Effects of Aluminum Exposure on the Adherence, Chemotaxis, and Phagocytosis Capacity of Peritoneal Macrophages in Rats

To investigate the effects of aluminum (Al) exposure on peritoneal macrophages of Wistar rats, four groups of ten rats each were orally exposed to 0, 13, 26, and 52?mg?kg(-1) Al(3+) in form of aluminum trichloride (AlCl(3)) in drinking water for 120?days. At the end of the experimental period, the Al concentration in serum, the adherence, chemotaxis, and phagocytosis capacity of peritoneal macrophages were determined. The results showed that the Al concentration in serum significantly increased in a dose-dependent manner; the adherence, chemotaxis, and phagocytosis capacity of peritoneal macrophages decreased with the increase of Al dose, and present a dose-effective relationship. Further, they were significantly lower in the high-dose groups (P?相似文献   

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.     

Models of Eukaryotic Gradient Sensing: Application to Chemotaxis of Amoebae and Neutrophils

Eukaryotic cells can detect shallow gradients of chemoattractants with exquisite precision and respond quickly to changes in the gradient steepness and direction. Here, we describe a set of models explaining both adaptation to uniform increases in chemoattractant and persistent signaling in response to gradients. We demonstrate that one of these models can be mapped directly onto the biochemical signal-transduction pathways underlying gradient sensing in amoebae and neutrophils. According to this scheme, a locally acting activator (PI3-kinase) and a globally acting inactivator (PTEN or a similar phosphatase) are coordinately controlled by the G-protein activation. This signaling system adapts perfectly to spatially homogeneous changes in the chemoattractant. In chemoattractant gradients, an imbalance between the action of the activator and the inactivator results in a spatially oriented persistent signaling, amplified by a substrate supply-based positive feedback acting through small G-proteins. The amplification is activated only in a continuous presence of the external signal gradient, thus providing the mechanism for sensitivity to gradient alterations. Finally, based on this mapping, we make predictions concerning the dynamics of signaling. We propose that the underlying principles of perfect adaptation and substrate supply-based positive feedback will be found in the sensory systems of other chemotactic cell types.     

Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells

Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells.     

Chemotaxis in Vitro: Quantitation of Human Granulocyte Movement Using a Stochastic Differential Equation

The quantitation of human granulocyte movement using a stochastic differential equation is described. The method has the potential to distinguish both positive and negative chemotaxis. Analysis and information concerning cell movements can be obtained for any point in time and distance for the duration of the experiment.     

热处理与冰温处理对板栗贮藏效应的影响

本试验研究了不同热处理和冰温处理对板栗生理指标和贮藏品质的影响。试验结果表明:热处理以60℃下60 min处理板栗保鲜效果最好,贮藏200 d时,鲜度指数为92%。冰温处理以-2℃下36 h处理板栗保鲜效果最好,贮藏200 d时,鲜度指数仍达94%。板栗贮前进行热处理或冰温预冷处理,贮后产品可达到有机食品级别。     

Suppression of Anti-Candida Activity of Murine and Human Neutrophils by Glucocorticoids

Effects of glucocorticoid (GC) compounds on inhibitory activity of neutrophils to mycelial growth of Candida albicans were examined by in vitro crystal violet staining method with 14 hr co-culture. Both GC hormones (hydrocortisone ≥6 × 10^–7 m and corticosterone ≥10^–6 m ) and anti-inflammatory GC agents (prednisolone ≥10^–7 m and dexamethasone ≥10^–8 m ) significantly suppressed anti-Candida activity of murine casein-induced neutrophils. Anti-Candida activity of human neutrophils prepared from peripheral blood was also suppressed by hydrocortisone (≥6 × 10^–7 m ). These GC compounds did not affect the Candida growth in the absence of neutrophils. Steroidal compounds without anti-inflammatory activity, cholesterol, cholic acid, aldosterone did not suppress neutrophil activity. These results suggest that GCs at their physiological or clinical concentration may suppress anti-Candida activity of neutrophils in vivo.     

