Humate-induced activation of human granulocytes

Naturally occurring humic substances are particular chemical compounds which are found in humus. They bind to carbohydrates, amino acids and steroids by means of hydrogen bonds, covalent bonds and epsilon donor-acceptor complexes. Three specimens of low-molecular humic substances were tested (two naturally occurring humates and one synthetically prepared humate). They were all capable of stimulating certain functions of human neutrophils (PMN), such as the respiratory burst which results in the production of toxic oxygen compounds. This PMN stimulation can be demonstrated with the help of chemiluminescence, as well as by cytochemistry and with the electron microscope. The main product of the humate-induced PMN response is H2O2. There was no activation of neutrophilic chemokinesis or chemotaxis. It is suggested that the low-molecular humic substances originating from decaying organic material contain chemical structures which can act as signals to change dormant PMN into activated cells.     

Independent regulation of human neutrophil chemotactic receptors after activation

The fluoresceinated chemotactic factors, C5a, formyl-methionyl-leucyl-phenylalanyl-lysine (FMLPL), and casein were used in conjunction with flow cytometry to examine chemotactic factor receptor expression on polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA), C5a, or formyl-methionyl-leucyl-phenylalanine. Activation with PMA resulted in a dose-dependent increase in binding of fluorescein-labeled (FL)-casein and (FL-FMLPL) over the range of PMA concentrations from 0.5 to 50 ng/ml. In contrast, activation of PMN with PMA resulted in a dose-dependent decrease in FL-C5a binding, and activation with concentrations above 5 ng/ml resulted in a complete loss of binding. This loss of binding was not caused by inactivation of the ligand or prevented by the addition of superoxide dismutase and catalase or protease inhibitors. Furthermore, incubation of PMN with supernatants from PMN stimulated to degranulate did not reduce the availability of C5a receptors. This pattern of increased FMLPL and casein binding with decreased C5a binding was also observed with cytochalasin B-pretreated PMN that were stimulated with chemotactic factors. Parallel studies of superoxide anion generation demonstrated that PMA-treated PMN were still responsive to formyl-methionyl-leucyl-phenylalanine, but not to C5a. These data demonstrate that the activation of PMN up-regulates formyl peptide and casein receptors whereas C5a receptors are down-regulated under similar conditions.     

A novel post-translational incorporation of tyrosine into multiple proteins in activated human neutrophils. Correlation with phagocytosis and activation of the NADPH oxidase-mediated respiratory burst.

Activation of human neutrophils by PMA causes a post-translational incorporation of 14C-labeled tyrosine into multiple neutrophil (PMN) proteins, that is distinctly different from the enzymatic tyrosinolation of tubulin in FMLP-stimulated PMN. Post-translational incorporation of other radiolabeled amino acids, including the structurally similar amino acid phenylalanine, does not occur under identical conditions of neutrophil activation, suggesting an involvement of the phenolic hydroxyl group of tyrosine in the PMA-mediated reaction. Similar to the stimulation of PMN tubulin tyrosinolation by FMLP, the PMA-induced incorporation of tyrosine into multiple PMN proteins is closely associated with activation of the NADPH oxidase-mediated respiratory burst in stimulated PMN and can be inhibited by a variety of reducing agents, inhibitors of peroxidase-mediated reactions, and intracellular scavengers of oxygen radicals. Moreover, the PMA-induced post-translational incorporation of tyrosine does not occur in PMN from patients with chronic granulomatous disease and is significantly reduced (50%) in PMN of an individual with myeloperoxidase deficiency. A similar stimulus-induced incorporation of tyrosine into multiple PMN proteins is also observed in PMN exposed to various phagocytic stimuli, and the incorporated radioactivity in cells undergoing phagocytosis is substantially enriched (40- to 50-fold) in isolated PMN phagolysosomes. Consistent with this latter observation, HPLC fractionation of stimulated PMN proteins and analysis of the incorporated radioactivity reveal that the 14C label is primarily associated with PMN membrane proteins. Furthermore, this post-translational incorporation of tyrosine, like that associated with PMA stimulation, is associated with production of oxygen radicals and the generation of protein carbonyl derivatives, which are indicative of oxidative protein modifications via mixed function oxidases. Our findings indicate that tyrosine incorporation into membrane proteins of stimulated PMN is functionally relevant to the physiologic host-defense responses of human neutrophils undergoing phagocytosis.     

