Chemotactic peptide induces cAMP elevation in human neutrophils by amplification of the adenylate cyclase response to endogenously produced adenosine

The transient increase in human neutrophil cAMP levels induced by the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) is shown to be caused by amplification of adenylate cyclase response to endogenously produced adenosine. The FMLP-stimulated increase in neutrophil cAMP was potentiated markedly by a nonmethylxanthine cAMP phosphodiesterase inhibitor (Ro 20-1724). By inhibiting the degradation of newly formed cAMP, Ro 20-1724 rendered the FMLP-induced cAMP elevation persistent rather than transient. The role of endogenously produced adenosine in this phenomenon is demonstrated by the ability of either adenosine deaminase or theophylline, an adenosine receptor antagonist, to prevent FMLP-stimulated cAMP elevation. The general nature of the FMLP-potentiated cAMP response is indicated by the finding that FMLP-treated neutrophils, in the presence of exogenously supplied adenosine deaminase, exhibited augmented cAMP generation in response to three different types of receptor agonists: 2-chloroadenosine, prostaglandin E1, and L-isoproterenol. Moreover, like the neutrophil cAMP increase caused by FMLP alone, the ability of FMLP to augment cAMP response to 2-chloroadenosine in adenosine deaminase-treated cells was short-lived and declined after 1.0 min of exposure to FMLP. Preincubation of neutrophil suspensions with the adenylate cyclase inhibitor SQ 22,536 completely prevented FMLP-induced cAMP generation. Furthermore, when neutrophil suspensions were preincubated with concentrations of Ro 20-1724, which apparently maximally inhibit cAMP phosphodiesterase, a 30-s incubation with FMLP still resulted in substantially elevated cAMP levels. It therefore appears that FMLP raises cAMP by activating adenylate cyclase rather than inhibiting cAMP phosphodiesterase.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or changing the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5'-(N-ethylcarboxamido)adenosine (NECA, 10 microM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 +/- 0.02 (S.E.) to 1.80 +/- 0.02 (p less than 0.001) while inosine (10 microM), a poor adenosine receptor agonist, had no effect (1.73 +/- 0.04, p = n.s. vs. control, p less than 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA greater than adenosine = N6-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 microM) did not significantly change the number or affinity of [3H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Neutrophil adherence to endothelium is enhanced via adenosine A1 receptors and inhibited via adenosine A2 receptors.

We have recently demonstrated that human neutrophils (PMN) possess two different classes of adenosine receptors (A1 and A2) that, when occupied, promote chemotaxis and inhibit the generation of reactive oxygen species (e.g., O2- and H2O2), respectively. We have previously demonstrated that adenosine protects endothelial cells (EC) from injury by stimulated neutrophils (PMN) both by diminishing generation of H2O2 and inhibiting adherence of PMN to EC. We therefore determined whether occupancy of A1 or A2 adenosine receptors regulated adherence of PMN to EC. At concentrations similar to those required to inhibit release of O2- by ligation of A2 receptors, both adenosine (IC50 = 56 nM) and 5'N-ethylcarboxamidoadenosine (NECA, IC50 = 8 nM), the most potent A2 agonist, inhibited adherence to EC by stimulated PMN (FMLP, 0.1 microM). In direct contrast, the specific A1 agonists N6-phenylisopropyladenosine and N6-cyclopentyladenosine (CPA) promoted PMN adherence to EC at concentrations of 1-100 nM. To further investigate the mechanisms by which adenosine receptor agonists affected the adherence of stimulated PMN we examined the effect of NECA (A2) and CPA (A1) on the adherence of PMN to fibrinogen (a ligand for the beta 2 integrin CD11b/CD18) and to gelatin. In a dose-dependent manner (IC50 = 2 nM), NECA inhibited the adherence of FMLP-treated PMN to fibrinogen- but not gelatin-coated plates. In contrast, CPA (A1) promoted adherence of stimulated PMN to gelatin-(EC50 = 13 pM) but not fibrinogen-coated plates. Theophylline (10 microM), an adenosine receptor antagonist, reversed the inhibition by NECA (0.3 microM) of stimulated neutrophil adherence to fibrinogen. These observations not only confirm the presence of A1 and A2 receptors on PMN but also suggest two opposing roles for adenosine in inflammation. Occupancy of A1 receptors promotes neutrophil adherence to endothelium and chemotaxis (a proinflammatory role) whereas occupancy of A2 receptors inhibits adherence and generation of toxic oxygen metabolites (an antiinflammatory role).     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Regulation of human neutrophil functions by adenine nucleotides

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.     

