects. The NCF activity in the BL fluid was inhibited approximately 40% by anti-C5 antiserum, and correlated with C5 content in the BL fluid. In the BAL fluids of patients with chronic airway diseases (CAD) and patients with idiopathic interstitial pneumonia (IIP), neutrophil number, NCF activity and C5 content were increased compared to those in BAL fluid from NV or CP. These results indicated that neutrophils are predominant in the bronchial region compared to the alveolar region, and that C5-derived NCF play important roles in the accumulation of neutrophils in the bronchial region. Also C5-derived NCF are thought to be related to, at least, a part of the neutrophil infiltration in the respiratory tract of patients with CAD and IIP.

Physiological Role of the Oxidative Stress-Sensitive TRPM2 Ca2+ Channel in Immunocytes

It is known that a large amount of reactive oxygen species (ROS) exist at inflamed sites. ROS induce chemokines responsible for the recruitment of inflammatory cells at inflamed sites. Here, we demonstrate that the plasma membrane Ca2+-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes/macrophages. In monocytes from Trpm2-defecient mice, H2O2-induced Ca2+ influx and production of the macrophage inflammatory protein-2 (CXCL2), which exhibit potent neutrophil chemotactic activity, were impaired. In the inflammation model dextran sulfate sodium-induced colitis, CXCL2 expression was attenuated by Trpm2 disruption. Interestingly, the number of recruited neutrophils was significantly reduced in DSS-treated TRPM2 KO mice, whereas that of DSS-induced macrophages after infiltration into inflamed sites, was indistinguishable in WT and TRPM2 KO mice. Importantly, TRPM2 deficiency failed to impair important aspects of CXCL2-evoked neutrophil chemotaxis, including Ca2+ response, in vitro migration, and in vivo infiltration after DSS administration. Thus, TRPM2-mediated Ca2+ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.

Tissue Inhibitor of Metalloproteinases 3 Regulates Resolution of Inflammation following Acute Lung Injury

Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits not only matrix metalloproteinases but also a disintegrin and metalloproteinase domain family members and thus contributes to controlling diverse processes mediated by proteolysis. We used Timp3(-/-) mice to assess the role of this inhibitor in acute lung injury. After bleomycin-induced injury, inflammation, as indicated by the influx of neutrophils in bronchoalveolar lavage (BAL), peaked at 7 days post-injury in the wild-type mice and began to wane thereafter; however, in Timp3(-/-) mice, inflammation persisted up to 28 days. Furthermore, although the level of chemokines in BAL and lung homogenate was similar in both genotypes, BAL from Timp3(-/-) mice 7, 14, and 28 days post-injury had increased neutrophil chemotactic activity compared with wild-type BAL. At day 14, a higher percentage of apoptotic neutrophils were present in wild-type mice compared with Timp3(-/-) mice, further suggesting that TIMP3 constrains continued neutrophil influx. In addition, total matrix metalloproteinase activity was increased in lungs from Timp3(-/-) mice, and treatment of mice with a synthetic inhibitor of metalloproteinases rescued the enhanced neutrophilia phenotype. These data demonstrate that TIMP3 regulates neutrophil influx in the lung following injury through its ability to inhibit metalloproteinase activity and indicates that TIMP3 functions to promote the resolution of inflammation in the lung.

C-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration

Cytoskeleton dynamics are regulated by Src-family tyrosine kinases (SFKs) and c-Abl. We found that the SFK members Hck and c-Fgr regulate tyrosine phosphorylation of c-Abl and c-Abl associates with β1 integrin-bound Hck or c-Fgr in murine macrophages. Studies with selective inhibitors and cells from SFK-deficient mice showed that c-Abl and SFK regulate migration and activation of the small GTPases Cdc42 and Rac in macrophages. Additionally, human neutrophil chemotactic activity was reduced by c-Abl inhibitors, and neutrophils from chronic myeloid leukaemia patients displayed an increased chemotactic ability. Hence, Src-family kinase and c-Abl cross-talk in the regulation of myeloid cell migration. Structured summary MINT- 7296608: Integrin beta-1 (uniprotkb: P09055) physically interacts (MI: 0914) with Hck (uniprotkb: P08103), Abl (uniprotkb: P00520) and Fgr (uniprotkb: P14234) by anti bait coimmunoprecipitation (MI: 0006) MINT- 7296596: Integrin beta-1 (uniprotkb: P09055) physically interacts (MI: 0914) with Fgr (uniprotkb: P14234) and Abl (uniprotkb: P00520) by anti bait coimmunoprecipitation (MI: 0006)

