Global analysis of neutrophil gene expression

A widespread, but incorrect, view of the neutrophil portrays it as a short-lived, terminally differentiated cell that has a highly condensed nucleus and hence is unable to induce gene expression. However, these cells express mRNA encoding phagocytic receptors, modulate RNA synthesis in response to lectin stimulation or glucocorticoid treatment, and upregulate genes involved in phagocytic function, such as respiratory burst activity and cytokine secretion. Most studies of neutrophil gene expression have examined cytokine stimulation and have focused on a few specific genes of known interest, rather than the global genetic repertoire of the cell. In part stimulated by the availability of gene and expressed sequence tag databases, several approaches have been developed to assess the levels of all mRNA species found in single RNA preparations. We have analyzed the regulation of gene expression in neutrophils using a gel-based method that displays 3' end fragments of cDNA generated by restriction enzymes. Our data indicate that neutrophils are capable of extensive, rapid, and complex changes in gene expression, involving at least several percent of all mRNAs present in the cell. The number and magnitude of mRNA responses are comparable to those measured on activation of normal T cells. The data also indicate that activated neutrophils are a source of newly synthesized, physiologically significant, intercellular signaling molecules.     

Adaptive response of Yersinia pestis to extracellular effectors of innate immunity during bubonic plague

Yersinia pestis causes bubonic plague, characterized by an enlarged, painful lymph node, termed a bubo, that develops after bacterial dissemination from a fleabite site. In susceptible animals, the bacteria rapidly escape containment in the lymph node, spread systemically through the blood, and produce fatal sepsis. The fulminant progression of disease has been largely ascribed to the ability of Y. pestis to avoid phagocytosis and exposure to antimicrobial effectors of innate immunity. In vivo microarray analysis of Y. pestis gene expression, however, revealed an adaptive response to nitric oxide (NO)-derived reactive nitrogen species and to iron limitation in the extracellular environment of the bubo. Polymorphonuclear neutrophils recruited to the infected lymph node expressed abundant inducible NO synthase, and several Y. pestis homologs of genes involved in the protective response to reactive nitrogen species were up-regulated in the bubo. Mutation of one of these genes, which encodes the Hmp flavohemoglobin that detoxifies NO, attenuated virulence. Thus, the ability of Y. pestis to destroy immune cells and remain extracellular in the bubo appears to limit exposure to some but not all innate immune effectors. High NO levels induced during plague may also influence the developing adaptive immune response and contribute to septic shock.     

黄连素作用鼠疫耶尔森菌的体外表达谱

目的 探讨黄连索抑制鼠疫耶尔森菌(简称鼠疫菌)作用的分子机制.方法 应用鼠疫菌全基因组芯片表达谱进行黄连素作用鼠疫菌研究.液体稀释法测定黄连索作用鼠疫菌的最小抑菌浓度(MIC).应用10倍MIC的黄连索作用于鼠疫菌30 min后,提取鼠疫菌总RNA,反转录合成eDNA,Cy3,Cy5荧光染料标记,芯片杂交,扫描仪扫描,SAM软件分析结果 .结果 在鼠疫菌全基因组范围内黄连素作用鼠疫菌的明显差异表达基因有360个.其中明显上调基因333个,下调基因27个.依据鼠疫菌CO92株基冈功能分类结果 ,这些差异基因分属24个不同的功能类群,其中主要有细胞膜相关基冈83个,未知功能基因75个,转运结合蛋白基因48个.发生明显改变的基因功能类群是代谢相关基因,共有40个.结论 获得了黄连素作用鼠疫菌的表达谱.阐述了黄连作用鼠疫菌的分子机制,主要机制在于影响代谢相关基因的上调.     

Evidence of early systemic activation and transendothelial migration of neutrophils in neonates with severe respiratory distress syndrome