Divergent effects of propranolol on neutrophil superoxide release: involvement of phosphatidic acid and diacylglycerol as second messengers.

Relatively high levels of propranolol (170 microM) markedly attenuated the generation of 1,2 diacylglycerol in neutrophils stimulated with either FMLP plus cytochalasin B or with 20.0 mM NaF. This effect resulted from inhibition of phosphatidic acid phosphohydrolase as it was accompanied by a corresponding increase in the recovery of phosphatidic acid in organic extracts of stimulated cells. Although propranolol enhanced phosphatidic acid levels in neutrophils treated with FMLP alone, the drug had only a slight inhibitory influence on diglyceride generation in these cells. The effect of propranolol on enhancement of PA levels in neutrophils treated with FMLP alone strongly correlated with enhancement of FMLP-induced O2- generation. However, propranolol induced a similar dose-dependent inhibition of O2- generation in neutrophils stimulated with either FMLP + cytochalasin B or with 20.0 mM NaF. These results are consistent with the hypothesis that both phosphatidic acid and diacylglycerol are required for optimal initiation of neutrophil O2- release.     

Adenosine; a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on human neutrophils

Adenosine specifically inhibits superoxide anion generation by N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils without affecting either degranulation or "aggregation." We present data that also supports the hypothesis that adenosine engages a specific cell surface receptor to mediate inhibition of stimulated neutrophils. Theophylline (10 and 100 mu M), a competitive antagonist at adenosine receptors, reversed the effects of adenosine (0.1 mu M) on superoxide anion generation by stimulated neutrophils. The adenosine analogue 5'N-ethylcarboxamidoadenosine (NECA) was a more potent inhibitor of superoxide anion generation than either N6-phenylisopropyladenosine (PIA) or adenosine, an order of potency consistent with that previously demonstrated for adenosine A2 receptors. 2-Chloroadenosine inhibited superoxide anion generation at concentrations similar to NECA. [3H]-NECA and [3H]-2-chloroadenosine bound to a single receptor on intact neutrophils. The characteristics of the receptors for [3H]-NECA and [3H]-2-chloroadenosine were similar (Kd = 0.22 and 0.23 mu M, respectively; number of binding sites = 9.31 and 11.1 X 10(3) sites/cell, respectively). NECA, 2-chloroadenosine, adenosine, and PIA inhibited binding of [3H]-NECA with a rank order similar to that for inhibition of superoxide anion generation (NECA = 2-chloroadenosine greater than adenosine greater than PIA). There was 50% inhibition of superoxide anion generation by NECA at approximately 20% receptor occupancy. Adenosine, derived from damaged tissues, may serve as a specific, endogenous modulator of superoxide anion generation by activated neutrophils through interaction at this newly described receptor on human neutrophils.     

Adenosine inhibits divalent cation influx across human neutrophil plasma membrane via surface adenosine A2 receptors.

Adenosine and its analogues inhibited increases in divalent cation influx stimulated by platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (FMLP) in a dose-dependent fashion. This effect was antagonized by theophylline, an adenosine receptor antagonist. When extracellular adenosine was removed by adenosine deaminase, the effect of adenosine was completely abolished. Two adenosine analogues with different affinities for adenosine receptor subtypes, 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyladenosine (PIA), also inhibited divalent cation influx, NECA being more potent than PIA. These results suggest that adenosine and its analogues inhibit divalent cation influx across neutrophil plasma membranes via surface adenosine A2 receptors. Adenosine had little effect on the initial peaks of intracellular free calcium rises induced by chemoattractants, but it inhibited the subsequent rise in free calcium. Since calcium influx through the divalent cation channels or neutrophil plasma membranes is responsible for maintaining free calcium concentration following the initial peaks, we suggest that adenosine modulates neutrophil function by interfering with this calcium influx.     

Effect of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) on human neutrophil function: novel inhibition of neutrophil stimulus-response coupling?

Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.     

Inhibition of neutrophil priming and tyrosyl phosphorylation by cepharanthine, a nonsteroidal antiinflammatory drug.

Receptor-mediated superoxide (O2.-)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimura, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism of O2.- generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated O2.- generation, on the priming of human peripheral neutrophils (HPPMN) was studied. Both enhancement of formyl-methionyl-leucyl- phenylalanine (FMLP)-mediated O2.- generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated O2.- generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.     

The antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action

Insulin antagonized the lipolytic actions of epinephrine in rat epididymal adipocytes when the phosphodiesterase inhibitor, Ro 20-1724, was present. Adipocytes were depleted of functional cAMP by inhibiting adenylate cyclase with N6-phenylisopropyladenosine in the presence of adenosine deaminase such that Ro 20-1724 no longer stimulated lipolysis. The cAMP analogs 8-thioisopropyl-cAMP or 8-thiomethyl-cAMP, which are resistant to phosphodiesterase hydrolysis, were subsequently added to bypass adenylate cyclase and phosphodiesterase action. Under these conditions, insulin antagonized the lipolytic effects of these analogs, even in the presence of Ro 20-1724.     

Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells

The effects of adenosine and two analogs, L-phenylisopropyladenosine (L-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet-derived growth factor (PDGF)-stimulated initiation of DNA synthesis in growth-arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose-dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA greater than adenosine greater than L-PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8-phenyltheophylline (8-PT). The stimulatory effect of adenosine was enhanced by 3 microM dipyridamole an adenosine-uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8-PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L-PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin-stimulated increase in cAMP was inhibited by L-PIA at nanomolar concentrations. L-PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1- and A2-subtype on SM-mediating bidirectional changes of cAMP and DNA synthesis.     

A role for soluble cAMP phosphodiesterases in differentiation of 3T3-L1 adipocytes

Differentiation of 3T3-L1 adipocytes, monitored by accumulation of neutral lipid and by increase in alpha-glycerophosphate dehydrogenase activity, is accelerated by incubation of confluent 3T3-L1 fibroblasts in media containing insulin, dexamethasone and isobutylmethylxantine (IBMX). IBMX inhibits cyclic nucleotide phosphodiesterases as well as the binding of adenosine to its receptor. Agents with relatively specific effects were utilized to examine the role of IBMX in differentiation. Ro 20-1724, a selective inhibitor of soluble cAMP phosphodiesterase activities, was as effective as IBMX in increasing alpha-glycerophosphate dehydrogenase activity and fat deposition. Neither cilostamide, which inhibits particulate but not soluble cAMP phosphodiesterase activities, 8-phenyltheophylline, an adenosine receptor antagonist with little inhibitory effect on phosphodiesterase activities, nor N6-(R phenyl-isopropyl) adenosine (PIA), a potent adenosine receptor agonist, were effective in promoting differentiation. In addition, we find that maximal increases in alpha-glycerophosphate dehydrogenase activity and lipid accumulation were observed when differentiation was initiated in the presence of 10 nM dexamethasone. These data suggest that inhibition of soluble cAMP phosphodiesterase activity and subsequent alterations in cAMP may play an important role in the mechanism whereby IBMX enhances differentiation of 3T3-L1 cells.     

Inhibition of prostaglandin E2-stimulated cAMP accumulation by lipopolysaccharide in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages with 100 nM prostaglandin E2 (PGE2) produced a rapid biphasic increase in intracellular cAMP that was maximal at 1 min and sustained through 20 min. Pretreatment of macrophages with 100 ng/ml of lipopolysaccharide (LPS) for 60 min prior to PGE2 decreased the magnitude of cAMP elevation by 50%, accelerated the decrease of cAMP to basal levels, and abolished the sustained phase of cAMP elevation. The effect of LPS was concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in the presence of 5% fetal calf serum and at 1 microgram/ml in the absence of fetal calf serum. LPS also inhibited cAMP accumulation in cells treated with 100 microM forskolin, but the decrease was about half that seen in cells treated with PGE2. LPS concentrations that inhibited cAMP accumulation produced a 30% increase in soluble low Km cAMP phosphodiesterase activity while having no effect on particulate phosphodiesterase activity. The nonspecific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram and Ro-20-1724 were effective in inhibiting soluble phosphodiesterase activity in vitro, producing synergistic elevation of cAMP in PGE2-treated cells, and blocking the ability of LPS to inhibit accumulation of cAMP. Separation of the phosphodiesterase isoforms in the soluble fraction by DEAE chromatography indicated that LPS activated a low Km cAMP phosphodiesterase. The enzyme(s) present in this peak could be activated 6-fold by cGMP and were potently inhibited by low micromolar concentrations of Ro-20-1724 and rolipram. Using both membranes from LPS-treated cells and membranes incubated with LPS, no decrease in adenylylcyclase activity could be attributed to LPS. Although effects of LPS on the rate of synthesis of cAMP cannot be excluded, the present evidence is most consistent with a role for phosphodiesterase activation in the inhibitory effects of LPS on cAMP accumulation in murine peritoneal macrophages.     

