5-OXO-EICOSANOIDS AND HEMATOPOIETIC CYTOKINES COOPERATE IN STIMULATING NEUTROPHIL FUNCTION AND THE MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY

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 Ca2+ transie nts 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 pre treated 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 ves icles from the cytosol to the plasmalemma, an effect not requiring cyt okine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated prot ein kinase (MAPK) and cytosolic phospholipase A(2) (cPLA(2)). PMN expr essed 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 prote ins' electrophoretic mobility and tyrosine phosphorylation of the MAPK s. However, the speed and duration of the MAPK response varied markedl y depending on the stimulus. 5-OxoETE caused a very rapid and transien t activation of MAPK. In contrast, the response to the cytokines was r ather slow and persistent. PMN pretreated with GM-CSF demonstrated a d ramatic increase in the extent of MAPK tyrosine phosphorylation and el ectrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxo ETE induced PMN to release some preincorporated [C-14]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 Ca2 + depletion, inhibited MAPK responses to 5-oxoETE and LTB(4), indicati ng that responses to both agents are coupled through G proteins but no t dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inacti ve while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potenc y than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB(4) antagonist, blocked the respons es 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 p otent activators of PMN that share some but not all activities with LT B(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.