Elevated levels of cyclooxygenase-2 in antigen-stimulated mast cells is associated with minimal activation of p38 mitogen-activated protein kinase.

We have investigated possible factors that underlie changes in the production of eicosanoids after prolonged exposure of mast cells to Ag. Ag stimulation of cultured RBL-2H3 mast cells resulted in increased expression of cyclooxygenase (COX-2) protein and message. Other eicosanoid-related enzymes, namely COX-1, 5-lipoxygenase, and cytosolic phospholipase A(2) were not induced. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein (MAP) kinase preceded the induction of COX-2, whereas phosphatidylinositol 3' kinase and its substrate, Akt, were constitutively activated in RBL-2H3 cells. Studies with pharmacologic inhibitors indicated that of these kinases, only p38 MAP kinase regulated expression of COX-2. The induction of COX-2 was blocked by the p38 MAP kinase inhibitor SB202190, even when added 12-16 h after stimulation with Ag when p38 MAP kinase activity had returned to near basal, but still minimally elevated, levels. Interestingly, expression of COX-2 as well as cytosolic phospholipase A(2) and 5-lipoxygenase were markedly reduced by SB202190 in unstimulated cells. Collectively, the results imply that p38 MAP kinase regulates expression of eicosanoid-related enzymes, passively or actively, at very low levels of activity in RBL-2H3 cells. Also, comparison with published data suggest that different MAP kinases regulate induction of COX-2 in inflammatory cells of different and even similar phenotype and suggest caution in extrapolating results from one type of cell to another.

Induction of telomerase activity during development of human mast cells from peripheral blood CD34+ cells: comparisons with tumor mast-cell lines.

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.

Characteristics of arachidonic acid generation in human basophils: relationship between the effects of inhibitors of secretory phospholipase A2 activity and leukotriene C4 release.

In human basophils, degranulation stimulated by receptor activation or Ca++ ionophores is accompanied by an increase in free arachidonic acid (AA) as determined by gas chromatography negative ion chemical ionization mass spectrometry. Previous studies suggested that there was more than one pool of AA generated during stimulation and indirectly suggested that the leukotriene (LTC4) generated in these reactions was dependent on only one of these pools of AA. Our studies further examined these issues. Preliminary studies demonstrated discordance in the generation of free AA and LTC4 release. Treatment of basophils with triacsin C, a reacylation inhibitor, led to a marked increase in N-formyl-L-methionyl-L-leucyl-L-phenylalanine-(fMLP) stimulated free AA generation with no effect on LTC4 release. Similarly, incubation of basophils with recombinant human secretory phospholipase A2 (sPLA2), before and during fMLP stimulation, led to the generation of extremely high levels of free AA with no effect on LTC4 release. Pretreatment of basophils with anti-14 kDa phospholipase A2 monoclonal antibody (mAb 3F10) inhibited fMLP-induced synthesis of LTC4 but did not attenuate the mass of AA measured nor histamine release. Treating human basophils with zileuton (an inhibitor of 5-lipoxygenase) inhibited the stimulated synthesis of LTC4 and in combination with triacsin C increased the amount of observable AA by an amount approximately equal to the loss in LTC4 mass. Monoclonal antibody 3F10 blocked only the enhanced AA production caused by the combination of zileuton and triacsin C. Monoclonal antibody 3F10 did not inhibit the increases in free AA produced by pretreatment with triacsin C alone. These findings were supported by experiments using another relatively specific inhibitor of sPLA2, SB 203347. In all respects, SB 203347 mimicked the addition of mAb 3F10. Taken together, these data indicate that not all pools of AA are well used for LTC4 synthesis. These experiments also suggest that LTC4 synthesis in human basophils stimulated with fMLP depends on a SB 203347- and monoclonal antibody 3F10-inhibitable deacylation activity, presumably a sPLA2 acting at or near the cell surface. Furthermore, under normal conditions, this pool of AA is not observable because it is efficiently coupled to 5-lipoxygenase. Other deacylating enzymes, which do not supply AA for 5-lipoxygenase metabolism, also appear to be activated by fMLP and these other enzymes appear responsible for the net free AA normally observed after stimulation.

Quantitation of 1-stearoyl-2-arachidonoyl-sn-3-glycerol in human basophils via gas chromatography-negative ion chemical ionization mass spectrometry.

