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ABSTRACT: Although indispensable in host defense against microbial pathogens, misdirected hyperacute and chronic activation of neutrophils presents the potential hazard of tissue damage, organ dysfunction, and carcinogenesis. In many clinical settings, particularly inflammatory disorders of the airways, over-reactivity of neutrophils is exacerbated by their relative resistance to conventional, pharmacological anti-inflammatory therapies, including, but not limited to, corticosteroids. Notwithstanding their sheer numbers, which can increase rapidly and dramatically during inflammatory responses, these cells are not only pre-programmed to release reactive oxygen species, proteinases, and eicosanoids/prostanoids immediately on exposure to pro-inflammatory stimuli but may also subsequently undergo the process of netosis, thereby enhancing and protracting their inflammatory potential. All of these mechanisms are likely to underpin the resistance of neutrophils to pharmacological control and have triggered the search for alternatives to corticosteroids. In addition to macrolides and adenosine 3',5'-cyclic adenosine monophospate-elevating agents, more recent innovations in the control of neutrophilic inflammation include activators of histone deacetylases and antagonists of chemokine receptors, as well as monoclonal antibodies which target neutrophil-activating cytokines and their receptors. These and other neutrophil-targeted strategies represent the focus of the current review.
Seminars in Immunopathology 03/2013; · 6.27 Impact Factor
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ABSTRACT: Although pro-inflammatory mechanisms have been implicated in the pathogenesis of manganese (Mn(2+))-related neurological and respiratory disorders, relatively little is known about the potential of this metal to interact pro-oxidatively with human phagocytes. The primary objective of the current study was to investigate the effects of Mn(2+) as MnCl(2) (0.5-100 µM) on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide (H(2)O(2)), and hypohalous acids by isolated human blood neutrophils and monocyte-derived macrophages following activation of these cells with the chemotactic tripeptide, FMLP (1 µM), or the phorbol ester, PMA (25 ng/mL). Generation of ROS was measured using the combination of oxygen consumption, lucigenin/luminol-enhanced chemiluminescence, spectrofluorimetric detection of oxidation of 2,7-dichlorodihydrofluorescein, radiometric assessment of myeloperoxidase (MPO)-mediated protein iodination, release of MPO by ELISA, and spectrophotometric measurement of nitrite formation. Treatment of activated neutrophils with either FMLP or PMA resulted in significantly decreased reactivity of superoxide in the setting of increased formation of H(2)O(2) and MPO-mediated iodination, with no detectable effects on either oxygen consumption or MPO release. Similar effects of the metal with respect to superoxide reactivity and H(2)O(2) formation were observed with activated macrophages, while generation of NO was unaffected. Taken together with the findings of experiments using cell-free ROS-generating systems, these observations are compatible with a mechanism whereby Mn(2+), by acting as a superoxide dismutase mimetic, increases the formation of H(2)O(2) by activated phagocytes. If operative in vivo, this mechanism may contribute to the toxicity of Mn(2+).
Inhalation Toxicology 08/2012; 24(10):634-44. · 1.92 Impact Factor
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ABSTRACT: The soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) is a useful marker of infection in patients with sepsis, but has not been adequately evaluated in patients with chemotherapy-associated febrile neutropenia (FN). The value of sTREM-1 in this setting has been tested in a retrospective, pilot study using stored serum from 48 cancer patients with documented FN. On presentation, patients were categorized according to the Talcott risk-index clinical score. Circulating soluble sTREM-1 was measured using an ELISA procedure, while procalcitonin (PCT) or interleukins 6 (IL-6) and 8 (IL-8), included for comparison, were measured using an immunoluminescence-based assay and Bio-Plex® suspension bead array system, respectively. Circulating concentrations of both sTREM-1 and PCT were significantly (P < 0.05) elevated in patients at high risk for complications or death, as predicted by the Talcott score and were significantly lower in patients who responded to empiric antimicrobial agents. Neither IL-6 nor IL-8 accurately predicted serious complications in patients with FN. These observations, albeit from a pilot study, demonstrate that sTREM-1 is indeed elevated in high-risk patients with FN and is potentially useful to predict their clinical course, either together with, or as an alternative to PCT.
