Human Basophils Histamine Release or IL-4 Production from Not Regulate Leukotriene C4 Generation, But Extracellular Signal-Regulated Kinases

2 Human basophils secrete histamine and leukotriene C4 (LTC 4 ) in response to various stimuli, such as Ag and the bacterial product, FMLP. IgE-mediated stimulation also results in IL-4 secretion. However, the mechanisms of these three classes of secretion are unknown in human basophils. The activation of extracellular signal-regulated kinases (ERKs; ERK-1 and ERK-2) during IgE- and FMLP-mediated stimulation of human basophils was examined. Following FMLP stimulation, histamine release preceded phosphorylation of ERKs, whereas phosphorylation of cytosolic phospholipase A2 (cPLA 2 ), and arachidonic acid (AA) and LTC 4 release followed phosphorylation of ERKs. The phosphorylation of ERKs was transient, decreasing to baseline levels after 15 min. PD98059 (MEK inhibitor) inhibited the phosphorylation of ERKs and cPLA 2 without inhibition of several other tyrosine phosphorylation events, including phosphorylation of p38 MAPK. PD98059 also inhibited LTC 4 generation (IC 50 5 ; 2 m M), but not histamine release. Stimulation with anti-IgE Ab resulted in the phosphorylation of ERKs, which was kinetically similar to both histamine and LTC 4 release and decreased toward resting levels by 30 min. Similar to FMLP, PD98059 inhibited anti-IgE-mediated LTC 4 release (IC 50 , ; 2 m M), with only a modest effect on histamine release and IL-4 production at higher concentrations. Taken together, these results suggest that ERKs might selectively regulate the pathway leading to LTC 4 generation by phosphorylating cPLA 2 , but not histamine release or IL-4 production, in human basophils. The Journal of The phosphorylation of ERKs was assessed using three different tech- niques: 1) anti-phospho-ERK Ab (38), 2) anti-phosphotyrosine Ab (4G10) (39, 40), and 3) electrophoretic mobility shift, using anti-ERK-1 and anti- ERK-2 Abs (23, 39). The phosphorylation of p38 MAPK was assessed using anti-phospho-p38 MAPK Ab or anti-phosphotyrosine Ab (41, 42). The phosphorylation of cPLA 2 was assessed using electrophoretic mobility shift with anti-cPLA 2 Abs (16, 26). After stimulating basophils in PAGCM buffer, reactions were stopped by adding ice-cold PAG and were microfuged for 5–10 s. Cell pellets were immediately lysed in lysis buffer (20 mM Tris (pH 7.5), 2 mM EDTA, 2 mM EGTA, 5 mM DTT, 1 mM PMSF, 10 mM benzamidine, 100 m g/ml aprotinin, 200 m g/ml leupeptin, 50 mM NaF, 5 mM Na 4 P 2 O 7 , 1 mM Na 3 VO 4 , and 1% Nonidet P-40). Extracts containing equal basophil cell numbers (5 3 10 5 cell equivalents/lane) were diluted with an equal volume of 2 3 loading buffer and subjected to 10% Tris glycine gel (NOVEX). After electrophoresis (160 V and 1.5 h for detection of MAPKs phosphorylation, and 160 V and 3.5 h for cPLA 2 phosphorylation), proteins were transferred to nitrocellulose membranes (as described above). The membranes were immersed overnight in TBST containing 2% BSA (for detection by 4G10) or in TBST containing 5% nonfat dry skim-milk (Carnation) for the others. Immunoreactive proteins were detected using 4G10 in TBST containing 1% BSA or anti-ERK-1 Ab,