Several observations imply that the early inflammatory response involving activated neutrophils, tissue macrophages, and cytokines plays an important role in the pathogenesis of neonatal respiratory distress syndrome (RDS) and progression to bronchopulmonary dysplasia (BPD). The aim of this study was to test the hypothesis that changes in circulating neutrophil number and function and plasma levels of cytokines, consistent with neutrophil activation and migration to the tissues, occur during the early stages of neonatal RDS. For this purpose we measured peripheral blood levels of certain immunological parameters that promote neutrophil activation and transendothelial migration. Twenty preterm neonates with severe RDS and 20 healthy infants matched for gestational age were the subjects. The absolute neutrophil count (ANC), and plasma levels of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and sL-selectin using an enzyme-linked immunosorbent assay (ELISA), neutrophil CD11b expression, and respiratory burst activity (RBA) using flow cytometry, were measured within 24 h after birth. The two groups were comparable regarding perinatal characteristics. None of the neonates studied had any clinical or laboratory evidence of infection by the time of blood sampling. The immunological investigation showed that the RDS neonates had significantly lower ANC (P = 0.032), higher expression of the CD11b on neutrophils (P = 0.0065), and higher G-CSF and IL-6 plasma levels (P = 0.0047 and P < 0.0001, respectively) in comparison to healthy preterm neonates. The neutrophil RBA and plasma sL-selectin levels did not differ significantly between the two groups. We conclude that in neonates with severe RDS, there is evidence of a systemic neutrophil activation early in the course of the disease, supporting the view of a contributing role of activated neutrophils in the pathogenesis of RDS.     

大黄抑制鼠疫耶尔森菌的体外转录组学研究

目的应用鼠疫菌全基因组芯片研究大黄抑制鼠疫菌的分子作用机制。方法液体稀释法测定大黄对鼠疫菌的最低抑菌浓度(MIC),以10倍MIC作用鼠疫菌30min,提取鼠疫菌总RNA,逆转录合成cDNA,荧光素标记,与芯片杂交,扫描仪扫描,应用SAM软件分析结果。结果获得了大黄作用鼠疫菌的表达谱。大黄作用鼠疫菌的明显差异基因为498个,其中上调基因358个,下调140个。结论蛋白质合成基因、细胞膜相关基因、转运结合蛋白基因以及部分热休克基因的改变是大黄抑制鼠疫菌的主要作用机制。     

Granulocyte-macrophage colony-stimulating factor enhances neutrophil function in acquired immunodeficiency syndrome patients.

We conducted a clinical trial of human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) in leukopenic patients with acquired immunodeficiency syndrome (AIDS) and analyzed neutrophil function before, during, and after in vivo administration of rGM-CSF. Prior to GM-CSF infusion, AIDS patients' neutrophil superoxide generation and neutrophil antibody-dependent cell-mediated cytotoxicity were enhanced normally by in vitro exposure to GM-CSF. Neutrophil phagocytosis and intracellular killing of Staphylococcus aureus were also normal in the majority of these patients. Two patients, however, had discrete neutrophil functional defects: one in phagocytosis and one in intracellular killing. During the period of GM-CSF infusion, these abnormalities were corrected. The number of circulating neutrophils increased in all patients treated with GM-CSF in a dose-dependent manner. Neutrophils produced in vivo in response to GM-CSF administration functioned normally and there was evidence for neutrophil priming and activation in vivo. We conclude that GM-CSF treatment of AIDS patients leads to the production of functionally active neutrophils, suggesting therapeutic potential for GM-CSF in the treatment of patients with impaired host defense.     

Tyrosine-specific protein phosphorylation during activation of human neutrophils.

The activation of human neutrophils by a variety of receptor-dependent and receptor-independent agonists induces the phosphorylation of a large number of proteins. Since we have previously shown that human neutrophils have at least two distinct tyrosine kinase activities, we examined protein tyrosine phosphorylation in human neutrophils stimulated with a variety of agonists. Using a monoclonal antibody specific for phosphotyrosine, the present study shows that the chemotactic peptides FMLP and leukotriene B4, the phorbol ester phorbol myristate acetate (PMA), and the calcium ionophore A23187 induce an increase in tyrosine phosphorylation of a number of neutrophil proteins. This increased protein tyrosine phosphorylation was dependent on the concentration of the agonist, as well as on the time of exposure to the agonist. Fractionation experiments showed that both a 150,000 g cytosolic and a particulate preparation showed increases in protein tyrosine phosphorylation with stimulation by FMLP or PMA, and showed that the pattern of protein tyrosine phosphorylation was slightly different in the FMLP- and PMA-stimulated cells. These data indicate that protein tyrosine phosphorylation is an early event in the activation of human neutrophils by a variety of receptor-dependent and receptor-independent agonists.     