Natural IgM Mediates Complement-Dependent Uptake of Francisella tularensis by Human Neutrophils via Complement Receptors 1 and 3 in Nonimmune Serum

A fundamental step in the life cycle of Francisella tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum nor the receptors that mediate infection of neutrophils have been defined. In this study, human neutrophil uptake of GFP-expressing F. tularensis strains live vaccine strain and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components, we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis, whereas C5 was not. Second, we used purification and immunodepletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-Ag and capsule as prominent targets of these Abs on the bacterial surface. Finally, we demonstrate using receptor-blocking Abs that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-Ag polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3 opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner.     

Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O₂^.-) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.     

Differential Interaction of the Two Related Fungal Species Candida albicans and Candida dubliniensis with Human Neutrophils

Candida albicans, the most common facultative human pathogenic fungus is of major medical importance, whereas the closely related species Candida dubliniensis is less virulent and rarely causes life-threatening, systemic infections. Little is known, however, about the reasons for this difference in pathogenicity, and especially on the interactions of C. dubliniensis with the human immune system. Because innate immunity and, in particular, neutrophil granulocytes play a major role in host antifungal defense, we studied the responses of human neutrophils to clinical isolates of both C. albicans and C. dubliniensis. C. dubliniensis was found to support neutrophil migration and fungal cell uptake to a greater extent in comparison with C. albicans, whereas inducing less neutrophil damage and extracellular trap formation. The production of antimicrobial reactive oxygen species, myeloperoxidase, and lactoferrin, as well as the inflammatory chemokine IL-8 by neutrophils was increased when stimulated with C. dubliniensis as compared with C. albicans. However, most of the analyzed macrophage-derived inflammatory and regulatory cytokines and chemokines, such as IL-1α, IL-1β, IL-1ra, TNF-α, IL-10, G-CSF, and GM-CSF, were less induced by C. dubliniensis. Similarly, the amounts of the antifungal immunity-related IL-17A produced by PBMCs was significantly lower when challenged with C. dubliniensis than with C. albicans. These data indicate that C. dubliniensis triggers stronger early neutrophil responses than C. albicans, thus providing insight into the differential virulence of these two closely related fungal species, and suggest that this is, in part, due to their differential capacity to form hyphae.     

The Influence of Superoxide on the Production of Hypochlorous Acid by Human Neutrophils

Human neutrophils stimulated with opsonized zyrnosan promoted hypochlorous acid (HOCl)-dependent loss of monochlorodimedon. Formation of HOCl was completely inhibited by catalase, and it was also inhibited up to 70% by SOD. There was no inhibition by desferal, DTPA, mannitol or dimethylsulphoxide. which excluded the involvement of -OH. Our results indicate that generation of O₂-by neutrophils enables these cells to enhance their production of HOCl. Furthermore, inhibition of neutrophil processes by SOD and catalase does not necessarily implicate -OH. We propose that O₂-may potentiate oxidant damage at inflammatory sites by boosting the rnyeloperoxidase-dependent production of HOCl     

人血清白蛋白荧光的红际激发效应

研究了人血清白蛋白中色氨酸的荧光红际激发效应,阐明了人血清白蛋白的红际激发效应与ＰＨ,温度及尿素浓度存在一定的关系,外界条件的改变使得色氨酸残基所处的微环境发生了变化,使得色氨酸残基的基态和激发态的能级产生不同的分布,从而得到不同的红际激发效应。     

Adhesion and Activation of Human Neutrophils onto Collagen Chains Separated by Electrophoresis

After separation of the various α chains of the collagens by SDS-PAGE, the binding of polymorphonuclear neutrophils (PMN) to these chains was detected by a double-antibody technique and the activation of PMN by nitro blue tetrazolium. All of the α chains tested were able to bind PMNs. The α1 chain of type I collagen activated the PMN when it had not been treated with pepsin. Pepsinized types II and VI collagens did not activate PMN. The pepsinized α1 (III) chains and all three α chains from pepsinized type V collagen were able to activate PMN.     

Toxic Effect of Heavy Metals on Human Blood Neutrophils

Activity of the peroxidase-hydrogen peroxide system is proposed as a biological marker of ecological risk of heavy metal contamination of the environment. The effect of lead, copper, mercury, and cadmium ions on peroxidase system activity in human peripheral blood neutrophils was studied using cytochemical methods. A possible mechanism of suppression of peroxidase system activity by metal ions is discussed.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 336–338.Original Russian Text Copyright © 2005 by Mushtakova, Fomina, Rogovin.     

