CGS 21680, dibutyryl cyclic AMP and rolipram attenuate the pro-inflammatory interactions of the Pseudomonas aeruginosa -derived pigment, 1-hydroxyphenazine, with human neutrophils.

The effects of the intracellular adenosine 3':5' cyclic monophosphate (cAMP)-elevating agents, CGS 21680 (0.01- 1 microM) and rolipram (0.01-1 microM), as well as those of dibutyryl cAMP (0. 05-4 mM) on the pro-inflammatory interactions of the P. aeruginosa -derived pigment, 1-hydroxyphenazine (1-hp, 3.1 and 12.5 microM), with human neutrophils have been investigated in vitro. Ca(2+)fluxes in FMLP-activated neutrophils were measured using a fura-2/AM spectrofluorimetric procedure, while a colourimetric method was used to measure release of the primary granule enzyme, elastase, from the cells. Treatment with 1-hp resulted in delayed clearance of Ca(2+)from the cytosol of N -formyl- L -methionyl- L -leucyl- L -phenylalanine (FMLP, 1 microM)-activated neutrophils and increased release of elastase. All 3 test agents caused dose-related antagonism of 1-hp-mediated potentiation of elastase release from activated neutrophils, which was associated with restoration of Ca(2+)homeostasis. These observations demonstrate the potential of cAMP-elevating agents, acting on Ca(2+)clearance mechanisms in activated neutrophils, to attenuate the potentially harmful pro-inflammatory effects of 1-hp.

Apparent involvement of the A(2A) subtype adenosine receptor in the anti-inflammatory interactions of CGS 21680, cyclopentyladenosine, and IB-MECA with human neutrophils.

This study was undertaken to identify the adenosine receptor (AR) subtypes which down-regulate the proinflammatory activities of human neutrophils, as well as the involvement of adenosine 3',5'-cyclic monophosphate (cAMP) and its relationship to cellular handling of Ca(2+) in mediating these effects. Neutrophils were treated with varying concentrations (0.01-1 microM) of AR agonists operative at A(1) (N(6)-cyclopentyladenosine, CPA), A(2A) (2(4-[(2-carboxyethyl)phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosi ne, CGS 21680), and A(3) (N(6)-(3-iodobenzyl-5'-N-methylcarbamoyladenosine, IB-MECA) receptors, after which they were activated with the chemoattractant, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 microM). Intracellular cAMP, superoxide, and elastase were assayed using radioimmunoassay, lucigenin-enhanced chemiluminescence (LECL), and colorimetric procedures, respectively, while changes in the concentrations of cytosolic Ca(2+) were monitored by fura-2-based spectrofluorimetry. CGS 21680, at all concentrations tested, inhibited superoxide production in a dose-related manner, while CPA and IB-MECA were effective only at the highest concentrations tested (0.5-1 microM). The release of elastase from activated neutrophils was also inhibited by all three AR agonists, but was more sensitive to CGS 21680 and IB-MECA than was superoxide production. The inhibitory effects of all 3 agonists on superoxide production and elastase release were associated with accelerated clearance of Ca(2+) from the cytosol of activated neutrophils, and were effectively neutralized by pretreatment of the cells with the highly selective A(2A)R antagonist, ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1, 2,4]triazolo[2,3-a][1,3,5]triazin-5yl amino]ethyl)phenol). Increased cAMP was detected in neutrophils treated with CGS 21680 and IB-MECA (1 microM). These data support the involvement of the A(2A)R subtype in the suppression of superoxide production and degranulation by activated human neutrophils, probably by cAMP-mediated alterations in Ca(2+) handling.

Accelerated resequestration of cytosolic calcium and suppression of the pro-inflammatory activities of human neutrophils by CGS 21680 in vitro.

