H. C. Steel

Clofazimine: current status and future prospects

Clofazimine, a lipophilic riminophenazine antibiotic, possesses both antimycobacterial and anti-inflammatory activities. However, its efficacy has been demonstrated only in the treatment of leprosy, not in human tuberculosis, despite the fact that this agent is impressively active in vitro against multidrug-resistant strains of Mycobacterium tuberculosis. Recent insights into novel targets and mechanisms of antimicrobial and anti-inflammatory activity coupled with the acquisition of innovative drug delivery technologies have, however, rekindled interest in clofazimine as a potential therapy for multidrug- and extensively multidrug-resistant tuberculosis in particular, as well as several autoimmune diseases. The primary objective of this review is to critically evaluate these recent developments and to assess their potential impact on improving the therapeutic efficacy and versatility of clofazimine.

Taming the neutrophil: calcium clearance and influx mechanisms as novel targets for pharmacological control

Neutrophils are relatively insensitive to the anti‐inflammatory actions of conventional chemotherapeutic agents, including corticosteroids, emphasizing the requirement for novel pharmacological strategies to control the potentially harmful proinflammatory activities of these cells. In the case of commonly‐occurring inflammatory diseases of the airways, the neutrophil is the primary mediator of inflammation in conditions such as chronic obstructive pulmonary disease, cystic fibrosis, acute respiratory distress syndrome, bronchiectasis and non‐eosinophilic bronchial asthma. Recent insights into the mechanisms utilized by neutrophils to restore Ca2+ homeostasis following activation with Ca2+‐mobilizing, proinflammatory stimuli have facilitated the identification of novel targets for anti‐inflammatory chemotherapy in these cells. The most amenable of these from a chemotherapeutic perspective, is the cyclic AMP‐dependent protein kinase‐modulated endomembrane Ca2+‐ATPase which promotes clearance of the cation from the cytosol of activated neutrophils. Second generation type 4 phosphodiesterase inhibitors and adenosine receptor agonists operative at the level of subtype A2A adenosine receptors, which are currently undergoing clinical and preclinical assessment respectively, hold promise as pharmacologic modulators during the restoration of Ca2+ homeostasis. If this promise is realized, it may result in novel chemotherapeutic strategies for the control of hyperacute and chronic inflammatory conditions in which neutrophils are primary offenders. Alternative, potential future targets include the Na+, Ca2+‐exchanger and store‐operated Ca2+ channels, which cooperate in the refilling of intracellular Ca2+ stores.

Comparison of the Anti‐inflammatory Activities of Imidazole Antimycotics in Relation to Molecular Structure

The objective of this study was to compare the anti‐inflammatory potencies of the imidazole antimycotics, fluconazole, itraconazole, ketoconazole and voriconazole (0.5 and 5u2003μm) in relation to their molecular structures. Anti‐inflammatory activity was determined according to the magnitude of inhibition of production of leukotriene B4 and influx of Ca2+ following activation of the cells with the chemo‐attractant platelet‐activating factor (200u2003nm), using enzyme‐linked immunosorbent assay and spectrofluorometric procedures, respectively. Treatment of platelet‐activating factor‐activated neutrophils with the imidazole antimycotics resulted in inhibition of production of leukotriene B4 and attenuation of Ca2+ influx, the order of potency being itraconazoleu2003>u2003ketoconazoleu2003> fluconazoleu2003=u2003voriconazole. These observations demonstrate the requirement for both the diazole/triazole moiety (all four agents), and the substituted phenylpiperazinyl ether side chain (itraconazole and ketoconazole only) for maximal anti‐inflammatory activity of this class of pharmacological agents.

Leukotrienes C4 and D4 sensitize human neutrophils for hyperreactivity to chemoattractants.

Abstract.Objective and design:To investigate the sensitizing effects of the cysteinyl leukotrienes (CysLTs) C4 and D4 on the proinflammatory responses of chemoattractant-activated human neutrophils in vitro.Materials:Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers.Treatment:Cells were exposed to LTC4 and LTD4 (50–300 nM) prior to activation with 1xa0μM of N-formyl-L-methionyl- L-leucyl-L-phenylalanine (fMLF).Methods:A fura-2/AM-based spectrofluorimetric procedure, lucigenin-enhanced chemiluminescence (LECL), a colourimetric method and an ELISA procedure, were used to measure Ca2+ mobilization, superoxide production, elastase and MMP-8 release respectively following activation of LTC4/ D4-primed neutrophils with fMLF. Superoxide generation was also measured in the presence and absence of the CysLT receptor 1 antagonist, montelukast (100 nM).Results:Exposure of neutrophils to either LTC4 or LTD4 alone had modest effects on Ca2+ mobilization, while superoxide generation and elastase release were unaffected. However, relative to the responses of neutrophils activated with fMLF in the absence of the CysLTs, pre-treatment of the cells with either LTC4or LTD4 resulted in significant, augmentation of fMLF-activated elastase and MMP-8 release and superoxide generation, which was attenuated by montelukast.ConclusionThese previously undocumented sensitizing interactions of LTs C4 and D4 with neutrophils may contribute to the activation of these cells in acute and chronic inflammation of both atopic and non-atopic aetiology, while identifying a role for montelukast in regulating neutrophil reactivity.