Polymorphonuclear neutrophils enhance anti-CD3-induced T cell activation: the role of polymorphonuclear FC-receptors.

We investigated the effect of polymorphonuclear neutrophils (PMN) on anti-CD3 mAb (OKT3 and anti-Leu4)-mediated T cell activation. In the absence of monocytes, purified E-rosette-positive cells (further referred to as "T cells") require either solid-phase bound anti-CD3 or the combination of both a high concentration of soluble anti-CD3 and exogenous recombinant interleukin 2 (rIL-2) to proliferate. PMN cannot sustain T cell proliferation with soluble anti-CD3, but they markedly boost proliferation in the presence of soluble anti-CD3 and rIL-2. When PMN were added to T cell cultures stimulated with anti-CD3, this resulted in IL-2 receptor (IL-2R) expression and CD3 modulation. The mechanism of enhancement of anti-CD3-induced IL-2-responsiveness by PMN was further analyzed. A cellular T cell-PMN interaction was found to play a critical role and this was mediated through PMN Fc receptors (FcR). PMN bear two types of low-affinity FcR (FcRII and FcRIII). FcRII is known to bind mIgG1 (e.g., anti-Leu4) and FcRIII binds mIgG2a (e.g., OKT3). FcR involvement was demonstrated by two observations. Anti-FcRII mAb IV.3 inhibited the PMN signal for T cell activation with anti-Leu4. PMN bearing the second variant of FcRII which is unable to bind mIgG1 failed to promote anti-Leu4/IL-2-mediated T cell proliferation. Thus, PMN potentiate T cell responsiveness to IL-2 in the presence of anti-CD3 mAb and this potentiation by PMN requires interaction of anti-CD3 with PMN-FcR.     

Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming.

The cytokine, TNF-alpha, interacts with human neutrophils (PMN) via specific membrane receptors and primes leukotriene B4 (LTB4) production in PMN for subsequent stimulation by calcium ionophores. We have further examined the effects of TNF-alpha on arachidonic acid (AA) release, LTB4 production, and platelet-activating factor (PAF) formation in PMN by prelabeling cells with either [3H]AA or [3H]lyso-PAF, priming with human rTNF-alpha, and then stimulating with the chemotactic peptide, FMLP. TNF-alpha, alone, had little effect; minimal AA release, LTB4 or PAF production occurred after PMN were incubated with 0 to 1000 U/ml TNF-alpha. However, when PMN were first preincubated with 100 U/ml TNF-alpha for 30 min and subsequently challenged with 1 microM FMLP, both [3H] AA release and LTB4 production were elevated two- to threefold over control values. Measurement of AA mass by gas chromatography and LTB4 production by RIA confirmed the radiolabeled results. TNF-alpha priming also increased PAF formation after FMLP stimulation. These results demonstrate that TNF-alpha priming before stimulation with a physiologic agonist can enhance activation of phospholipase A2 (PLA2) resulting in increased AA release and can facilitate the activities of 5-lipoxygenase (LTB4 production) and acetyltransferase (PAF formation). Reports in the literature have hypothesized that the priming mechanism involves either production of PLA2 metabolites, increased diglyceride (DG) levels, or enhanced cytosolic calcium levels induced by the priming agent. We investigated these possibilities in TNF-alpha priming of PMN and report that TNF-alpha had no direct effect on PLA2 activation or metabolite formation. Treatment of PMN with TNF-alpha did not induce DG formation and, in the absence of cytochalasin B, no increased DG production (measured by both radiolabel techniques and mass determinations) occurred after TNF-alpha priming followed by FMLP stimulation. TNF-alpha also had no effect on basal cytosolic calcium and did not enhance intracellular calcium levels after FMLP stimulation. These results suggest that an alternative, as yet undefined, mechanism is active in TNF-alpha priming of human PMN.     

