5-Oxo-eicosanoids and Hematopoietic Cytokines Cooperate in Stimulating Neutrophil Function and the Mitogen-activated Protein Kinase Pathway

Abstract

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B 4 (LTB 4 ). All three eicosanoids stimulate Ca 2؉ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB 4 , 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A 2 (cPLA 2 ). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB 4 , GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA 2 , as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [ 14 C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca 2؉ depletion, inhibited MAPK responses to 5-oxoETE and LTB 4 , indicating that responses to both agents are coupled through G proteins but not dependent upon Ca 2؉ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3-and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB 4 antagonist, blocked the responses to LTB 4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB 4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB 4 . The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.

Expression of the antiapoptotic MCL1 gene product is regulated by a mitogen activated protein kinase-mediated pathway triggered through microtubule disruption and protein kinase C

“…It therefore seemed likely that exposure to TPA, and possibly also the microtubule-disrupting agents, might result in an increase in ERK phosphorylation in ML-1 cells. To test this possibility, an antibody speci®c for the phosphorylated forms of ERK1 and -2 was used (Kuo et al, 1996;O'Flaherty et al, 1996). Total ERK1/2 protein levels were monitored in parallel with an antibody that recognizes both the phosphorylated and non-phosphorylated forms.…”

Oxidative Stress Stimulates the Synthesis of the Eosinophil Chemoattractant 5-Oxo-6,8,11,14-eicosatetraenoic Acid by Inflammatory Cells

“…However, tBuOOH (100 M) stimulated the formation of 5-oxo-ETE by about 3-fold (p Ͻ 0.01), and reduced the amount of 5-HETE detected (p Ͻ 0.05). In contrast, tBuOOH had little effect on the formation of LTB 4 by PBMC. DISCUSSION U937 cells served as an excellent model to investigate the effects of oxidative stress on the formation of 5-oxo-ETE.…”

“…This demonstrates that oxidative stress can stimulate 5-oxo-ETE formation not only from exogenous 5-HETE, but also from 5-HETE generated by 5-lipoxygenase. The effect of tBuOOH was specific for 5-oxo-ETE, as it had no effect on the formation of the related 5-lipoxygenase product LTB 4 .…”

“…To determine whether oxidative stress could also stimulate the formation of 5-oxo-ETE when the substrate is provided by 5-lipoxygenase rather than being added directly, we investigated the effects of tBuOOH on the formation of 5-lipoxygenase products by PBMC stimulated with A23187 (5 M) in the presence of arachidonic acid (20 M). Under these conditions the formation of 5-lipoxygenase products is dependent on the synthesis of 5-HETE and LTA 4 by monocytes, and their conversion to 5-oxo-ETE and LTB 4 by both monocytes and lymphocytes. The major 5-lipoxygenase product formed after stimulation of PBMC with A23187 and arachidonic acid was 5-HETE (Fig.…”

5-Oxo-ETE regulates tone of guinea pig airway smooth muscle via activation of Ca2+pools and Rho-kinase pathway

“…2D, a and b), is further supported by the inhibitory effect of 2-APB, which has been presented as an inhibitor of inositol 1,4,5-trisphosphate (InsP 3 )-activated Ca 2ϩ release channels (23,24). Despite the fact that 2-APB was reported to interfere with nonselective channels and other transmembrane processes (26), addition of 2-APB alone resulted in an initial transient increase in ASM tension, possibly due to the activation of PLC␥ (20).…”