Participation of cofilin in opsonized zymosan-triggered activation of neutrophil-like HL-60 cells through rapid dephosphorylation and translocation to plasma membranes. (49/3362)

We studied the roles of cofilin, an actin-binding phosphoprotein, in superoxide production of neutrophil-like HL-60 cells triggered by opsonized zymosan (OZ). OZ caused dephosphorylation of cofilin as well as a transient increase of F-actin. Both reactions were complete within 30 s. Okadaic acid (OA) magnified the OZ-triggered O2--production 3.3-fold at 1 microM, but inhibited it completely at 5 microM. We used these critical concentrations to study the effects of OA on changes in phosphorylation and intracellular localization of cofilin. The OZ-induced dephosphorylation of cofilin was inhibited by 5 microM OA but not by 1 microM OA. Subcellular fractionation and immunoblotting revealed that 1 microM OA increased cofilin on the phagosomal membranous fraction but 5 microM OA decreased it. At 1 microM, OA increased translocation of p47phox to membranes, which may explain in part the enhancing effect of 1 microM OA. Confocal laser scanning microscopy showed that: (i) Cofilin diffused throughout the cytosol of resting cells, but accumulated at the plasma membranes forming phagocytic vesicles in activated cells. (ii) At 1 microM, OA had little effect on the OZ-evoked translocation of cofilin, whereas 5 microM OA suppressed it completely. (iii) OA alone, which could not trigger the phagocytic respiratory burst, did not cause any change in the distribution of cofilin at such concentrations. Furthermore, in a superoxide-producing cell-free system employing membranous and cytosolic fractions, affinity-purified anti-cofilin antibody showed an enhancing effect. These results suggest that cofilin participates in the superoxide production of the OZ-activated phagocytes through dephosphorylation and translocation. The roles of cofilin in the activated leukocytes will be discussed.  (+info)

Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. (50/3362)

A major function of Rac2 in neutrophils is the regulation of oxidant production important in bacterial killing. Rac and the related GTPase Cdc42 also regulate the dynamics of the actin cytoskeleton, necessary for leukocyte chemotaxis and phagocytosis of microorganisms. Although these GTPases appear to be critical downstream components of chemoattractant receptor signaling in human neutrophils, the pathways involved in direct control of Rac/Cdc42 activation remain to be determined. We describe an assay that measures the formation of Rac-GTP and Cdc42-GTP based on their specific binding to the p21-binding domain of p21-activated kinase 1. A p21-binding domain glutathione S-transferase fusion protein specifically binds Rac and Cdc42 in their GTP-bound forms both in vitro and in cell samples. Binding is selective for Rac and Cdc42 versus RhoA. Using this assay, we investigated Rac and Cdc42 activation in neutrophils and differentiated HL-60 cells. The chemoattractant fMet-Leu-Phe and the phorbol ester phorbol myristate acetate stimulate formation of Rac-GTP and Cdc42-GTP with distinct time courses that parallel cell activation. We also show that the signaling pathways leading to Rac and Cdc42 activation in HL-60 cells involve G proteins sensitive to pertussis toxin, as well as tyrosine kinase and phosphatidylinositol 3-kinase activities.  (+info)

Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages. (51/3362)

In previous reports we described a 22-kb Legionella pneumophila chromosomal locus containing 18 genes. Thirteen of these genes (icmT, -R, -Q, -P, -O, -M, -L, -K, -E, -C, -D, -J, and -B) were found to be completely required for intracellular growth and killing of human macrophages. Three genes (icmS, -G, and -F) were found to be partially required, and two genes (lphA and tphA) were found to be dispensable for intracellular growth and killing of human macrophages. Here, we analyzed the requirement of these genes for intracellular growth in the protozoan host Acanthamoeba castellanii, a well-established important environmental host of L. pneumophila. We found that all the genes that are completely required for intracellular growth in human macrophages are also completely required for intracellular growth in A. castellanii. However, the genes that are partially required for intracellular growth in human macrophages are completely required for intracellular growth in A. castellanii. In addition, the lphA gene, which was shown to be dispensable for intracellular growth in human macrophages, is partially required for intracellular growth in A. castellanii. Our results indicate that L. pneumophila utilizes the same genes to grow intracellularly in both human macrophages and amoebae.  (+info)

Ehrlichia chaffeensis and E. sennetsu, but not the human granulocytic ehrlichiosis agent, colocalize with transferrin receptor and up-regulate transferrin receptor mRNA by activating iron-responsive protein 1. (52/3362)