A novel role of growth hormone and insulin-like growth factor-I. Priming neutrophils for superoxide anion secretion.

Growth hormone (GH) and the GH-dependent growth promoting peptide, insulin-like growth factor-I (IGF-I), are both potent signals for priming human and porcine polymorphonuclear neutrophils (PMN) to secrete superoxide anion (O2-). PMA, opsonized-zymosan, or FMLP could all be used as triggering stimuli to demonstrate priming by GH or IGF-I. As positive controls, IFN-gamma and LPS also primed both human and porcine PMN for enhanced O2- release. However, only the LPS-mediated enhancement was inhibited by polymyxin B, which demonstrates that the priming induced by GH, IGF-I, or IFN-gamma was not caused by LPS contamination. Furthermore, a specific antibody to GH abrogated priming induced by this molecule. In contrast to IGF-I, the closely related molecule insulin was unable to prime PMN even at pharmacologic levels. Insulin, at pharmacologic levels, antagonized the priming mediated by IGF-I but had no effect on GH priming. A mAb directed against the human IGF-I receptor blocked the enhanced secretion of O2- by human PMN that was caused by IGF-I, but not GH, indicating that neutrophil priming induced by GH was not mediated by inducing extracellular release of IGF-I. However, priming PMN by both GH and IGF-I required de novo protein synthesis, because cycloheximide completely abrogated enhanced O2- secretion that was caused by these growth factors. These data show that a classic pituitary hormone (GH), as well as its widely recognized growth promoting peptide (IGF-I), are involved in regulating an important functional activity of both porcine and human PMN. Inasmuch as GH and IGF-I have recently been demonstrated to be synthesized by leukocytes, these data support the possibility that both of these proteins could act in a paracrine fashion as cytokines to prime PMN for an enhanced respiratory burst.     

Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation.

Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.     

Studies on the mechanism of PMN activation III. by lymphokines

The influence of a guinea pig lymphokine preparation on the oxidative metabolism of human and guinea pig granulocytes of various sources was investigated. A dose-dependent increase of the oxidative burst following lymphokine challenge was observed. It occurred in unstimulated guinea pig peripheral polymorphonuclear leukocytes (PMN) and in prestimulated PMN obtained from the peritoneal cavity after glycogen injection as well. The lymphokine effect on the oxidative metabolism is not species-restricted because the guinea pig lymphokine preparation elicits an oxidative burst in human PMN, too. The increase caused by lymphokines is nearly of the same order of magnitude as that obtained with zymosan.     

Growth inhibition of Candida albicans by human polymorphonuclear neutrophils: activation by interferon-gamma and tumor necrosis factor

This study was designed to determine whether anti-fungal activity in human polymorphonuclear neutrophils (PMN) might be under the regulation of cytokines such as tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma). By using a radiolabel microassay developed in our laboratory that makes use of the incorporation of [3H]glucose into residual candida, we demonstrated that PMN were better able to inhibit Candida albicans growth in vitro than peripheral blood lymphocytes (PBL). PMN from normal volunteers added to C. albicans for 24 hr at 37 degrees C in a 96-well microplate inhibited fungal growth almost completely at the 300:1 effector/target ratio and frequently at 100:1. Significant activity was still detected at 10:1. In contrast, PBL from the same donors had less activity than PMN at all the ratios tested and lost all function at the 30:1 ratio. TNF and IFN-gamma added to the PMN/candida cultures additionally enhanced PMN to inhibit candida growth. Both cytokines effectively activated PMN down to 0.1 to 0.01 U/ml, and neither cytokine interfered directly with fungal growth, even up to 1000 U/ml. Concentrations of TNF and IFN-gamma below the level that enhanced PMN function when added together to PMN acted synergistically to significantly enhance their anti-fungal activity. Therefore, TNF and IFN-gamma which are active on lymphoid cells, also appear to have the ability to directly activate PMN, and the synergistic action of the two cytokines at low doses that may be below the toxic range may prove to be of clinical importance in protection of immunocompromised host against opportunistic infections.     

Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil

Lipid rafts are cholesterol-rich membrane microdomains that are thought to act as coordinated signaling platforms by regulating dynamic, agonist-induced translocation of signaling proteins. They have been described to play a role in multiple prototypical cascades, among them the lipopolysaccharide pathway, and to host multiple signaling proteins, including kinases and low molecular weight G-proteins. Here we report lipopolysaccharide-induced activation of the Rho family GTPase Cdc42, and we show its activation in the human neutrophil to be mediated by a p38 mitogen-activated protein kinase-dependent mechanism. Subcellular fractionation reveals that lipopolysaccharide induces translocation of Cdc42 to lipid rafts, where it and p38 are both found to be activated. By contrast, lipopolysaccharide causes translocation of Rac from the polymorphonuclear leukocyte (PMN) rafts and does not induce its activation. With the use of methyl-beta-cyclodextrin, a cholesterol-depleting agent that reversibly disrupts rafts, we confirm an important regulatory role for rafts in the activation state of p38 and Cdc42 and in the Rho GTPase-dependent functions superoxide anion production and actin polymerization. Methyl-beta-cyclodextrin induces activation of p38 and Cdc42, but not Rac, in the nonstimulated PMN, yet inhibits subsequent lipopolysaccharide-induced activation of p38 and Cdc42. In parallel, methyl-beta-cyclodextrin primes the human PMN for subsequent superoxide release triggered by the formylated bacterial tripeptide formyl-Met-Leu-Phe, and induces actin polymerization in a subcellular distribution distinct from that induced by lipopolysaccharide. In sum, these findings provide evidence for an important regulatory role of cholesterol in both transmission of the lipopolysaccharide signal and the inflammatory phenotype of the human neutrophil.     

Two roles for Ia in antigen-specific T lymphocyte activation

In this study we examined the mechanism by which a PPD-specific murine T cell hybridoma, 8B2, recognized PPD associated with antigen-presenting cells (APC) in a manner genetically restricted by I-Ad. It was found that PPD-pulsed APC that were glutaraldehyde-fixed and treated with anti-Ia monoclonal antibody (abbreviated as PGM) were unable to stimulate the 8B2 T cells, as expected, due to inhibition caused by antibody binding to the Ia. However, addition of non-antigen-treated, glutaraldehyde-fixed APC (abbreviated as G) to cultures containing 8B2 T cells and PGM restored T cell activation, as determined by IL 2 production. This second non-antigen-specific function provided by the additional APC, G, was attributed to Ia and could be substituted by APC plasma membranes and by soluble membrane extracts. Genetic restriction analysis in which a variety of Ia-positive and Ia-negative cell lines and B cell blasts from different mouse strains were used as PGM or as G showed that each APC provided different Ia determinants that were specifically recognized by the T cells. PGM cells had to express I-Ad in order to present the PPD determinant, whereas the non-antigen-specific function was specific for I-Ad or I-Ab. These results suggest that the anti-Ia antibody does not interfere with the PPD/I-Ad-specific determinant bound by the antigen-specific T cell receptor, but prevents a second non-antigen-specific interaction with another region of the Ia molecule, which is provided by G. These two roles for Ia (antigen-specific and non-antigen-specific) were also found for activation of normal polyclonal PPD-specific T cell responses; thus they are not unique to the 8B2 T cell, but are generally applicable. In addition, T cell interactions with PGM and with G each provide different intracellular activation signals. This was determined by substituting the PGM or the G with either the tumor promoter phorbol 12-myristate 13-acetate (PMA) or the Ca++ ionophore, ionomycin. It was found that 8B2 T cells cultured with PGM and ionomycin, but not with PGM and PMA, were activated for IL 2 production. Neither PMA nor ionomycin in conjunction with G resulted in T cell activation. Taken together, these results indicate that 8B2 T cell activation involves APC Ia antigens in two different ways: one is to contribute to the presentation of the foreign PPD antigen, and a second is a non-antigen-specific Ia-T cell interaction necessary to provide additional intracellular activation signals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

Triggering receptor expressed on myeloid cells-1 in neutrophil inflammatory responses: differential regulation of activation and survival

