Faculty Bibliography

Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases

Leukotriene B4 [LTB4, (5S,12R)-hydroxyeicosa-6,14-cis-8,10-trans-tetraenoic acid], a potent mediator of inflammation, is released from neutrophils by agonists that provoke degranulation of the cell. To examine whether degranulation is a necessary requirement for synthesis and metabolism of LTB4 (or of other arachidonate metabolites), we prepared neutrophil-derived cytoplasts (neutroplasts), organelle-depleted vesicles of cytoplasm surrounded by the plasma membrane. In the presence of extracellular Ca2+ with or without exogenous arachidonic acid (150 microM), neutroplasts were exposed to the Ca2+ ionophore A23187 (10 microM) and the resultant lipoxygenation products of arachidonate were determined. Neutrophils metabolize arachidonic acid to 5-HETE greater than 15-HETE greater than LTB4 greater than all-trans-LTB4 isomers. Neutroplast products of arachidonate lipoxygenation were 15-HETE greater than 5-HETE greater than LTB4 greater than all-trans-LTB4 isomers. Neutroplasts, like neutrophils, were capable of converting LTB4 into its 20-hydroxy and 20-carboxy metabolites. Finally, neutroplasts could utilize intrinsic arachidonate, since the neutroplasts synthesized LTB4 (30 pmol/mg of protein) in the absence of added arachidonic acid. The data demonstrate that neutrophil degranulation is not required for synthesis or metabolism of LTB4 by neutrophils

Current dogma holds that nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibition of the synthesis and release of prostaglandins. However, NSAIDs also inhibit the activation of neutrophils, which provoke inflammation by releasing products other than prostaglandins. We now report that NSAIDs (e.g., indomethacin, piroxicam) inhibit activation of neutrophils by inflammatory stimuli, such as C5-derived peptides and leukotriene B4, even when cyclooxygenase products generated in suspensions of stimulated neutrophils (prostaglandin E and thromboxanes) are present. Sodium salicylate (3 mM) greatly inhibited aggregation of neutrophils but had no effect on aggregation of platelets or production of thromboxane induced by arachidonate. Sodium salicylate and other NSAIDs also inhibit calcium movements (45Ca uptake, changes in fluorescence of chlortetracycline and quin-2). Aspirin, sodium salicylate, indomethacin, and piroxicam also enhanced the poststimulation increase in intracellular cyclic AMP. NSAIDs therefore inhibit early steps in neutrophil activation as reflected by their capacity to inhibit movements of Ca and to enhance intracellular levels of cyclic AMP

Episodes of fever, serositis, and arthritis in familial Mediterranean fever (FMF) suggested circulating mediators of acute inflammation (e.g., neutrophil activation). The mean serum neutrophil-aggregating activity of 51 FMF patients was 2.5 +/- 0.2 cm2/min, compared to 1.0 +/- 0.1 cm2/min in 20 normal controls (P less than 0.0002). Lipid extracts of FMF sera retained neutrophil-aggregating activity and had UV absorbance peaks at 269 and 279 nm, indicating the presence of lipids with a conjugated triene structure. Chromatography of extracts yielded peaks that were coeluted with reference dihydroxyicosatetraenoic acids, had UV absorbance peaks at 259, 269, and 279 nm, and possessed neutrophil-aggregating activity. The presence of leukotriene B4 was excluded by chromatography following methyl-esterification. Monohydroxy compounds identified in FMF extracts by gas chromatography/mass spectrometry included 5-hydroxyicosatetraenoic acid, and 9- and 13-hydroxyoctadecadienoic acids. Hydroxy acids were present in 19 of 31 FMF sera and absent in extracts of sera from 8 patients with active systemic lupus erythematosus, 7 with fever from infection, and 12 normal controls. The finding of circulating mono- and dihydroxy fatty acids in FMF suggests that defects in the formation or elimination of these compounds might play a role in the pathogenesis of FMF.

Dissociated cells of the marine sponge, Microciona prolifera, aggregate in response to a species-specific aggregation factor (MAF) and Ca ionophores. We now report that leukotriene B4, a 5-lipoxygenase product, also causes aggregation of sponge cells. No other lipoxygenase products provoked aggregation. However, nordihydroguaiaretic acid (NDGA), colchicine, indomethacin, piroxicam and ibuprofen inhibited MAF-induced aggregation; the latter three agents inhibited Ca movements. Inhibition of prostaglandin release cannot be the mechanism whereby these agents inhibit aggregation of sponge cells, since the cells do not respond to exogenous cyclooxygenase products