Ehrlichia chaffeensis and E. sennetsu are genetically divergent obligatory intracellular bacteria of human monocytes and macrophages, and the human granulocytic ehrlichiosis (HGE) agent is an obligatory intracellular bacterium of granulocytes. Infection with both E. chaffeensis and E. sennetsu, but not HGE agent, in the acute monocytic leukemia cell line THP-1 almost completely inhibited by treatment with deferoxamine, a cell-permeable iron chelator. Transferrin receptors (TfRs) accumulated on both E. chaffeensis and E. sennetsu, but not HGE agent, inclusions in THP-1 cells or the cells of the promyelocytic leukemia cell line HL-60. Reverse transcription-PCR showed an increase in the level of TfR mRNA 6 h postinfection which peaked at 24 h postinfection with both E. chaffeensis and E. sennetsu infection in THP-1 or HL-60 cells. In contrast, HGE agent in THP-1 or HL-60 cells induced no increase in TfR mRNA levels. Heat treatment of E. chaffeensis or the addition of monodansylcadaverine, a transglutaminase inhibitor, 3 h prior to infection inhibited the up-regulation of TfR mRNA. The addition of oxytetracycline 6 h after E. chaffeensis infection caused a decrease in TfR mRNA which returned to the basal level by 24 h postinfection. These results indicate that both internalization and continuous proliferation of ehrlichial organisms or the production of ehrlichial proteins are required for the up-regulation of TfR mRNA. Results of electrophoretic mobility shift assays showed that both E. chaffeensis and E. sennetsu infection increased the binding activity of iron-responsive protein 1 (IRP-1) to the iron-responsive element at 6 h postinfection and remained elevated at 24 h postinfection. However, HGE agent infection had no effect on IRP-1 binding activity. This result suggests that activation of IRP-1 and subsequent stabilization of TfR mRNA comprise the mechanism of TfR mRNA up-regulation by E. chaffeensis and E. sennetsu infection.  (+info)

Relationship between cell surface carbohydrates and intrastrain variation on opsonophagocytosis of Streptococcus pneumoniae. (53/3362)

Streptococcus pneumoniae undergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci.  (+info)

Avidity as a determinant of the protective efficacy of human antibodies to pneumococcal capsular polysaccharides. (54/3362)

Antibodies reactive with capsular polysaccharides are considered the principal mediators of immunity against invasive diseases caused by Streptococcus pneumoniae. In this study, we tested the hypothesis that anti-pneumococcal capsular polysaccharide (PPS) antibody avidity can influence protective efficacy. We measured the avidities of individual adult postvaccination immunoglobulin G2 (IgG2) antibodies to PPS serotypes 6B and 23F and examined the relationship between avidity and opsonophagocytic and mouse-protective activities. The avidities of PPS 6B- and PPS 23F-specific IgG2 antibodies ranged from 6 to 31 nM-1 and from 3 to 20 nM-1, respectively. We observed an inverse correlation between the magnitude of avidity and the amount of antibody required to protect mice against lethal bacteremia caused by serotype 6B pneumococci. Similarly, higher-avidity antibodies were more effective than lower-avidity antibodies in vitro in mediating complement-dependent opsonophagocytosis of both 6B and 23F pneumococci. These data suggest that in adults, PPS antibodies are sufficiently polymorphic to possess biologically significant variations in avidity. We conclude that avidity functions as an important determinant of anticapsular antibody protective efficacy against pneumococci.  (+info)

Annexin V delays apoptosis while exerting an external constraint preventing the release of CD4+ and PrPc+ membrane particles in a human T lymphocyte model. (55/3362)

Phosphatidylserine exposure in the exoplasmic leaflet of the plasma membrane is one of the early hallmarks of cells undergoing apoptosis. The shedding of membrane particles carrying Ags testifying to their tissue origin is another characteristic feature. Annexin V, a protein of as yet unknown specific physiologic function, presents a high Ca2+-dependent affinity for phosphatidylserine and forms two-dimensional arrays at the membrane surface. In this study, we report the delaying action of annexin V on apoptosis in the CEM human T cell line expressing CD4 and the normal cellular prion protein (PrPc), two Ags of particular relevance to cell degeneration and with different attachments to the membrane. The effect of annexin V was additive to that of z-Val-Ala-Asp-fluoromethyl ketone, a potent caspase inhibitor. Annexin V significantly reduced the degree of proteolytic activation of caspase-3, and totally blocked the release of CD4+ and PrPc+ membrane particles. z-Val-Ala-Asp-fluoromethyl ketone was a more powerful antagonist of caspase-3 processing, but prevented the shedding of CD4+ vesicles only partially and had no effect on that of PrPc+ ones. These results suggest that an external membrane constraint, such as that exerted by annexin V, has important consequences on the course of programmed cell death and on the dissemination of particular Ags. In vivo, annexin V had a significant protective effect against spleen weight loss in mice treated by an alkylating agent previously shown to induce lymphocyte apoptosis.  (+info)

G-protein-stimulated phospholipase D activity is inhibited by lethal toxin from Clostridium sordellii in HL-60 cells. (56/3362)

Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton. In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells. We show that guanosine 5'-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a time- and dose-dependent manner after an overnight treatment with LT. A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4, 5)P2). Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol. Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P2 to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity. Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity. In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity. Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase Calpha was decreased after LT treatment. We conclude that in HL-60 cells, lethal toxin from C. sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity. Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells.  (+info)