Protrusive and Contractile Forces of Spreading Human Neutrophils

Human neutrophils are mediators of innate immunity and undergo dramatic shape changes at all stages of their functional life cycle. In this work, we quantified the forces associated with a neutrophil’s morphological transition from a nonadherent, quiescent sphere to its adherent and spread state. We did this by tracking, with high spatial and temporal resolution, the cell’s mechanical behavior during spreading on microfabricated post-array detectors printed with the extracellular matrix protein fibronectin. Two dominant mechanical regimes were observed: transient protrusion and steady-state contraction. During spreading, a wave of protrusive force (75 ± 8 pN/post) propagates radially outward from the cell center at a speed of 206 ± 28 nm/s. Once completed, the cells enter a sustained contractile state. Although post engagement during contraction was continuously varying, posts within the core of the contact zone were less contractile (−20 ± 10 pN/post) than those residing at the geometric perimeter (−106 ± 10 pN/post). The magnitude of the protrusive force was found to be unchanged in response to cytoskeletal inhibitors of lamellipodium formation and myosin II-mediated contractility. However, cytochalasin B, known to reduce cortical tension in neutrophils, slowed spreading velocity (61 ± 37 nm/s) without significantly reducing protrusive force. Relaxation of the actin cortical shell was a prerequisite for spreading on post arrays as demonstrated by stiffening in response to jasplakinolide and the abrogation of spreading. ROCK and myosin II inhibition reduced long-term contractility. Function blocking antibody studies revealed haptokinetic spreading was induced by β₂ integrin ligation. Neutrophils were found to moderately invaginate the post arrays to a depth of ∼1 μm as measured from spinning disk confocal microscopy. Our work suggests a competition of adhesion energy, cortical tension, and the relaxation of cortical tension is at play at the onset of neutrophil spreading.     

High-Molecular-Weight Hyaluronic Acids Inhibit Chemotaxis and Phagocytosis but Not Lysosomal Enzyme Release Induced by Receptor-Mediated Stimulations in Guinea Pig Phagocytes

The effects of high-molecular-weight (HMW) hyaluronic acids (HAs) of 1.9 × 10⁶ Da, 8 × 10⁵ Da and 3 × 10⁵ Da on the receptor-mediated functions of guinea pig peritoneal phagocytes were studied. HMW-HAs of 1.9 × 10⁶ Da (HA190) and 8 × 10⁵ Da (HA80) effectively inhibited the chemotactic activity of polymorphonuclear leukocytes (PMNs) for formyl-Met-Leu-Phe (fMLP). The degree of inhibition was dose-dependent and the concentrations of HA190 and HA80 required for 50% inhibition were 0.5–1.5 mg/ml and 1.5–2.5 mg/ml, respectively. HMW-HA of 3 × 10⁵ Da (HA30) hardly affected the chemotaxis within a concentration range of 0.5–5.0 mg/ml. The phagocytic activities of PMNs and macrophages (Møs) for serum-opsonized zymosan (SOZ) and polystyrene latex particles were also inhibited by these HAs in a dose- and molecular-weight-dependent manner and HA190 was again the most inhibitory. By contrast, the release of lysosomal enzyme from Møs stimulated with SOZ was not significantly affected by HMW-HAs at any concentration used. Furthermore, the binding of [³H]fMLP with PMNs and the rosette formation of Møs with SOZ were not influenced by the presence of HMW-HAs. These findings suggested that the binding of HMW-HAs to the HA receptors on PMNs and Møs might produce certain intracellular signals which would be responsible for the suppression of the chemotaxis and the phagocytosis but not for the release of lysosomal enzyme. For the generation of such signals, higher-molecular-weight HMW-HAs would be more effective than lower one.     