Galectin-9 ameliorates immune complex-induced arthritis by regulating FcγR expression on macrophages

Galectin-9 up-regulated Fc gamma RIIb expression of mouse peritoneal macrophages in vitro but down-regulated Fc gamma RIII expression. Galectin-9-treated macrophages stimulated with immune complexes (IC) produced less TNFalpha and IL-1 beta but more IL-10 than PBS-treated macrophages. Macrophage enhancing effects on IC-induced C5a and neutrophil chemotactic activity were also diminished for galectin-9-treated macrophages. In galectin-9-treated mice, the severity of IC-induced arthritis was reduced, as were pro-inflammatory cytokine levels in inflamed joints and serum C5a. Fc gamma RIIb expression of macrophages from galectin-9-treated mice was up-regulated, whereas Fc gamma RIII expression was down-regulated. Macrophages from galectin-9-treated mice produced less TNFalpha and IL-1 beta but more IL-10 than PBS-treated mice. Disease severity of galectin-9-transgenic mice was milder than wild-type mice, whereas that of galectin-9-deficient mice was exaggerated. Furthermore, macrophage Fc gamma RIIb expression in galectin-9-deficient mice was down-regulated, while Fc gamma RIII expression was up-regulated. These results suggest that galectin-9 suppresses IC-induced inflammation partly by regulating Fc gamma R expression on macrophages.

Leukotriene B4 Contributes to Exhaled Breath Condensate and Sputum Neutrophil Chemotaxis in COPD

Neutrophils have been implicated in the pathogenesis of COPD. Several chemoattractants for neutrophils have been measured in samples of exhaled breath condensate (EBC) and induced sputum (IS) from patients with COPD. The aims of this study were to compare EBC and IS supernatant neutrophil chemotactic activity (NCA) from ex-smoking subjects with COPD and healthy ex-smokers, and to assess the contribution of leukotriene B(4) (LTB(4)) to this activity. Thirty-four subjects with COPD were compared to 24 control subjects. EBC and IS chemotactic activity for neutrophils was assessed by using Boyden microchambers. The chemotactic index was used to evaluate cell migration. LTB(4) was measured by a specific enzyme immunoassay. The contribution of LTB(4) to EBC and sputum neutrophil chemotaxis was assessed by an LTB(4) receptor antagonist (U-75302; Cayman Chemical Company; Ann Arbor, MI). EBC and IS samples from both COPD patients and healthy subjects displayed significant NCA, but this activity was raised in COPD patients compared to healthy subjects. The chemotactic activity contained in sputum, however, failed to correlate with that in EBC. In COPD patients, there was a significant correlation between EBC NCA and sputum neutrophil counts. LTB(4) levels were raised in EBC samples, but not in sputum samples, from COPD subjects compared to those from healthy subjects. LTB(4) receptor antagonist concentrations (2.5 x 10(-4) mol/L) reduced by 44.6% and by 44.4%, respectively, the chemotactic activity contained in the EBC and sputum samples. EBC and IS from COPD patients have a raised NCA to which LTB(4) contributes.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ LIGANDS 15-DEOXY-δ12,14-PROSTAGLANDIN J2 AND PIOGLITAZONE INHIBIT HYDROXYL PEROXIDE-INDUCED TNF-α AND LIPOPOLYSACCHARIDE-INDUCED CXC CHEMOKINE EXPRESSION IN NEONATAL RAT CARDIAC MYOCYTES