We have investigated the effects of the adenosine A(2A) receptor agonist CGS 21680 (0.01 - 1 microM) on reactive oxidant production by, and elastase release from FMLP-activated human neutrophils, as well as on cytosolic Ca(2+) fluxes and intracellular concentrations of cyclic AMP. Oxidant production, elastase release and cyclic AMP were assayed using lucigenin-enhanced chemiluminescence, colourimetric and radioimmunoassay procedures respectively, while cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures which distinguish between net efflux and influx of the cation. Treatment of neutrophils with CGS 21680 did not affect the FMLP-activated release of Ca(2+) from intracellular stores, but resulted in dose-related acceleration of the rate of decline in fura-2 fluorescence, as well as decreases in both efflux and store-operated influx of Ca(2+), compatible with enhancement of resequestration of the cation by the endo-membrane Ca(2+)-ATPase. These effects on neutrophil Ca(2+) handling were associated with increased intracellular cyclic AMP and with inhibition of oxidant production and release of elastase. In contrast, treatment of neutrophils with the selective A(2A) receptor antagonist, ZM 241385 (2.5 microM), prevented the transient increase in cyclic AMP in FMLP-activated neutrophils which was associated with delayed sequestration of incoming Ca(2+) during store-operated influx. The CGS 21680-mediated reduction of Ca(2+) efflux from FMLP-activated neutrophils was also antagonized by pretreatment of the cells with ZM 241385 (2.5 microM), as well as by thapsigargin (1 microM), an inhibitor of the endo-membrane Ca(2+)-ATPase. ZM 241385 also neutralized the cyclic AMP-elevating and anti-inflammatory interactions of CGS 21680 with neutrophils. We conclude that A(2A) receptors regulate the pro-inflammatory activities of human neutrophils by promoting cyclic AMP-dependent sequestration of cytosolic Ca(2+).

Exposure of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-activated human neutrophils to the Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine is associated with impaired calcium efflux and potentiation of primary granule enzyme release.

The effects of pathologically relevant concentrations (0.38 to 12.5 microM) of the proinflammatory, Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine (1-hp) on Ca2+ metabolism and intracellular cyclic AMP (cAMP) in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM)-activated human neutrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro. Ca2+ fluxes were measured by the combination of a fura-2/AM-based spectrofluorimetric method and radiometric procedures, which together enable distinction between net efflux and influx of the cation, while radioimmunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively. Coincubation of neutrophils with 1-hp did not affect intracellular cAMP levels or the FMLP-activated release of Ca2+ from intracellular stores but did retard the subsequent decline in the chemoattractant-induced increase in the concentration of cytosolic free Ca2+. These effects of 1-hp on the clearance of Ca2+ from the cytosol of activated neutrophils were associated with decreased efflux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was unaffected by the pigment. The plasma membrane Ca2+-ATPase rather than a Na+-Ca2+ exchanger appeared to be the primary target of 1-hp. These observations suggest that the proinflammatory interactions of 1-hp with activated human neutrophils are a consequence of interference with the efflux of cytosolic Ca2+ from these cells.

Effect of rolipram and dibutyryl cyclic AMP on resequestration of cytosolic calcium in FMLP-activated human neutrophils.

1. We have investigated the effects of the selective phosphodiesterase (PDE) type 4 inhibitor, rolipram (0.01-1 microM) on cytosolic Ca2+ fluxes in FMLP-activated human neutrophils, as well as on superoxide production by, and release of elastase from, these cells. 2. Cytosolic Ca2+ fluxes were measured by use of fura-2 spectrofluorimetry in combination with a radiometric procedure that enables distinction between net efflux and influx of the cation. Superoxide production and elastase release were measured by lucigenin-enhanced chemiluminescence and a colorimetric procedure, respectively. 3. Pretreatment of neutrophils with rolipram did not affect the FMLP-activated release of Ca2+ from intracellular stores, but was associated with dose-related acceleration of the rate of decline in fura-2 fluorescence and with decreased efflux, as well as store-operated influx of 45Ca2+, indicative of enhancement of resequestration of the cation by the endo-membrane Ca2+-ATPase. 4. Inhibition of superoxide production and elastase release was observed at concentrations of rolipram which accelerated the clearance of Ca2+ from the cytosol of FMLP-activated neutrophils. 5. These effects of rolipram on FMLP-activated Ca2+ fluxes, superoxide generation and elastase release were mimicked by pretreatment of neutrophils with dibutyryl cyclic AMP (0.5-4 mM), while theophylline (10-150 microM), a non-specific PDE inhibitor, as well as the beta2-agonist, salbutamol, were less effective. 6. We conclude that rolipram deactivates FMLP-stimulated human neutrophils by enhancement of cyclic AMP-dependent resequestration of cytosolic Ca2+.

Roxithromycin, clarithromycin, and azithromycin attenuate the injurious effects of bioactive phospholipids on human respiratory epithelium in vitro.

