技术资料

BACKGROUND: Basophils are increasingly utilized as indicators of allergic inflammation and as primary allergic effector cells to study signalling pathways. However,until the present,their enrichment has been time consuming,costly and limited to relatively few specialized laboratories. OBJECTIVE: We have therefore devised a reproducible and rapid method for the purification of human basophils from small quantities of peripheral blood within 1.5 h,which does not require the use of specialized equipment such as elutriators. METHODS: Human basophils were obtained from healthy volunteers undergoing venipuncture. Heparinized or K3-ethylenediaminetetraacetic acid blood samples were first subjected to centrifugation in Hetasep,directly followed by negative selection using immunomagnetic beads. Basophil morphology and purity were assessed by May-Grünwald staining of cytospins. IgE-mediated histamine release was analysed spectrofluorometrically and IL-4 and IL-13 production by quantitative RT-PCR. CD203c and CD63 surface expression was measured using flow cytometry before and after activation with anti-IgE. RESULTS: Using this protocol,basophils were enriched close to homogeneity in most cases with a mean purity of 99.34+/-0.88% (range 97-100%,n=18) and a mean recovery of 75.6 (range 39-100%,n=8). Basophil viability following purification was 99.6+/-0.89% using Trypan blue exclusion. The purification procedure gave rise to basophils with normal functional responses to anti-IgE regarding histamine release as well as IL-4 and IL-13 mRNA expression. Moreover,constitutive cell-surface CD203c/CD63 expressions were not elevated before anti-IgE stimulation. CONCLUSION: The rapidity,simplicity and reproducibility of this method will facilitate the employment of basophils in high-output ex vivo studies. View Publication

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene,which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding,induction of endoplasmic reticulum (ER) stress,activation of the unfolded protein response (UPR),and ultimately a block in granulocytic differentiation. To test this model,we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice,though without significant basal UPR activation,are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover,inaction of protein kinase RNA-like ER kinase (Perk),one of the major sensors of ER stress,either alone or in combination with G193X Elane,had no effect on basal granulopoiesis. However,inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN. View Publication

Under steady-state conditions,aged neutrophils are removed from the circulation in bone marrow,liver,and spleen thereby maintaining myeloid cell homeostasis. The fate of these aged immune cells under inflammatory conditions,however,remains largely obscure. Here,we demonstrate that in the acute inflammatory response during endotoxemia aged neutrophils cease returning to the bone marrow and instead rapidly migrate to the site of inflammation. Having arrived in inflamed tissue,aged neutrophils were found to exhibit a higher phagocytic activity as compared to the subsequently recruited non-aged neutrophils. This distinct behavior of aged neutrophils under inflammatory conditions is dependent on specific age-related changes in their molecular repertoire that enable these 'experienced' immune cells to instantly translate inflammatory signals into immune responses. In particular,aged neutrophils engage toll-like receptor-4- and p38 mitogen-activated protein kinases-dependent pathways to induce conformational changes in β2 integrins which allow these phagocytes to effectively accomplish their mission in the front line of the inflammatory response. Hence,ageing in the circulation might represent a critical process for neutrophils that enables these immune cells to properly unfold their functional properties for host defense. View Publication

Type 2 innate lymphoid cells (ILC2) share cytokine and transcription factor expression with CD4+ Th2 cells,but functional diversity of the ILC2 lineage has yet to be fully explored. Here,we show induction of a molecularly distinct subset of activated lung ILC2,termed ILC210. These cells produce IL-10 and downregulate some pro-inflammatory genes. Signals that generate ILC210 are distinct from those that induce IL-13 production,and gene expression data indicate that an alternative activation pathway leads to the generation of ILC210. In vivo,IL-2 enhances ILC210 generation and is associated with decreased eosinophil recruitment to the lung. Unlike most activated ILC2,the ILC210 population contracts after cessation of stimulation in vivo,with maintenance of a subset that can be recalled by restimulation,analogous to T-cell effector cell and memory cell generation. These data demonstrate the generation of a previously unappreciated IL-10 producing ILC2 effector cell population. View Publication

Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury,we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)),embryonic endothelial progenitor cells,mouse embryonic fibroblasts (control),or saline were then injected into the pulmonary circulation. At 21 days of age,saline-treated mice had enlarged alveoli,reduced septation,and a reduction in vascular density. In contrast,mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia. View Publication