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma) such as prostaglandin metabolite 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) or thiazolidinedione pioglitazone have been identified as a new class of anti-inflammatory compounds with possible clinical applications. Reactive oxygen species play an important role in the generation of cellular damage by induction of proinflammatory cytokines and chemokines during myocardial I/R. These events were preceded by activation of the transcription factors nuclear factor (NF)-kappaB pathway. It has been suggested that myocardium overproduces TNF-alpha after I/R, and locally produced TNF-alpha is sufficient to cause severe impairment of cardiac function. LPS-induced CXC chemokine (LIX) is a rodent chemokine with potent neutrophil-chemotactic activity. Based on this concept, we examined the effects of 15d-PGJ2 and pioglitazone on oxidative stress-induced TNF-alpha and LIX expression in neonatal rat cardiac myocytes. Pretreatment of myocytes with 15d-PGJ2 or pioglitazone decreased hydrogen peroxide-induced TNF-alpha and LIX production (mRNA and protein) in a concentration-dependent manner. The beneficial effects of both ligands were associated with reduction of hydrogen peroxide-induced NF-kappaB activation. Treatment with 15d-PGJ2, but not pioglitazone, caused dose-dependent activation of heat shock factor 1, which could render cells unresponsive to stimulation of NF-kappaB. The cytoprotection afforded by pioglitazone was attenuated by the PPAR-gamma antagonist GW9662, which failed to affect the beneficial effects afforded by 15d-PGJ2. Taken together, these results demonstrate that treatment with these chemically distinct ligands of PPAR-gamma results in diverse anti-inflammatory mechanisms.

Polymeric Osteopontin Employs Integrin α9β1 as a Receptor and Attracts Neutrophils by Presenting a de Novo Binding Site

Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin alpha9beta1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because alpha9beta1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, alpha9beta1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with alpha9-transfected cells and by detection of direct binding of recombinant soluble alpha9beta1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a ligand for alpha9beta1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-alpha9beta1 antibody. Unexpectedly, mutagenesis studies showed that alpha9beta1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel alpha9beta1-binding site and that the interaction of this site with the alpha9beta1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.

Monocyte and Neutrophil Chemotactic Activity at the Site of Interstitial Inflammation in Patients on High-Flux Hemodialysis or Hemodiafiltration

Background/Aims: We have observed a difference between patients on low-flux hemodialysis (HD) or peritoneal dialysis and patients on hemodiafiltration (HDF) or high-flux HD in the capacity of transmigrated leukocytes to mobilize CD11b in response to inflammatory stimuli compared with healthy subjects. This could be due to different interstitial chemokine concentrations. Methods: We measured concentrations of circulating and interstitial macrophage inflammatory protein-1α (MIP-1α), matrix metalloproteinase-9 (MMP-9)/neutrophil gelatinase-associated lipocalin (NGAL), monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in 10 patients on HDF or high-flux HD and 11 healthy subjects by using immunoassay. Results: The interstitial concentrations of MIP-1α, MMP-9/NGAL and IL-8 were similar in patients and healthy subjects, while the corresponding concentration of MCP-1 was significantly higher in patients on HDF or high-flux HD as compared with healthy subjects (p < 0.01). Conclusion: We suggest that an equal or higher concentration of chemokines in the interstitium in patients with HDF or high-flux HD might be one mechanism responsible for the preserved function of transmigrated leukocytes.