Polymorphonuclear neutrophils (PMN) are crucial in the innate host defense by their ability to rapidly accumulate in inflamed tissues and clear a site of infection from microbial pathogens by their potent effector mechanisms. The triggering receptor expressed on myeloid cells (TREM)-1 is a recently described activating receptor on PMN with an important role in inflammation. However, the effects of TREM-1 stimulation on a cellular level remain to be further defined. To characterize TREM-1-mediated activation of human PMN, we evaluated the effect of receptor ligation on PMN effector functions. Activation via TREM-1 induces immediate degranulation of neutrophilic granules resulting in the release of IL-8, respiratory burst, and phagocytosis. TREM-1 ligation synergizes with the activation by the Toll-like receptors (TLR) ligands LPS, Pam(3)Cys, and R-848. In contrast, no synergy between TREM-1- and TLR-mediated stimulation was observed concerning PMN survival, whereas TLR-mediated stimuli protect PMN from apoptosis, concurrent TREM-1 activation neutralizes these anti-apoptotic effects. These results give a new perspective for the regulation of neutrophil inflammatory responses emphasizing the importance of TREM-1 in innate immunity.     

Immune complex-induced integrin activation and L-plastin phosphorylation require protein kinase A.

Integrins in resting leukocytes are poorly adhesive, and cell activation is required to induce integrin-mediated adhesion. We recently demonstrated a close correlation between phosphorylation of Ser(5) in L-plastin (LPL), a leukocyte-specific 67-kDa actin bundling protein, and activation of alpha(M)beta(2)-mediated adhesion in polymorphonuclear neutrophils (PMN) (Jones, S. L., Wang, J., Turck, C. W., and Brown, E. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9331-9336). However, the kinase that phosphorylates LPL Ser(5) has not been identified. We found that cAMP-dependent protein kinase (PKA), but not a variety of other serine kinases, can specifically phosphorylate LPL and LPL-derived peptides on Ser(5) in vitro. The cell-permeable cAMP analog 8-bromo-cAMP and the adenylate cyclase activator forskolin both induce LPL phosphorylation in cells. Two PKA inhibitors, H89 and KT5720, inhibited immune complex (IC)-stimulated LPL phosphorylation as well as IC-induced activation of alpha(M)beta(2)-mediated adhesion in PMN. The dose response of H89 inhibition of PMN adhesion correlated with its inhibition of LPL phosphorylation in response to IC. IC stimulation also transiently increased intracellular cAMP concentration in PMN. Thus, PKA functions in an integrin activation pathway initiated by IC binding to Fcgamma receptors in addition to its better known role as a negative regulator of cell activation by G protein-coupled receptors. In contrast, LPL Ser(5) phosphorylation and PMN adhesion induced by formylmethionyl-leucylphenylalanine or phorbol myristate acetate were not affected by PKA inhibitors, suggesting that a different kinase(s) is responsible for LPL phosphorylation in response to these agonists. Phosphoinositidyl 3-kinase also is required for FcgammaR but not formylmethionyl-leucylphenylalanine- or phorbol myristate acetate-induced LPL phosphorylation and activation of alpha(M)beta(2). Two phosphoinositidyl 3-kinase inhibitors blocked FcgammaR-induced cAMP accumulation, demonstrating that this kinase acts upstream of PKA. These data demonstrate a necessary role for PKA in IC-induced integrin activation and LPL phosphorylation.     

Involvement of intermediary metabolites in the pathway of extracellular Ca2+-induced mitogen-activated protein kinase activation in human fibroblasts.

Human fibroblasts in culture will grow in serum-free medium containing serum replacement factors, but without protein growth factors, as long as the Ca2+ level is 1.0-2.0 mM. When the Ca2+ is reduced to 0.1 mM, the cells stop cycling, but they can be reinduced to cycle by raising the Ca2+ level to 1.0 mM Ca2+ or to higher concentrations that result in activation of mitogen-activated protein kinase (MAPK). We now report that exposure of human fibroblasts to extracellular Ca2+ increased the level of inositol (1,4,5)-trisphosphate in the cytoplasm and caused a transient rise in the concentration of intracellular free Ca2+. Ca2+-induced MAPK activation was partly abolished by treatment of the cells with pertussis toxin. It was also decreased by treatment of cells with thapsigargin, which depletes intracellular Ca2+ stores; with phorbol 12-myristyl 13-acetate (PMA), which down-regulates protein kinase C (PKC); with the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide HCl (W-7), and calmidazolium (24571); as well as with lanthanum, a Ca2+ channel inhibitor. Ca2+ stimulation did not result in phosphorylation of the c-raf-1 protein. Our results suggest that extracellular Ca2+ stimulates MAPK activation through a pathway(s) involving a pertussis toxin-sensitive G protein, phospholipase C, intracellular free Ca2+, calmodulin, and PKC.     

Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors

Hypertonic saline (HS) holds promise as a novel resuscitation fluid for the treatment of trauma patients because HS inhibits polymorphonuclear neutrophil (PMN) activation and thereby prevents host tissue damage and associated posttraumatic complications. However, depending on conditions of cell activation, HS can increase PMN degranulation, which could exacerbate tissue damage in trauma victims. The cellular mechanism by which HS increases degranulation is unknown. In the present study, we tested whether HS-induced ATP release from PMN and feedback via P1 and/or P2 receptors may be involved in the enhancement of degranulation by HS. We found that HS enhances elastase release and ERK and p38 MAPK activation when HS is added after activation of PMN with formyl peptide (fMLP) or phorbol ester (PMA). Agonists of P2 nucleotide and A3 adenosine receptors mimicked these enhancing effects of HS, whereas antagonists of A3 receptors or removal of extracellular ATP with apyrase diminished the response to HS. A1 adenosine receptor antagonists increased the enhancing effect of HS, whereas A1 receptor agonists inhibited elastase release. These data suggest that HS upregulates degranulation via ATP release and positive feedback through P2 and A3 receptors. We propose that these feedback mechanisms can serve as potential pharmacological targets to fine-tune the clinical effectiveness of HS resuscitation. resuscitation; inflammation; osmotic stimulation; nucleotide receptor signaling     

The assessment of relative surface hydrophobicity as a factor involved in the activation of human polymorphonuclear leukocytes by uropathogenic strains of Escherichia coli

The initiation of the respiratory burst and the degranulation of human polymorphonuclear leukocytes (PMN) in response to stimulation by uropathogenic strains of Escherichia coli is dependent on the expression of Type 1 fimbriae by those strains. These PMN responses correlate with an increasing tendency of the interacting E. coli strain to be retained on hydrophobic columns. The present work assessed the measurement of relative surface hydrophobicity in relation to PMN activation. Type 1 fimbriate organisms bound most readily to Octyl-Sepharose columns and were strongly agglutinated in the salt aggregation test. In contrast, the same organisms partitioned to the dextran-rich (hydrophilic) phase of aqueous two-polymer phase systems. Electron microscopic observation of the organisms eluted from the Octyl-Sepharose columns and of the organisms recovered from both phases of the aqueous two-phase systems demonstrated, however, that both Type 1 and P-fimbriate organisms were retained on the columns and partitioned into the dextran-rich phase as a consequence of their being fimbriate and failed to identify this as a major factor in the activation of PMN. In addition electron microscopy demonstrated that each P-fimbriate population had fewer organisms expressing fimbriae than did Type 1 fimbriate populations, confirming the importance of phase variation as a factor affecting the physicochemical characteristics of a bacterial population.     

Dissection of the functions of antigen-presenting cells in the induction of T cell activation