Annals of Hematology 10/2011; 91(4):605-11. · 2.62 Impact Factor
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ABSTRACT: Soluble triggering receptor expressed on myeloid cells (s-TREM-1) is upregulated on the surface of inflammatory cells in the presence of bacterial infections, apparently excluding those due to Mycobacterium tuberculosis. Therefore, sputum concentrations of s-TREM-1 may be of value in distinguishing bacterial pneumonia from pulmonary tuberculosis (PTB) in patients with respiratory infections. The current pilot study was designed to evaluate whether s-TREM-1 concentrations measured in the sputum of patients with suspected community-acquired pneumonia (CAP) allowed differentiation of those patients with PTB from other causes of pneumonia and to correlate s-TREM-1 with CURB-65, a marker of disease severity. Soluble s-TREM-1 concentrations were measured in sputum samples from patients admitted to a tertiary hospital with CAP or PTB by means of an ELISA procedure. Soluble-TREM-1 was readily detectable and quantifiable in sputum samples from patients with both CAP and PTB, with concentrations of 234±47 and 178±36 pg/ml respectively, but did not differ significantly between the two groups. However, patients with PTB had significantly lower leukocyte counts, 9±1.3 vs 15±1.4 × 10(9)/l compared with those without PTB. Interestingly, sputum s-TREM-1 concentrations correlated significantly with the CURB-65 pneumonia severity score calculated at the time of admission. Soluble-TREM-1 expression is upregulated in patients with both CAP and PTB, but does not differentiate between these two conditions. Sputum concentrations of s-TREM-1 may predict the severity of disease in patients with CAP.
European Journal of Clinical Microbiology 05/2011; 31(1):73-6. · 2.86 Impact Factor
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ABSTRACT: The research question addressed in the current study was: do formoterol (1 and 10 nM) and montelukast (2 μM) possess interactive inhibitory effects on activated human neutrophils, particularly in relation to alterations in cyclic AMP and cytosolic Ca²(+) fluxes? Isolated human blood neutrophils were activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 μM) in combination with cytochalasin B (CB; 3 μM). Fura-2-acetoxymethyl ester-based spectrofluorimetry, lucigenin-enhanced chemiluminescence, colorimetric and flow cytometric procedures were used to measure cytosolic Ca²(+) fluxes, production of superoxide, elastase release and beta-2 integrin (CR3) expression, respectively, while cyclic AMP and leukotriene (LT)B₄ were assayed using competitive binding ELISA procedures. Activation of the cells with fMLP/CB resulted in abrupt and sustained increases in cytosolic Ca²(+), as well as release of elastase and production of superoxide and LTB₄, and expression of CR3, all of which were attenuated by formoterol and montelukast individually, and especially by the combination of these agents. These anti-inflammatory effects of each agent, as well as the combination, were associated with significant increases in cyclic AMP. The findings of the current study may explain the efficacy of montelukast and formoterol when used in combination with inhaled corticosteroids in the treatment of severe asthma, possibly by controlling neutrophil-driven inflammation of the airways.
European Respiratory Journal 04/2010; 36(6):1417-24. · 5.89 Impact Factor
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ABSTRACT: This study was designed to investigate the neutrophil-targeted anti-inflammatory potential of posaconazole (0.1-5 microM, equivalent to 0.7-3.9 mg/L) by measuring the effects of this agent on the release of leukotriene B(4) (LTB(4)) and store-operated uptake of Ca(2+) following stimulation of human neutrophils with platelet-activating factor (200 nM).
LTB(4) release and uptake of Ca(2+) by the cells were measured using an enzyme immunoassay and fura-2/AM-based spectrofluorimetric procedures, respectively.
Treatment of neutrophils with posaconazole resulted in dose-related attenuation of PAF-activated release of LTB(4) and influx of Ca(2+), which attained statistical significance at 1 microM of the antimycotic.