Mediators of neutrophil recruitment in human abdominal aortic aneurysms

AimsNeutrophils/platelet interactions are involved in abdominal aortic aneurysm (AAA). The intraluminal thrombus (ILT) is a human model of platelet/neutrophil interactions. The present study focused on mediators involved in neutrophil recruitment in AAA.Methods and resultsConditioned media from luminal, intermediate, and abluminal layers of 29 human ILTs were analysed for neutrophil markers [elastase/α1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase (MPO), and α-defensin peptides], RANTES, platelet factor 4 (PF4), and interleukin-8 (IL-8). Their time-dependent release into serum from clots generated in vitro and their plasma concentrations in AAA patients and controls were determined. Immunohistochemistry for neutrophils, platelets, IL-8, PF4, and RANTES on AAA sections was performed; and molecules involved in ILT neutrophil chemotactic function were analysed in vitro. Neutrophils and platelets colocalized in the luminal layer of the thrombus. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were released predominantly by the luminal thrombus pole, where their concentrations were significantly correlated. The luminal ILT layer was also the main source of IL-8, whose immunostaining colocalized with neutrophils. All were also released time dependently from clots and were increased in plasma of AAA patients. Luminal ILT layers displayed potent neutrophil chemotactic activity in vitro, which was inhibited by RANTES- and IL-8-blocking antibodies as well as by reparixin, an antagonist of the IL-8 receptors CXCR1 and CXCR2.ConclusionTaken together, these results suggest that platelet-derived RANTES and neutrophil-derived IL-8 are involved in attracting neutrophils to the luminal layer of AAA ILT.

Differential inflammatory activity across human abdominal aortic aneurysms reveals neutrophilderived leukotriene B4 as a major chemotactic factor released from the intraluminal thrombus

Development and progression of acquired abdominal aortic aneurysms (AAAs) have been associated with different inflammatory mediators. The aim of the present study was to elucidate the topology and the potential mechanisms linking the leukotriene pathway to human AAAs. Human aneurysmal lesions were obtained from 24 patients undergoing surgery, and the intraluminal thrombus was separated from the vascular wall. Histological examination revealed major expression of the leukotriene-producing enzymes 5-lipoxygenase and LTA(4) hydrolase, as well as the two receptors for leukotriene B(4) (BLT1R and BLT2R), corresponding to neutrophils in the luminal part of the thrombus. In contrast, in the vascular wall, the leukotriene pathway mainly localized in macrophage-rich adventitial areas. Furthermore, conditioned media of the intraluminal thrombus contained significantly higher concentrations of leukotriene B(4) than that derived from the vascular wall, which were significantly correlated to other neutrophil-derived mediators, such as elastase/alpha(1)-antitrypsin complexes, myeloperoxidase, and MMP9/NGAL complexes. Finally, the neutrophil-chemotactic activity of the conditioned media from the intraluminal thrombus exhibited major inhibition by antagonists of the leukotriene B(4) receptors. Taken together, these results indicate neutrophil-derived leukotriene B(4) as a major neutrophil chemotactic factor released from the intraluminal thrombus of human AAAs and suggest that targeting BLT receptors may represent a potential medical therapeutic strategy in the prevention of AAA progression in humans.

Biological Markers in Induced Sputum of Patients with Different Phenotypes of Chronic Airway Obstruction

Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by a combination of 3 different disorders, namely chronic asthma, chronic bronchitis and pulmonary emphysema, sometimes simultaneously present in the same subject. Objectives: The aim of our study was to compare sputum inflammatory markers in patients with different phenotypes of chronic airway obstruction. Methods: Forty-five subjects (forced expiratory volume in 1 s/vital capacity, FEV₁/VC: 58.8 ± 12.2%; FEV₁: 49.8 ± 11.5% of predicted) were classified as chronic asthma (n = 10) or COPD patients (n = 35); the latter were further divided into patients with prevalent chronic bronchitis (n = 24) or prevalent pulmonary emphysema (n = 11) according to clinical history and functional evaluation, and underwent sputum induction and analysis of inflammatory cell and soluble mediators. Results: Patients with chronic asthma showed higher sputum eosinophil percentages and eosinophilic cationic protein levels, and lower neutrophil percentages and neutrophil elastase levels than COPD patients. Neutrophil chemotactic activity in sputum supernatant was higher than the pool of normal subjects both in chronic asthma and COPD patients. No difference in sputum cell composition and levels of soluble mediators was observed between patients with chronic bronchitis and patients with pulmonary emphysema. Conclusions: The pattern of airway inflammation in induced sputum of patients with chronic asthma is different from that of COPD patients with a similar FEV₁. Among COPD patients, however, the pattern of airway inflammation shows no difference between chronic bronchitis and patients with pulmonary emphysema, suggesting that these two clinically and functionally distinct phenotypes share a common inflammatory pattern as detected by induced sputum.