Calcium-dependent potentiation of the pro-inflammatory functions of human neutrophils by tigecycline in vitro

OBJECTIVESnTigecycline is the prototype of the recently introduced, intravenously administered glycylcycline class of antibiotics, developed in response to the increasing problem of antibiotic resistance in Gram-positive bacteria, especially Staphylococcus aureus, as well as Gram-negative bacteria and anaerobes. However, relatively little is known about the immunomodulatory potential of tigecycline, specifically its interactions with human neutrophils. In the current study we investigated the effects of tigecycline at therapeutically relevant concentrations and greater (0.625-10 mg/L) on alterations in cytosolic Ca(2+) concentrations, generation of antimicrobial reactive oxygen species (ROS) and release of granule proteases [elastase, matrix metalloproteinase-8 (MMP-8) and matrix metalloproteinase-9 (MMP-9)] by human blood neutrophils activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 μM).nnnMETHODSnCytosolic Ca(2+) concentrations were measured using fura-2/AM-based spectrofluorimetry and radiometric procedures, generation of ROS by oxygen consumption and myeloperoxidase-mediated auto-iodination, and protease release by ELISA procedures.nnnRESULTSnExposure of the cells to fMLP resulted in activation of the generation of ROS, as well as release of the granule proteases, all of which were significantly increased by pre-incubation of the cells with tigecycline in a dose-dependent manner. Tigecycline-mediated enhancement of these neutrophil functions was associated with elevations in the concentrations of cytosolic Ca(2+), which appeared to result from the Ca(2+) ionophore activity of tigecycline.nnnCONCLUSIONSnTigecycline, by functioning as a Ca(2+) ionophore, and independent of antimicrobial activity, potentiates the pro-inflammatory functions of human neutrophils in vitro.

Manganese promotes increased formation of hydrogen peroxide by activated human macrophages and neutrophils in vitro

Although pro-inflammatory mechanisms have been implicated in the pathogenesis of manganese (Mn2+)-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 Mn2+ as MnCl2 (0.5–100 µM) on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide (H2O2), 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 (25u2009ng/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 H2O2 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 H2O2 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 Mn2+, by acting as a superoxide dismutase mimetic, increases the formation of H2O2 by activated phagocytes. If operative in vivo, this mechanism may contribute to the toxicity of Mn2+.

Characterization of the interactions of the pneumolysoid, Δ6 PLY, with human neutrophils in vitro

The pneumolysin toxoid, Δ6 PLY, is a prototype pneumococcal protein vaccine candidate. However, its potentially detrimental residual pro-inflammatory interactions with human neutrophils are unknown. In the current study the effects of the toxoid (8-1000 ng/ml) have been compared with those of wild-type pneumolysin (WT/PLY, 8 ng/ml) on neutrophil cytosolic Ca(2+) fluxes, generation of leukotriene B(4) (LTB(4)), and release of matrix metalloproteinase-9 (MMP-9), using spectrofluorimetric, and ELISA procedures (LTB(4) and MMP-9) respectively. Exposure of neutrophils to WT/PLY resulted in influx of Ca(2+) and significant (P<0.05) release of MMP-9 and generation of LTB(4). However, treatment of the cells with Δ6 PLY at concentrations of up to 1000 ng/ml had only trivial effects on Ca(2+) influx and no effects on either release of MMP-9 or LTB(4) production. The observed absence of pro-inflammatory interactions of Δ6 PLY with neutrophils is clearly an important property of this pneumococcal protein vaccine candidate.

Posaconazole attenuates leukotriene B4 release and uptake of calcium by chemoattractant-activated human neutrophils: a potential strategy to control neutrophil-mediated inflammation

OBJECTIVESnThis 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).nnnMETHODSnLTB(4) release and uptake of Ca(2+) by the cells were measured using an enzyme immunoassay and fura-2/AM-based spectrofluorimetric procedures, respectively.nnnRESULTSnTreatment 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.nnnCONCLUSIONSnAlthough primarily an antimycotic, posaconazole possesses secondary anti-inflammatory activities, which may contribute to the therapeutic efficacy of this agent in patients with sepsis.

Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

BackgroundThe role of protein kinase C (PKC) in regulating the activity of phospholipase C (PLC) in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM), was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM) and staurosporine (400 nM).MethodsAlterations in cytosolic Ca2+, Ca2+ influx, inositol triphosphate (IP3), and leukotriene B4 production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively.ResultsActivation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca2+ followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP3 production with associated enhanced Ca2+ release from storage vesicles, prolongation of the peak cytosolic Ca2+ transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB4. The alterations in Ca2+ fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca2+-resequestering endomembrane ATPase.Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP3 production and down-regulation of Ca2+ mediated pro-inflammatory responses of PAF-activated neutrophils.ConclusionAlthough generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.