Although primarily an antimycotic, posaconazole possesses secondary anti-inflammatory activities, which may contribute to the therapeutic efficacy of this agent in patients with sepsis.
Journal of Antimicrobial Chemotherapy 10/2009; 64(5):1008-12. · 5.07 Impact Factor
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ABSTRACT: The primary objective of this study was to probe the involvement of leukotriene B(4) (LTB(4)) in itraconazole (0.1-5 microM)-mediated inhibition of Ca(2+) uptake by chemoattractant-activated human neutrophils. Following exposure of the cells to platelet-activating factor (PAF, 200 nM), LTB(4) was measured by immunoassay, while neutrophil cytosolic Ca(2+) concentrations were determined by a fura-2/AM-based spectrofluorimetric procedure. Activation of neutrophils was accompanied by an abrupt and sustained (for about 1 min) elevation in cytosolic Ca(2+) which was associated with increased generation of LTB(4), both of which were attenuated significantly by itraconazole at 0.5 microM and higher. The inhibitory effect of the anti-mycotic on Ca(2+) uptake by PAF-activated cells was mimicked by an LTB(4) antibody, as well as by LY255283 (1 microM) and MK886 (0.5 microM), an antagonist of LTB(4) receptors and an inhibitor of 5'-lipoxygenase-activating protein, respectively, while addition of itraconazole to purified 5'-lipoxygenase resulted in inhibition of enzyme activity. A mechanistic relationship between itraconazole-mediated inhibition of LTB(4) production and Ca(2+) influx was also supported by the observation that pulsed addition of purified LTB(4) to PAF-activated neutrophils caused substantial restoration of Ca(2+) uptake by cells treated with the anti-mycotic. Taken together, these observations suggest that the potentially beneficial anti-inflammatory interactions of itraconazole with activated neutrophils result from interference with production of LTB(4), with consequent attenuation of a secondary LTB(4)-mediated wave of Ca(2+) uptake by the cells.
Clinical & Experimental Immunology 11/2007; 150(1):144-50. · 3.36 Impact Factor
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ABSTRACT: To identify the involvement of adenosine in restoration of Ca2+ homeostasis to activated human neutrophils.
Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers.
The cells were exposed to adenosine deaminase (ADA, 0.1-2 units/ml) for 10 min at 37 degrees C prior to activation with N-formyl-L-methionyl-L-leucyl-L-phenylala-nine (FMLP, 1 microM).
Cytosolic Ca2+ concentrations and transmembrane fluxes of the cation in FMLP-activated neutrophils +/- ADA were measured using spectrofluorimetric and radiometric procedures respectively, while intracellular cAMP and inositol triphosphate were measured by radioassay, and superoxide production and elastase release by, chemiluminescence and colourimetric methods respectively. Levels of statistical significance were calculated using the Mann-Whitney U-test and ANOVA.
Although FMLP-activated generation of inositol triphosphate and mobilisation of Ca2+ from neutrophil internal stores, as well as the magnitude of the subsequent efflux and store-operated influx of the cation were unaffected by ADA, there was a prolonged elevation in cytosolic Ca2+ in the presence of the enzyme, which was associated with failure to activate adenylate cyclase and with increased production of superoxide and release of elastase. These effects of ADA were attenuated by dibutyryl cAMP (4 mM), CGS 21680 (1 microM) and rolipram (0.5 microM), as well as by EGTA (10 mM).
These results are compatible with a physiological role for adenosine in promoting deactivation of neutrophils, possibly by promoting cAMP-dependent clearance of Ca2+ from the cytosol of the cells by the endo-membrane Ca2+-ATPase.
Inflammation Research 01/2003; 51(12):594-602. · 2.11 Impact Factor
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ABSTRACT: The objectives of the current study were to: (i) present an integrated model for the restoration of calcium homeostasis in activated human neutrophils based on current knowledge and recent research; and (ii) identify potential targets for the modulation of calcium fluxes in activated neutrophils based on this model and to investigate the effects of intracellular probes which target key processes involved in calcium homeostasis and pro-inflammatory activity in these cells.