Cocaine and its derivatives blunt neutrophil functions without influencing phosphorylation of a 47-kilodalton component of the reduced nicotinamide-adenine dinucleotide phosphate oxidase

Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses.     

Differential inhibition of human neutrophil activation by cyclosporins A, D, and H. Cyclosporin H is a potent and effective inhibitor of formyl peptide-induced superoxide formation.

Cyclosporin (Cs)A but not CsH inhibits activation of human lymphocytes. We studied the effects of CsA, CsD, and CsH on human neutrophil activation induced by chemoattractants and by various substances that circumvent receptor stimulation. CsH inhibited superoxide (O2-) formation induced by the chemotactic peptide, FMLP (30 nM), with a half-maximal effect at 40 nM. O2- formation was abolished by CsH at 1 microM. CsH increased the concentration of FMLP causing half-maximal activation of O2- formation from 30 nM to 0.8 microM and substantially reduced the stimulatory effect of FMLP at supra-maximally effective concentrations. The inhibitory effect of CsH on O2- formation was evident immediately after addition to neutrophils. CsH also markedly inhibited the increase in cytosolic Ca2+ ([Ca2+]i), beta-glucuronidase, and lysozyme release and aggregation stimulated by FMLP. CsA and CsD were considerably less effective than CsH to inhibit FMLP-induced O2- formation. CsA and CsD were without effect on exocytosis, rises in [Ca2+]i, and aggregation induced by the chemotactic peptide. Cyclosporines inhibited FMLP-induced O2- formation in an additive manner, indicating that they acted through a mechanism they had in common. Cyclosporines only slightly inhibited O2- formation and lysozyme release induced by C5a. Aggregation and rises in [Ca2+]i stimulated by C5a were not affected by cyclosporines, and they did not inhibit O2- formation and exocytosis induced by platelet-activating factor and leukotriene B4. Cyclosporines partially inhibited O2- formations induced by NaF and gamma-hexachlorocyclohexane. CsA marginally inhibited PMA-induced O2- formation and lysozyme release. CsA, CsD, and CsH did not inhibit arachidonic acid-induced O2- formation and its potentiation by NaF or stable guanine nucleotides in a cell-free system from DMSO-differentiated HL-60 cells. CsH partially inhibited binding of FML [3H]P to formyl peptide receptors in membranes from DMSO- or dibutyryl cAMP-differentiated HL-60 cells. Our data show that: 1) cyclosporines differentially inhibit activation of human neutrophils; and 2) CsH is, indeed, not immunologically inactive but is a potent and effective inhibitor of FMLP-induced O2- formation. 3) CsH interferes with agonist binding to formyl peptide receptors and in addition, cyclosporines may also act at sites distal to chemoattractant receptors.     

Lysophosphatidylcholine modulates neutrophil oxidant production through elevation of cyclic AMP

Lysophosphatidylcholine (LPC) is an oxidized phospholipid present in micromolar concentrations in blood and inflamed tissues. The effects of LPC on neutrophil functions remain incompletely understood, because conflicting reports exist for its stimulatory and inhibitory roles. We report in this study that LPC inhibits superoxide generation in fMLP- and PMA-stimulated neutrophils without affecting fMLP-induced Ca(2+) mobilization and cell viability. This effect was observed with LPC dissolved in ethanol, but not with LPC stock solutions prepared in water or in BSA-containing aqueous solution with sonication. Under the same experimental conditions, platelet-activating factor primed neutrophils for superoxide generation. The inhibitory effect of LPC was observed within 30 s after its application and was maximal at LPC concentrations between 0.1 and 1 muM. Inhibition of superoxide generation was accompanied by a 2.5-fold increase in the intracellular cAMP concentration. In addition, LPC reduced fMLP-stimulated phosphorylation of ERK and Akt and membrane translocation of p67(phox) and p47(phox). The protein kinase A inhibitors H-89 and adenosine 3'5'-cyclic monophosphorothioate Rp-isomer (Rp-cAMP) partially restored superoxide production in LPC-treated neutrophils, indicating involvement of protein kinase A in LPC-mediated inhibition. Using an ex vivo mouse lung perfusion model that measures lung weight change and capillary filtration coefficient, we found that LPC prevented lung vascular injury mediated by fMLP-activated neutrophils. Taken together, these results suggest that LPC-induced elevation of intracellular cAMP is partially responsible for its inhibition of neutrophil NADPH oxidase activation. A similar mechanism of inhibition may be used for the control of neutrophil-mediated tissue injury.     