The effects of the bioactive phospholipids (PL), platelet-activating factor (PAF), lyso-PAF, and lysophosphatidylcholine (LPC) on the beat frequency and structural integrity of human ciliated respiratory epithelium were studied in vitro, in the presence or absence of polymorphonuclear leukocytes (PMNL), the antimicrobial agents, roxithromycin, clarithromycin, and azithromycin and the antioxidative enzymes catalase and superoxide dismutase (SOD). All three PL caused dose-dependent slowing of ciliary beat frequency (CBF) and epithelial damage (ED) at concentrations > or = 1 microgram/ml, which were unaffected by inclusion of the antimicrobial agents and antioxidative enzymes. When epithelial strips were exposed to the combination of PMNL and PL, there was significant potentiation of ciliary dysfunction and ED, which was ameliorated by pretreatment of the PMNL with the antimicrobial agents or by inclusion of catalase, but not SOD. These results demonstrate that LPC, PAF, and lyso-PAF cause epithelial damage by direct mechanisms which are oxidant-independent, as well as by indirect mechanisms involving phagocyte-derived reactive oxidants. Macrolides and azalide antimicrobial agents may have beneficial effects on airway inflammation in asthma and microbial infections by protecting ciliated epithelium against oxidative damage inflicted by PL-sensitized phagocytes.

Membrane stabilizing, anti-oxidative interactions of propranolol and dexpropranolol with neutrophils.

We have investigated the effects of the beta-adrenoreceptor-blocking agent, propranolol (9-300 microM) on several pro-inflammatory activities of human neutrophils in vitro. Superoxide production by calcium ionophore (A23187)-activated neutrophils was particularly sensitive to inhibition by low concentrations (9-18.7 microM) of this drug. However, inhibition of superoxide generation by neutrophils activated with phorbol myristate acetate (PMA), opsonized zymosan (OZ), and arachidonate (AA) only occurred with higher concentrations of propranolol, and coincided with decreased intracellular calcium fluxes, phospholipase A2 (PLA2) activity and synthesis of platelet-activating factor (PAF). Propranolol possessed neither cytotoxic nor superoxide-scavenging properties but, using a haemolytic assay of membrane-stabilizing activity, this agent neutralized the membrane-disruptive effects of the bioactive phospholipids, lysophosphatidylcholine (LPC), PAF, and lysoPAF(LPAF). A mechanistic relationship between the anti-oxidative and membrane-stabilizing properties of propranolol was suggested by the observation that pretreatment of neutrophils with LPC or PAF eliminated the inhibitory effects of the drug on superoxide generation by PMA-activated neutrophils. Dexpropranolol, a stereoisomer with minimal beta-blocking activity, and propranolol were equally effective with respect to their membrane-stabilizing and anti-oxidative interactions with neutrophils, but several other beta-blocking agents (atenolol, metoprolol, sotalol, and timolol) did not possess these activities. Inhibition of oxidant generation is, therefore, not a common property of beta-blocking agents and, in the case of propranolol, appears to occur as a consequence of membrane-stabilization rather than by beta-receptor-directed effects.

Anti-inflammatory, membrane-stabilizing interactions of salmeterol with human neutrophils in vitro.

1. We have investigated the effects of salmeterol (0.3-50 microM) on several pro-inflammatory activities of human neutrophils in vitro. 2. Oxidant production by FMLP- and calcium ionophore (A23187)-activated neutrophils was particularly sensitive to inhibition by low concentrations (0.3-3 microM) of salmeterol, while the responses of phorbol myristate acetate- and opsonised zymosan-stimulated cells were affected only by higher concentrations (3-50 microM) of the drug. At these concentrations salmeterol is not cytotoxic, nor does it act as a scavenger of superoxide. 3. These anti-oxidative interactions of salmeterol with neutrophils were insensitive to propranolol but could be eliminated by washing the cells, or by pretreatment with low concentrations (1-2 microM) of the pro-oxidative, membrane-destabilizing phospholipids, lysophosphatidylcholine (LPC), platelet activating factor (PAF) and lysoPAF (LPAF). 4. At concentrations of 6.25-50 microM salmeterol interfered with several other activities of stimulated neutrophils, including intracellular calcium fluxes, phospholipase A2 activity and synthesis of PAF. 5. In an assay of membrane-stabilizing activity, salmeterol (25 and 50 microM) neutralized the haemolytic action of LPC, PAF and LPAF. 6. Of the other commonly used beta 2-adrenoceptor agonists, fenoterol, and formoterol, but not salbutamol, caused moderate inhibition of neutrophil oxidant generation by a superoxide-scavenging mechanism. However, unlike salmeterol, these agents possessed only weak membrane stabilizing properties. 7. We conclude that salmeterol antagonizes the pro-inflammatory, pro-oxidative activity of several bioactive lipids implicated in the pathogenesis of bronchial asthma, by a mechanism related to the membrane-stabilizing, rather than to the beta 2-agonist properties of this agent.