Laboratory-based experimental research using purified human neutrophils from healthy, adult human volunteers.
Calcium metabolism and pro-inflammatory activity of neutrophils.
Modulation of calcium fluxes in activated human neutrophils can be achieved by cAMP-dependent upregulation of the activity of the endomembrane Ca(2+)-ATPase which resequesters cytosolic Ca2+. Formoterol, a long-acting beta 2-agonist, elevates intracellular cAMP levels, accelerates Ca2+ restoration in activated neutrophils and downregulates the pro-inflammatory responses of these cells. Alterations in the membrane potential of activated neutrophils may play a role in regulating calcium reuptake into the cells as attenuation of the membrane depolarisation response is associated with accelerated calcium influx.
Modulation of the activity of the endomembrane Ca(2+)-ATPase in human neutrophils represents an important target for anti-inflammatory
South African medical journal = Suid-Afrikaanse tydskrif vir geneeskunde 01/2003; 92(12):990-6. · 2.04 Impact Factor
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ABSTRACT: This study was designed to evaluate the effects of epinephrine (0.01-1 microM) on superoxide production by, and release of elastase from human neutrophils activated with the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 microM) in vitro, and to relate alterations in these responses to changes in adenosine 3,5' cyclic monophosphate (cAMP) and cytosolic free Ca(2+). Cyclic AMP, superoxide production and elastase release were measured by radioimmunoassay, lucigenin-enhanced chemiluminescence, and a colorimetric procedure respectively. Cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures that enable distinction between net efflux and influx of the cation. Epinephrine treatment of neutrophils resulted in increased cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, and attenuated by propranolol, but not by selective beta(1)-, alpha(1)- or alpha(2)-adrenoreceptor antagonists. Although epinephrine did not affect the FMLP-activated abruptly-occurring increase in fura-2 fluorescence intensity, indicating no effects on the release of Ca(2+) from neutrophil intracellular stores, this agent accelerated the rate of decline in fluorescence in the setting of decreased efflux and a reduction in store-operated influx of Ca(2+). These effects of epinephrine on the clearance of Ca(2+) from the cytosol of FMLP-activated neutrophils were attenuated by propranolol, and are compatible with enhancement of the activity of the cAMP-dependent Ca(2+) sequestering/resequestering endo-membrane Ca(2+)-ATPase. We conclude that epinephrine down-regulates the pro-inflammatory activities of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca(2+).
Biochemical Pharmacology 06/2001; 61(10):1319-28. · 4.70 Impact Factor
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ABSTRACT: The relationship between activation of NADPH-oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from four subjects with chronic granulomatous disease (CGD). Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, and net efflux and influx of Ca2+ by radiometric procedures. Exposure of normal neutrophils to the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM) was accompanied by an abrupt increase in cytosolic Ca2+ coincident with membrane depolarization and efflux of the cation. These events terminated at around 30 s after the addition of FMLP and were followed by membrane repolarization and store-operated influx of Ca2+, both of which were superimposable and complete after about 5 min. Activation of CGD neutrophils was also accompanied by an increase in cytosolic Ca2+, which, in spite of an efficient efflux response, was prolonged in relation to that observed in normal cells. This prolonged increase in cytosolic Ca2+ in activated CGD neutrophils occurred in the setting of trivial membrane depolarization and accelerated influx of Ca2+, and was associated with hyperactivity of the cells according to excessive release of elastase and increased activity of phospholipase A2. Treatment of CGD neutrophils with the type 4 phosphodiesterase inhibitor, rolipram (1 microM) restored Ca2+ homeostasis and attenuated the increase in elastase release. These findings support the involvement of NADPH-oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses and granuloma formation which are characteristic of CGD.
Clinical & Experimental Immunology 03/2001; 123(2):254-63. · 3.36 Impact Factor