Effect of cetiedil on the superoxide-generating system of porcine neutrophils

Cetiedil, alpha-cyclohexyl-3-thiopheneacetic acid 2-(hexahydro-1H-azepin-1-yl)-ethyl ester, was found to inhibit the generation of superoxide (O2-) by porcine neutrophils exposed to various stimulators. The concentration of cetiedil required for 50% inhibition was about 45 microM when neutrophils were stimulated by phorbol myristate acetate. Cetiedil not only decreased the rate of generation of O2-, but prolonged the lag time prior to the production of O2-. The inhibitory effect of cetiedil on the O2(-)-generating activity of the NADPH oxidase in the membrane vesicles was less than that on whole cells; the concentration of cetiedil necessary for 50% inhibition was about 250 microM. To study the mechanism of cetiedil's effect on the membrane, the transmembrane potential of neutrophils and the intracellular free Ca2+ concentration were monitored by using fluorescence probes, diS-C3-(5), and quin-2, respectively. Cetiedil caused depolarization of the membrane potential and increased the intracellular free Ca2+. These results indicate that integrity of ionic distribution is necessary to activate the O2(-)-generating system of neutrophils.     

The antiinflammatory effects of adenosine receptor agonists on the carrageenan-induced pleural inflammatory response in rats

Adenosine and adenosine receptor agonists have a variety of inhibitory effects on the generation of inflammatory mediators by neutrophils and other cell types. In human neutrophils stimulated with the chemotactic peptide FMLP, adenosine agonists inhibit O2- generation and degranulation. Because these findings suggest that the agonists may have potential as antiinflammatory agents, several compounds were evaluated for effects on the exudative and cellular phases of carrageenan-induced pleural inflammation in rats. All of the agonists tested inhibited both parameters of the inflammatory response. Inhibition appeared to correlate better with binding to the A1 than to the A2 receptor and was reversible by a known adenosine receptor antagonist, 8-phenyltheophylline. In mechanistic studies, R-N-(1-methyl-2-phenylethyl)adenosine, a standard A1 selective agonist, reversed the drop in circulating neutrophil count that occurs after injection of carrageenan. These results suggest that the agonists may prevent cell emigration by inhibiting adhesion to the endothelium or diapedesis. In addition (R)-N-(1-methyl-2-phenylethyl)adenosine had weak inhibitory effects on superoxide production by FMLP-stimulated rat neutrophils. Control studies showed that the effects of the agonists were not the result of agonist-induced hypotension nor corticosterone production by the adrenal glands. These findings indicate that adenosine receptor agonists are effective new pharmacologic tools for the study of inflammatory processes.     

Nitrite generation and antioxidant effects during neutrophil apoptosis

Neutrophil apoptosis is important for the resolution of airway inflammation in a number of lung diseases. Inflammatory mediators, endogenous reactive oxygen and nitrogen species, and intracellular and extracellular antioxidants may all influence neutrophil apoptosis. This study investigated the involvement of these factors during apoptosis of neutrophils cultured in vitro. Neutrophils undergoing spontaneous apoptosis in culture as assessed by annexin V binding generated significant amounts of nitrite. Incubation with agonistic anti-Fas monoclonal antibody or tumor necrosis factor-alpha (TNF-alpha) enhanced neutrophil apoptosis at 6 h, although it decreased nitrite accumulation. Although granulocyte-macrophage colony-stimulating factor significantly reduced neutrophil apoptosis, this was also associated with decreased nitrite accumulation. In contrast, inhibition of apoptosis at 16 h by dibutyryl cyclic adenosine monophosphate was associated with increased nitrite accumulation. Exogenous glutathione (GSH) or N-acetylcysteine significantly enhanced neutrophil apoptosis at 6 h and stimulated the production of H(2)O(2), which may mediate apoptosis through intracellular hydroxyl radical production. Intracellular GSH concentrations decreased in neutrophils undergoing apoptosis, and this was more marked in neutrophils treated with anti-Fas or TNF-alpha. These results suggest a causal association between reduced endogenous nitric oxide production, reduced intracellular GSH, and Fas- and TNF-alpha-mediated neutrophil apoptosis, whereas enhanced neutrophil survival mediated by dibutyryl cyclic adenosine monophosphate is associated with increased nitrite generation and maintenance of intracellular GSH. The interaction of endogenous reactive oxygen species with extracellular antioxidants such as GSH could also contribute to the complex processes regulating neutrophil apoptosis and hence the resolution of inflammation in the lung.