Role of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in the development of an acute neutrophil inflammatory response in mice

The intraperitoneal injection into mice of casein preparations containing bacteria induced a rapid accumulation of neutrophils within 3 hours due to selective release of mature cells from the bone marrow. Significant increases in the concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) occurred in the peritoneal cavity during the process, but the intraperitoneal injection of neither CSF induced a significant accumulation of neutrophils and the coinjection of G-CSF and casein failed to enhance the neutrophil response. The lack of involvement of either CSF in the neutrophil migration was confirmed by the development of typical neutrophil exudates when casein was injected into mice with inactivation of the genes encoding GM-CSF, G-CSF, or the beta-common chain of the GM-CSF receptor. However, preinjection of G-CSF increased the number of marrow neutrophils available for migration and did result in increased numbers of neutrophils in the peritoneal cavity after casein injection. Typical eosinophil inflammatory responses to the injection of casein or thioglycollate occurred in GM-CSF -/ -mice but not in beta c -/- mice, suggesting that interleukin-5 was necessary for this response.     

Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro

The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll‐like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane‐associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N‐terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan‐associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose‐dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism.     

Adrenal deficiency alters mechanisms of neutrophil mobilization

Deficiency of adrenal hormones promotes exacerbated neutrophil influx into inflammatory sites. We investigated the effect of adrenal deficiency on neutrophil mobilization comparing adrenalectomized (ADX) male Wistar rats to sham-operated (SO) or non-manipulated (N) animals, as controls. Seven days after surgeries, the number of neutrophils in peripheral blood was increased in ADX rats, by accelerating neutrophil maturation steps in the bone marrow. The investigation of adhesive properties on neutrophil membranes indicated reduced and increased expressions of L-selectin on cells present in the bone marrow and circulating blood, respectively. Similar levels of L-selectin mRNA in both cells from ADX or non-manipulated rats suggest that these effects do not depend on gene expression. Even though no differences in the expression of beta(2) integrin by neutrophils were detected, modulation on subsequent PMN activation may occur by adrenal hormones, since circulating neutrophils from ADX exhibit lower in vitro adherence to the endothelium. We conclude that adrenal hormones control the adhesive interactions of neutrophils with the bone marrow microenvironment and with the vascular endothelium chiefly by modulation of L-selectin on PMN membrane in a mechanism independent of L-selectin gene expression.     

Neutrophil elastase depends on serglycin proteoglycan for localization in granules

Granule proteins play a major role in bacterial killing by neutrophils. Serglycin proteoglycan, the major intracellular proteoglycan of hematopoietic cells, has been proposed to play a role in sorting and packing of granule proteins. We examined the content of major neutrophil granule proteins in serglycin knockout mice and found neutrophil elastase absent from mature neutrophils as shown by activity assay, Western blotting, and immunocytochemistry, whereas neutrophil elastase mRNA was present. The localization of other neutrophil granule proteins did not differ between wild-type and serglycin knockout mice. Differential counts and neutrophil ultrastructure were unaffected by the lack of serglycin, indicating that defective localization of neutrophil elastase does not induce neutropenia itself, albeit mutations in the neutrophil elastase gene can cause severe congenital neutropenia or cyclic neutropenia. The virulence of intraperitoneally injected Gram-negative bacteria (Klebsiella pneumoniae) was increased in serglycin knockout mice compared with wild-type mice, as previously reported for neutrophil elastase knockout mice. Thus, serglycin proteoglycan has an important role in localizing neutrophil elastase in azurophil granules of neutrophils, while localization of other granule proteins must be mediated by other mechanisms.     

Monoclonal antibody AHN-1 inhibits phagocytosis by human neutrophils

The granulocyte-specific monoclonal antibody, AHN-1, immunoprecipitates two major surface-iodinated proteins of 105,000 and 145,000 to 150,000 daltons from normal human neutrophils. In this study, the effect of AHN- 1 on a number of neutrophil functions was evaluated in vitro. Both complement- and antibody-mediated phagocytosis were inhibited when human neutrophils were pretreated with AHN-1 and opsonized bacteria were used as targets. The inhibition of phagocytosis was specific, in that lysosomal enzyme release and chemotaxis were not altered by treatment with AHN-1. AHN-1 did inhibit superoxide production by neutrophils in response to particulate stimuli, but not in response to the soluble stimulus, 12-O-tetradecanoylphorbol-13-acetate. The data indicate that one or both of these surface proteins may be important in the process of phagocytosis. AHN-1 should be useful in isolating and further characterizing the nature of these molecules.     

Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis

Neutrophil functions of phagocytosis and intracellular killing of bacteria were examined in 40 patients with alcoholic cirrhosis of whom 18 had a superimposed acute alcoholic hepatitis. In 65% of these, defective neutrophil phagocytosis was demonstrable, and in 62.5% there was a defect of intracellular killing of either Staphylococcus aureus or Escherichia coli. Studies of the patients' serum failed to reveal inhibitors of neutrophil function. Additional assays of superoxide (O2-) and hydrogen peroxide production, hexose monophosphate shunt activity, degranulation and cellular levels of granule enzymes and glutathione revealed that these neutrophil defects are caused by both reduced production of superoxide and defects of degranulation. The hydrogen peroxide/superoxide molar ratio was raised in patients' neutrophils, and the strong inverse correlation found between the value of this ratio and intracellular levels of reduced glutathione would be consistent with the hypothesis that the neutrophils from patients with cirrhosis are unable to detoxify hydrogen peroxide effectively and that this is a result of reduced levels of glutathione in the cells. The consequent increase in oxidant stress, both intra- and extracellularly, may be the cause of phagocytic and degranulation defects. The reduced responses of patients' neutrophils may be caused by previous exposure of the cells to activating stimuli in circulation, as evidenced by depleted intracellular levels of granule enzymes and glutathione. Neutrophils from the patients with a superimposed acute alcoholic hepatitis had depressed phagocytosis in the early stages of incubation but, on the whole, neutrophils from these patients had a greater capacity for ingestion and killing of bacteria than neutrophils from patients with cirrhosis alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Very late antigen-4 in CD18-independent neutrophil emigration during acute bacterial pneumonia in mice

This study tested the hypothesis that very late antigen (VLA)-4 mediates CD18-independent neutrophil emigration into the airspaces induced by either Streptococcus pneumoniae, a stimulus that induces primarily CD18-independent neutrophil emigration, or Escherichia coli, toward which only 20-30% of the total number of neutrophils emigrate through CD18-independent pathways. In wild-type (WT) mice, VLA-4 expression was less on neutrophils that emigrated into the airspaces than on circulating neutrophils. Vascular cell adhesion molecule-1 (VCAM-1) mRNA, the major endothelial cell ligand for VLA-4, increased more in E. coli than in S. pneumoniae pneumonia. VCAM-1 protein expression was not detected in capillaries, the major site of neutrophil emigration. Neutrophil emigration during E. coli or S. pneumoniae pneumonia was similar in mice given antibodies against both CD18 and VLA-4 compared with mice given the anti-CD18 antibody and a control antibody. However, in hematopoietically reconstituted mice with both WT and CD18-deficient neutrophils in their blood, the migration of CD18-deficient neutrophils in response to S. pneumoniae was slightly but significantly less in animals pretreated with the anti-VLA-4 antibody than in those receiving a control antibody. These data suggest that VLA-4 plays a small role in CD18-independent neutrophil emigration, but the majority of CD18-independent neutrophil emigration induced by bacteria in the lungs occurs through VLA-4-independent mechanisms.     

Antigen-induced acute and late-phase responses in primates.

We have examined the proinflammatory cell influx as well as the levels of eosinophil and neutrophil-derived granule proteins in BAL fluid obtained from monkeys undergoing acute and late-phase (dual) or single acute bronchoconstriction following antigen inhalation. Prior to antigen inhalation, there was a significantly higher number (and percentage) of eosinophils in BAL fluid from dual responder monkeys as compared with single responders. The late-phase response (LPR) (6 to 8 h postantigen) was associated with a decrease in the number of BAL eosinophils and an increase in the levels of BAL fluid EPO that returned to baseline levels by 24 h postantigen inhalation. In contrast, the number of BAL neutrophils prior to antigen inhalation were low. Concurrent with the LPR, the number of BAL neutrophils and the concentration of EPO in BAL fluid were significantly increased above that occurring in single responders. Chronic treatment (7 days) with dexamethasone significantly reduced the number of BAL eosinophils and the BAL levels of EPO prior to antigen inhalation in dual responder (LPR) monkeys and significantly blocked the dual response and both the associated neutrophil influx into the airways and an increase in BAL fluid EPO during the LPR. We conclude that, in this primate model, eosinophil activation and a large influx of neutrophils into the airways is associated with the occurrence of the antigen-induced late-phase airway obstructive response.     

Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies

Antineutrophil cytoplasmic autoantibodies (ANCA) have been described in sera of patients with several forms of systemic vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. The two main targets of ANCA in vasculitis are proteinase 3 (PR3) and myeloperoxidase (MPO). ANCA are capable of activating neutrophils primed by tumor necrosis factor-alpha (TNF-alpha) in vitro, which may be relevant for the induction of the vascular inflammation observed in vivo. Recently, it has been suggested that engagement of Fc gamma receptor IIa (Fc gamma RIIa) on the neutrophils is involved in the activation by ANCA. In the present study, we show that activation of the neutrophil respiratory burst by anti-PR3 and anti-MPO is strongly enhanced after TNF priming and lost on removal of the Fc parts of the antibodies. Similar results were obtained when the neutrophils were activated with antibodies against known membrane antigens without major changes in the expression of the target antigens. The TNF-induced enhancement of the neutrophil activation was not observed when adherence of the cells was prevented by continuous stirring of the suspension or by the addition of CD18 antibodies before TNF exposure. Hence, our results indicate that engagement of both Fc gamma RIIa and beta 2 integrins is instrumental in neutrophil activation induced by ANCA.     

Regulation of neutrophil function by Rac GTPases

Rac plays a central role in regulating neutrophil responses to inflammatory signals, including actin remodeling, chemotaxis, and superoxide production by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Rac-GTP is a component of the membrane-assembled NADPH oxidase complex, and new evidence suggests that Rac-GTP interacts directly with the oxidase flavocytochrome, in addition to binding to the regulatory p67 subunit, to regulate electron transfer both independently and cooperatively from NADPH to molecular oxygen. Other new studies suggest that Rac-GTP plays a dual role in NADPH oxidase activation, and can initiate signaling pathways leading to translocation of cytosolic oxidase subunits in addition to functioning in the assembled enzyme complex. Rac activation in response to neutrophil chemoattractants may be regulated in large part by a newly identified guanine nucleotide exchange factor, P-Rex1, which is activated by either phosphatidylinositols or Gbetagamma subunits. Multiple Rac GTPase activating proteins are present in neutrophils and may also modulate levels of Rac-GTP. The importance of Rac in a broad range of neutrophil functions is shown by the variety of defects seen in neutrophils from Rac2 knockout mice and from a patient with recurrent infections and a dominant-negative mutation in Rac2.     

Granulocyte-macrophage colony-stimulating factor enhances phagocytosis of bacteria by human neutrophils

In order to determine whether human granulocyte-macrophage colony- stimulating factor (GM-CSF) can enhance phagocytosis, neutrophils were combined with Staphylococcus aureus (S aureus), and both the number of bacteria per neutrophil and the percent of neutrophils phagocytizing were assessed in the absence and presence of GM-CSF. Exposure to GM-CSF did not enable neutrophils to ingest unopsonized bacteria. When bacteria were opsonized with serum, both the number of bacteria per neutrophil and the percent of cells phagocytizing were increased by treatment with GM-CSF. Digestion of extracellular organisms by lysostaphin was used to substantiate phagocytosis. These results indicate that another effect of GM-CSF on the mature neutrophil is the enhancement of phagocytosis.     

Decreased expression of the common acute lymphoblastic leukaemia antigen (CALLA/CD10) on neutrophils from patients with thermal injury

Summary. The common acute lymphoblastic leukaemia antigen (CALLA/CD10) is a normal component of the circulating neutrophil cell surface membrane. In order to examine the potential functional significance of CALLA/CD10 we analysed the expression of this molecule on neutrophils isolated from thermal injury patients, since these patients have a well-documented constellation of neutrophil defects affecting their microbicidal functions. Expression of neutrophil CALLA/CD10 was monitored by indirect immunofluorescence and flow cytometry. We observed that CALLA/CD10 expression was quantitatively reduced on burn patient neutrophils, compared to healthy donors ( P < 0.001). In contrast, burn patient neutrophils expressed normal levels of class I HLA molecules and the C3bi receptor. Reduced expression of CALLA/CD10 was not associated with neutrophil activation or exposure to plasma 'factor(s)' in vivo . Analysis of normal bone marrow neutrophils by cell sorting indicated that expression of CALLA/CD10 occurs late in neutrophil maturation, since 25% of polymorphonucleated bone marrow neutrophils did not express cell surface CALLA/CD10. Attempts to examine the chemotactic responses of CALLA/CD10 positive and negative neutrophils from burn patients were hampered by previous exposure of these cells to chemoattractants in vivo . Collectively, our findings suggest that burn patient peripheral blood neutrophils may be deficient in CALLA/CD10 due to insufficient maturation time in the bone marrow following thermal injury.