Inflammation is characterized by the recruitment of leukocytes from the bloodstream. The rapid arrival of neutrophils is followed by a wave of inflammatory lymphocyte antigen 6 complex (Ly6C)-positive monocytes. In contrast Ly6C(low) monocytes survey the endothelium in the steady state,but their role in inflammation is still unclear. Here,using confocal intravital microscopy,we show that upon Toll-like receptor 7/8 (TLR7/8)-mediated inflammation of mesenteric veins,platelet activation drives the rapid mobilization of Ly6C(low) monocytes to the luminal side of the endothelium. After repeatedly interacting with platelets,Ly6C(low) monocytes commit to a meticulous patrolling of the endothelial wall and orchestrate the subsequent arrival and extravasation of neutrophils through the production of proinflammatory cytokines and chemokines. At a molecular level,we show that cysteine-rich protein 61 (CYR61)/CYR61 connective tissue growth factor nephroblastoma overexpressed 1 (CCN1) protein is released by activated platelets and enables the recruitment of Ly6C(low) monocytes upon vascular inflammation. In addition endothelium-bound CCN1 sustains the adequate patrolling of Ly6C(low) monocytes both in the steady state and under inflammatory conditions. Blocking CCN1 or platelets with specific antibodies impaired the early arrival of Ly6C(low) monocytes and abolished the recruitment of neutrophils. These results refine the leukocyte recruitment cascade model by introducing endothelium-bound CCN1 as an inflammation mediator and by demonstrating a role for platelets and patrolling Ly6C(low) monocytes in acute vascular inflammation. View Publication

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1),suggesting a role in neutrophil migration. However,CD177pos neutrophils exhibit no clear migratory advantage in vivo,despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system,we found that CD177pos and CD177neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177pos neutrophils,an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly,CD177 ligation enhanced its interaction with β2 integrins,as revealed by fluorescence lifetime imaging microscopy,leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity,impaired internalization of integrin attachments,and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. View Publication

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9),neutrophil elastase (NE),and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment,where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis,HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly,HPC mobilization by G-CSF was normal in MMP-9-deficient mice,NE x CG-deficient mice,or mice lacking dipeptidyl peptidase I,an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover,combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE x CG-deficient mice,indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12 alpha protein expression in the bone marrow of Ne x CG-deficient mice,indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively,these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization. View Publication

Chimerism analysis has become an important tool to manage patients in the peri-transplant period of allogenic stem cell transplantation. During this period,cells of donor and host origin can coexist and increasing proportion of cells of host origin is considered as a recurrence of the underlying disease. We currently performed chimerism analysis on separate peripheral blood cell subsets,lymphocytes and granulocytes. To improve our isolation method,a new automated device from Stem Cell Technology Roboseptrade mark was tested and compared to our manual separation technique. The results obtained on T cell purification showed an improvement of the purity (98.42% with Robosep vs. 92.42% with the manual technique Rosettesep) and of the recovery (63.43% with Robosep and 38% with Rosettesep). The results were significantly improved on patient samples with less than 10% CD3 positive cells (purity: 90% vs. 44.44%; recovery: 73.79% vs. 43.98%). Granulocytes separation was based on CD15 expression. The results showed an improvement of the purity with Robosep (96.90% vs. 86.20% with the manual technique Polymorphprep) but the recovery was impaired (35.2% vs. 52.30%). Using a myeloid (CD66/CD33) cocktail,recovery was improved with the Robosep device (64.04% with the myeloid cocktail vs. 22.4% with the CD15 cocktail). Our data demonstrated that Robosep allowed a performant cell purification in the early period post-transplantation even for populations representing less than 10% of the peripheral blood cells. View Publication