Impact of MK-886 on H2O2 Generation by Human Neutrophils and Cell Degranulation

Metabolic inhibitors can clearly affect different aspects of the functional activity of cells. This property was studied in the present work with respect to MK-886, a well-known inhibitor of the 5´-lipoxygenase-activating protein. It was found that this inhibitor in a micromolar concentration range (2-20 µM) induced in a dose-dependent manner H₂O₂ generation by human neutrophils and the release of lysozyme from the cells. The MK-886-induced activation of neutrophils was accompanied by a significant decrease in N-(1-pyrene)maleimide-accessible SH-groups in the cells. According to its activity, MK-886 can be considered an agonist that causes up-regulation of inherent neutrophil functions. In summary, the results indicate that during the application of MK-886 as a 5´-lipoxygenase inhibitor in neutrophils, the impact of the compound on the functional activity of the cells should be taken into consideration.     

Deregulation of Collagen Phagocytosis in Aging Human Fibroblasts: Effects of Integrin Expression and Cell Cycle

Intracellular degradation of collagen by phagocytosis in fibroblasts is essential for physiological remodeling of the extracellular matrix in a wide variety of connective tissues but imbalances between degradation and synthesis can lead to loss of tissue collagen. As aging is associated with loss of dermal and periodontal collagen and with increased lysomomal enzyme content in fibroblasts, we examined the regulation of collagen phagocytosis by integrin expression and the cell cycle in anin vitrofibroblast aging model. Two different fibroblast lines (CL1; CL2) at the fourth subculture were passaged up to replicative senescence to model aging processesin vitro.Cells were incubated with collagen-coated or BSA-coated green fluorescent beads for 3 h to assess α₂β₁-integrin-mediated or nonspecific phagocytosis, respectively. Single-cell suspensions were stained with DAPI and sulforhodamine 101 to separate cycling G₁and noncycling G₀cells. Staining for α₂-integrin, bead internalization, and bivariate analyses of DNA/protein content were measured by three-color flow cytometry. Serum deprivation was used to induce increases in the proportion of G₀cells. For G₁cells, the proportion of collagen phagocytic cells was >50% for all passages and collagen beads were internalized >5-fold more frequently than BSA beads. In contrast, G₀cells with diploid DNA content but low protein content exhibited greatly reduced phagocytic capacity (<10% of cells internalized collagen or BSA beads), the number of beads per cells was 4-fold less, and α₂integrin expression was very low compared to G₁cells. The proportion of collagen phagocytic cells and the proportion of α₂-integrin-positive cells increased with transit through the cell cycle. At higher passage numbers mean cell volume and cytoplasmic granularity were reduced 30% but at replicative senescence cells with large surface area and subdiploid DNA predominated. The proportion of collagen and BSA phagocytic G₁cells increased 1.5- and 5-fold, respectively, and the number of beads per cell increased <3-fold. However, surface α₂-integrin staining remained unchanged. These data indicate that the collagen and nonspecific internalization pathways were greatly upregulated, independent of cell cycle phase, and that cellular agingin vitrostrongly influences the specificity and rate of phagocytic processes in fibroblasts. We suggest that age-related loss of collagen in connective tissues undergoing turnover may be a manifestation of a deregulated increase of collagen phagocytosis in which the net loss of degraded collagen exceeds new synthesis.     

Human Red Cells Enhance the Formation of 5-Lipoxygenase-Derived Products by Neutrophils

Upon activation, human neutrophils generate 5-lipoxygenase products which are involved in inflammation as well as other physiological and pathophysiological processes. We have examined the influence of red cells on the generation of lipoxygenase-derived products by neutrophils utilizing high pressure liquid chromato-graphy system which permitted quantitation of SHETE, leukotriene B₄ (and its isomers) and the omega oxidation products of leukotriene B₄ (20-hydroxyleukotriene B₄, 20-carboxyleukotriene B₄) within the same sample. Co-incubation of red cells with neutrophils (50:1, red cells:neutrophils) resulted in a 722 percent increase in 5-hydroxyeicosatetraenoic acid production and a slight increase in leukotriene B₄ and its omega oxidation products which were not accompanied by increases in 15-hydroxyeicosatetraenoic acid production. The role of the sulfhydryl status of the red cell and its ability to scavenge hydrogen peroxide were assessed in relationship to the interaction of red cells on the neutrophil-derived lipoxygenase products. Together, these findings indicate that red cells can regulate the levels of lipid-derived mediators produced by neutrophils. Moreover, they suggest that red cell-neutrophil interactions may be of importance in inflammatory reactions.