Faculty Bibliography

Nonsteroidal antiinflammatory drugs (NSAID) are thought to act in part by inhibiting prostaglandin H (PGH) synthase which diminishes release of inflammatory prostaglandins (PG). Paradoxically, PG of the E series also have antiinflammatory properties. We therefore studied the combined effects of NSAID and PGE1 on neutrophil activation. Incubation of neutrophils with a PGE1 analog, misoprostol (miso; 1 microM; 5 min, 37 degrees), reduced superoxide anion generation in response to the chemoattractant fmet-leu-phe (FMLP) to 70.7 +/- 7% of control (p less than 0.01). Piroxicam (10 microM) independently reduced FMLP dependent superoxide anion generation to 63.7 +/- 7.4% (p less than 0.01) of control. Addition of miso to piroxicam reduced superoxide anion production to 37.4 +/- 1.9%, an inhibition that exceeded that observed with either drug alone. Similarly, the addition of miso enhanced the inhibitory effects of indomethacin and sodium salicylate on superoxide anion generation, and of all 3 NSAID on other neutrophil functions (degranulation, aggregation and global rises in cytosolic calcium). Miso (1 microM) and NSAID, alone or in combination, did not inhibit superoxide anion generation in response to the calcium ionophore A23187 or phorbol myristate acetate, agents that bypass G protein depending signaling pathways, and that do not induce a rise in cytosolic cyclic AMP (cAMP). Therefore, our data clearly show that miso at micromolar concentrations, augments the inhibitory effects of NSAID on neutrophil activation via a mechanism dependent upon signal transduction across the plasma membrane

Arachidonic acid (20:4) and other cis-unsaturated fatty acids exert direct effects on a variety of cells, effects that do not depend on the metabolism of fatty acids via cyclooxygenase or lipoxygenase pathways. In these studies arachidonic acid and other cis-unsaturated fatty acids (but not trans-unsaturated or saturated fatty acids) increased the specific binding of the nonhydrolyzable analog of GTP, [35S]GTP gamma S, to purified neutrophil membrane preparations and elicited superoxide anion generation from intact neutrophils. There was a positive correlation (r = 0.70) between the capacity of fatty acids to increase nucleotide binding and to elicit the respiratory burst. Scatchard plot analysis of binding at equilibrium demonstrated an increase in the number of available GTP binding sites in the presence of 50 microM arachidonic acid. Nonsteroidal antiinflammatory agents interfered with the arachidonic acid effect on [35S]GTP gamma S binding. ADP-ribosylation of the pertussis toxin substrate Gi alpha within the plasmalemma-reduced specific [35S]GTP gamma S binding and blocked arachidonate-dependent enhancement of binding. Moreover, pertussis toxin treatment of intact neutrophils inhibited arachidonic acid-induced superoxide anion generation. The data indicate that arachidonic acid directly activates a GTP binding protein in the neutrophil plasma membrane and may thereby act as a second messenger in signal transduction

OBJECTIVE: To determine the frequency of unexplained reversible hypoxemia in patients with systemic lupus erythematosus and to assess the relation between hypoxemia and elevated plasma levels of complement split products. DESIGN: Cohort study. SETTING: Inpatient and outpatient facilities of the New York University Medical Center/Bellevue Hospital and the Hospital for Joint Diseases. PATIENTS: Case patients were 22 patients hospitalized with disease exacerbation and no evidence of parenchymal lung disease on chest roentgenogram. Four patients with stable disease were followed in the outpatient clinic, and five healthy normal volunteers served as controls. MEASUREMENTS: Plasma levels of complement split products (C3a, factor Bb fragment), alveolar-arterial (A-a) Po2 gradients, and pulmonary function were measured. MAIN RESULTS: Nine episodes of hypoxemia or hypocapnia (mean A-a gradient, 30.4 +/- 4.8 mm Hg) or both (despite normal chest roentgenogram results) were noted in six hospitalized patients (group 1). Gas exchange improved within 72 hours of steroid therapy (mean A-a gradient, 11.6 +/- 4.3 mm Hg; P less than 0.01). These patients had an elevated initial mean C3a level (938.4 +/- 246.8 ng/mL) that decreased within 72 hours (407.8 +/- 80.9 ng/mL; P less than 0.01), concomitant with improved oxygenation. Ventilation-perfusion scans, obtained for four of six group 1 patients, excluded pulmonary emboli. Four hospitalized patients (group 2) had a normal A-a gradient (mean, 7.5 +/- 2.7 mm Hg). The mean C3a level of this group (358.3 +/- 39.2 ng/mL) was lower than that of group 1 (P less than 0.05). Four patients with stable disease (group 3) had a mean A-a gradient and a mean C3a level of 3.3 +/- 2.7 mm Hg and 237.8 +/- 105.7 ng/mL, respectively, similar to values found in five normal volunteers, in whom the mean A-a gradient was 3.7 +/- 1.7 mm Hg and the mean C3a level was 124.8 +/- 9.2 ng/mL. CONCLUSION: A syndrome of reversible hypoxemia, unassociated with parenchymal lung disease, is unexpectedly common in acutely ill, hospitalized patients with systemic lupus erythematosus. The pathogenesis of this syndrome is unclear, although the data are compatible with the hypothesis that hypoxemia may be related to pulmonary leukoaggregation

Sodium salicylate and other non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neutrophil functions via unknown mechanisms. To examine their site of action in the neutrophil we have studied discrete events within the plasma membrane which depend upon the normal function of a GTP binding protein (G protein). We demonstrated that sodium salicylate and piroxicam inhibit neutrophil activation in response to stimuli which require signal transduction via a G protein (e.g. formyl-methionine-leucine-phenylalanine) but have no effect on stimuli which do not (e.g. phorbol myristate acetate, ionomycin). NSAIDs blocked the ADP-ribosylation of the pertussis toxin substrate in human neutrophils. This effect was associated with the capacity of NSAIDs to block pertussis toxin-dependent inhibition of neutrophil functions. Finally, NSAIDs inhibited the binding of GTP gamma S, a stable analog of GTP, to purified neutrophil membrane preparations. The data indicate that salicylate and other NSAIDs interact with a G protein in the neutrophil plasmalemma and thereby uncouple post-receptor signaling events

Nonsteroidal anti-inflammatory drugs have anti-inflammatory, analgesic, and antipyretic properties. Although most nonsteroidal anti-inflammatory drugs inhibit prostaglandin synthesis, these agents also have important pharmacologic actions unrelated to their effects on prostaglandins. Among these properties is the ability to inhibit the release of mediators of inflammation from neutrophils and macrophages. These effects are due to the ability of nonsteroidal anti-inflammatory drugs to intercalate into the lipid bilayer of the plasma membrane and thereby disrupt protein-protein and protein-lipid interactions critical for cell responses (eg, calcium translocations, membrane phospholipid turnover). Our data, for example, indicate that nonsteroidal anti-inflammatory drugs exert direct inhibitory actions at the regulatory GTP protein (G protein) within the plasma membrane. This exhibit examines the evidence for diverse mechanisms of action of nonsteroidal anti-inflammatory drugs including effects directed at the G protein as well as new evidence that nonsteroidal anti-inflammatory drugs regulate the expression of the cyclooxygenase enzyme at the level of gene transcription

Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments

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