Sepsis represents uncontrolled inflammation due to an infection. Cold-inducible RNA-binding protein (CIRP) is a stress-induced damage-associated molecular pattern (DAMP). A subset of neutrophils expressing ICAM-1+ neutrophils was previously shown to produce high levels of reactive oxygen species. The role of CIRP for the development and function of ICAM-1+ neutrophils during sepsis is unknown. We hypothesize that CIRP induces ICAM-1 expression in neutrophils causing injury to the lungs during sepsis. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis,we found increased expression of CIRP and higher frequencies and numbers of ICAM-1+ neutrophils in the lungs. Conversely,the CIRP-/- mice showed significant inhibition in the frequencies and numbers of ICAM-1+ neutrophils in the lungs compared to wild-type (WT) mice in sepsis. In vitro treatment of bone marrow-derived neutrophils (BMDN) with recombinant murine CIRP (rmCIRP) significantly increased ICAM-1+ phenotype in a time- and dose-dependent manner. The effect of rmCIRP on increasing frequencies of ICAM-1+ neutrophils was significantly attenuated in BMDN treated with anti-TLR4 Ab or NF-κB inhibitor compared,respectively,with BMDN treated with isotype IgG or DMSO. The frequencies of iNOS producing and neutrophil extracellular traps (NETs) forming phenotypes in rmCIRP-treated ICAM-1+ BMDN were significantly higher than those in ICAM-1- BMDN. Following sepsis the ICAM-1+ neutrophils in the lungs showed significantly higher levels of iNOS and NETs compared to ICAM-1- neutrophils. We further revealed that ICAM-1 and NETs were co-localized in the neutrophils treated with rmCIRP. CIRP-/- mice showed significant improvement in their survival outcome (78% survival) over that of WT mice (48% survival) in sepsis. Thus,CIRP could be a novel therapeutic target for regulating iNOS producing and NETs forming ICAM-1+ neutrophils in the lungs during sepsis. View Publication

Immune recognition of pathogen-associated ligands leads to assembly and activation of inflammasomes,resulting in the secretion of inflammatory cytokines IL-1β and IL-18 and an inflammatory cell death called pyroptosis. Inflammasomes are important for protection against many pathogens,but their role during chronic infectious disease is poorly understood. Pseudomonas aeruginosa is an opportunistic pathogen that persists in the lungs of cystic fibrosis (CF) patients and may be responsible for the repeated episodes of pulmonary exacerbation characteristic of CF. P. aeruginosa is capable of inducing potent inflammasome activation during acute infection. We hypothesized that to persist within the host during chronic infection,P. aeruginosa must evade inflammasome activation,and pulmonary exacerbations may be the result of restoration of inflammasome activation. We therefore isolated P. aeruginosa from chronically infected CF patients during stable infection and exacerbation and evaluated the impact of these isolates on inflammasome activation in macrophages and neutrophils. P. aeruginosa isolates from CF patients failed to induce inflammasome activation,as measured by the secretion of IL-1β and IL-18 and by pyroptotic cell death,during both stable infection and exacerbation. Inflammasome evasion likely was due to reduced expression of inflammasome ligands and reduced motility and was not observed in environmental isolates or isolates from acute,non-CF infection. These results reveal a novel mechanism of pathogen adaptation by P. aeruginosa to avoid detection by inflammasomes in CF patients and indicate that P. aeruginosa-activated inflammasomes are not involved in CF pulmonary exacerbations. View Publication

Clinical observations suggest that in chronic myelogenous leukemia (CML),the Philadelphia chromosome (Ph+) clone has a growth advantage over normal hematopoiesis. Patients with CML have high levels of neutrophil elastase,which has recently been shown to antagonize the action of granulocyte-colony-stimulating factor (G-CSF) and other growth factors. We therefore compared the effect of elastase on the growth of normal and CML progenitor cells. In 10-day suspension cultures of normal or CML CD34+ cells supplemented with G-CSF,stem cell factor (SCF),and granulocyte macrophage-colony-stimulating factor (GM-CSF),CML cells had diminished sensitivity to the growth inhibitory effect of elastase. When equal numbers of CML and normal CD34+ cells were cocultured for 10 days,there was no change in the relative proportions of normal and leukemic cells (measured by fluorescence in situ hybridization [FISH] or flow cytometry). However,when elastase was added,CML cells predominated at the end of the culture period (78% vs 22% with 1 microg/mL and 80% vs 20% with 5 microg/mL elastase). CML neutrophils substituted effectively for elastase in suppressing the proliferation of normal CD34+ cells,but this effect was abrogated by serine protease inhibitors. These results suggest that elastase overproduction by the leukemic clone can change the growth environment by digesting growth factors,thereby giving advantage to Ph+ hematopoiesis. View Publication