The functions of antigen-presenting cells (APC) in the initiation of T cell activation was examined by culturing antigen-bearing guinea pig macrophages (M phi) with T cells obtained from antigen-primed animals. Although such antigen-bearing M phi stimulated primed syngeneic T cell DNA synthesis, as assessed by tritiated thymidine incorporation, paraformaldehyde fixation (0.15% for 1 min at 37 degrees C) abolished this capacity. Analysis with acridine orange staining indicated that fixed antigen-bearing M phi could not trigger primed syngeneic T cells to progress from the G0 to the G1 phase of the cell cycle. The addition of control non-antigen-bearing syngeneic or allogeneic M phi but not interleukin 1 or 2 to cultures of T cells and fixed APC permitted a proliferative response. Although the interaction between fixed antigen-bearing M phi and responding T cells was genetically restricted, there was no similar restriction for the supplemental control M phi. In fact, completely Ia-negative endothelial cells (EC) and fibroblasts (FB) could restore antigen responsiveness to cultures of fixed antigen-bearing M phi and syngeneic responding T cells, although they could not directly present antigen. Moreover, metabolically intact accessory cells, including Ia-negative EC and FB, could take up and process antigen to an immunogenic moiety, which fixed Ia-positive M phi could present to primed T cells. These data indicate that recognition of the antigen-Ia complex on an APC is necessary but not sufficient to trigger proliferation of freshly obtained primed T cells. The results additionally support the conclusion that APC carry out at least two separate functions necessary for the initiation of antigen-induced T cell activation. Not only must the APC display the antigen-Ia complex, but it must also convey another required effect. This influence, which apparently involved the establishment of cell to cell contact, was neither Ia nor antigen dependent and could only be provided by a metabolically intact cell. By contrast, genetically restricted antigen presentation could be accomplished by a fixed Ia-positive cell. Only when both the antigen-Ia complex and the influence of an intact accessory cell were provided by the same or different accessory cell were T cells triggered to enter the cell cycle.     

Ceramide-dependent caspase 3 activation is prevented by coenzyme Q from plasma membrane in serum-deprived cells

Coenzyme Q (CoQ) is the key factor for the activity of the eukaryotic plasma membrane electron transport chain. Consequently, CoQ is essential in the cellular response against redox changes affecting this membrane. Serum withdrawal induces a mild oxidative stress, which produces lipid peroxidation in membranes. In fact, apoptosis induced by serum withdrawal can be prevented by several antioxidants including CoQ. Also, CoQ can maintain cell growth in serum-limiting conditions, whereas plasma membrane redox system (PMRS) inhibitors such as capsaicin, which compete with CoQ, inhibit cell growth and induce apoptosis. To understand how plasma membrane CoQ prevents oxidative stress-induced apoptosis we have studied the induction of apoptosis by serum withdrawal in CEM cells and its modulation by CoQ. Serum-withdrawal activates neutral sphingomyelinase (N-SMase), ceramide release and caspase-3-related proteases. CoQ addition to serum-free cultures inhibited a 60% N-SMase activation, an 80% ceramide release, and a 50% caspase-3 activity induced by serum deprivation. Caspase activation dependent on ceramide release since C 2 -ceramide was only able to mimic this effect in 10% foetal calf serum cultured cells but not in serum-free cultures. Also, in vitro experiments demonstrated that C 2 -ceramide and ceramide-rich lipid extracts directly activated caspase-3. Taken together, our results indicate that CoQ protects plasma membrane components and controls stress-mediated lipid signals by its participation in the PMRS.     

Soluble fibrinogen modulates neutrophil functionality through the activation of an extracellular signal-regulated kinase-dependent pathway

The integrin family not only mediates the recruitment of polymorphonuclear leukocytes (PMN) to sites of inflammation but also regulates several effector functions by binding to specific ligands. We have recently demonstrated that soluble fibrinogen (sFbg) is able to trigger an activating signal in PMN through an integrin-dependent mechanism. This activation results in degranulation, phagocytosis enhancement, and apoptosis delay. The aim of the present work was to further elucidate the molecular events that follow sFbg interaction with CD11b in human PMN, and the participation of this signaling pathway in the regulation of neutrophil functionality. We demonstrate that sFbg triggers a cascade of intracellular signals that lead to focal adhesion kinase and extracellular signal-regulated kinase 1/2 tyrosine phosphorylation. The activation of this mitogen-activated protein kinase pathway plays a central role in the sFbg modulation of secondary granule degranulation, Ab-dependent phagocytosis, and apoptosis. However, fibrinogen-induced secretory vesicle degranulation occurs independently of the signaling transduction pathways investigated herein. In the context of an inflammatory process, the intracellular signal pathway activated by sFbg may be an early event influencing the functionality of PMN.