Investigation of the role of diacylglycerol molecular species in signal transduction in human basophils has been impeded by the lack of an assay method with adequate sensitivity and selectivity. Conversion of 1-stearoyl-2-arachidonoyl-sn-3-glycerol to the pentafluorobenzoyl ester conveys electron-capture properties to the diacylglycerol. The electron-capture derivative of the diacylglycerol is amenable to gas chromatographic analysis and undergoes limited fragmentation under negative ion mass spectrometric conditions with generation of an intense molecular anion at m/z 838. Monitoring m/z 838 for detection of 1-stearoyl-2-arachidonoyl-sn-3-glycerol and m/z 841 for detection of 1-trideuterostearoyl-3-arachidonoyl-sn-2-glycerol employed as the internal standard provides the analytical basis for GC-MS quantitation of the endogenous diacylglycerol in human basophils. The assay displays excellent reproducibility over a wide range of concentrations with variations < or = 10%. The GC-MS assay is highly selective and exquisitely sensitive with a detection limit of < or = 0.20 pg (approximately 30 fmol) for endogenous 1-stearoyl-2-arachidonoyl-sn-3-glycerol per injection. Approximately 400 fmol of the diacylglycerol were extracted from 10(5) stimulated human basophils.

Carbachol stimulation of triacylglycerol lipase activity in pancreatic acinar cells.

Carbachol (CCh) reduced the levels of [3H]arachidonic acid in triacylglycerol (TG) of pancreatic acinar cells. In cells prelabeled with [14C]glycerol, CCh reduced [14C]TG and increased [14C]diacylglycerol levels. Using [3H]triolein as exogenous substrate, CCh enhanced TG lipase activity 3-fold in a particulate fraction derived from intact acinar cells. These results portray a mechanism for generating diacylglycerol and arachidonic acid in exocrine pancreas involving agonist stimulation of TG hydrolysis.

Regulation of diacylglycerol levels in carbachol-stimulated pancreatic acinar cells: relationship to the breakdown of phosphatidylcholine and metabolism to phosphatidic acid.

Rat pancreatic acinar cells prelabeled with [14C]palmitic acid and then exposed to carbachol (CCh) exhibited a time-dependent increase in 1,2-[14C]diacylglycerol ([14C]DAG) levels, which was first detected at 2 min and then continued to rise in a linear manner. There was a concomitant increase in [14C]phosphatidic acid, which plateaued after 2 min and then remained at steady-state levels. CCh also promoted the release of phosphocholine, but not choline, within 60 s and caused a decrease in [14C]phosphatidylcholine in cells prelabeled with [14C]glycerol after 15 min. The inability to detect a rise in [14C]phosphatidylethanol accumulation and a fall in [14C]phosphatidate levels in [14C]palmitate prelabeled cells after exposure to CCh plus ethanol documented the absence of a phospholipase D-mediated pathway. The rapid phosphorylation of diglyceride in homogenates from unstimulated and carbachol-treated cells increased with increasing concentrations of exogenous substrate, thereby affirming that carbachol stimulates the phosphorylation of DAG by promoting the accumulation of the diglyceride. These collective findings provide evidence for the existence of an integrative control mechanism for regulating endogenous DAG levels during pancreatic acinar cell activation involving phosphatidylcholine-specific phospholipase C and DAG kinase.

Hepatic reticuloendothelial system activation in autoimmune mice: differences between (NZB X NZW)F1 and MRL-lpr/lpr strains.

5'-Nucleotidase (5'N) activity and Ia expression of hepatic nonparenchymal cells (NPCs) of the autoimmune (NZB X NZW)F1 and MRL-lpr/lpr and nonautoimmune C3H/FeJ, DBA/2J, and A/J strains were assayed to determine endogenous activation of the hepatic reticuloendothelial system (RES). Pretreatment of the nonautoimmune strains with Corynebacterium parvum resulted in decreases in Fc receptor expressor and 5'N and an increase in Ia expression of NPCs. Endogenous hepatic RES activation, as measured by low 5'N and high Ia expression, was observed in both the autoimmune MRL-lpr/lpr and (NZB X NZW)F1 strains even without C. parvum pretreatment. However, in the MRL-lpr/lpr strain, age is a dependent variable in activation and correlates with delayed clearance from the circulation of soluble IgG immune complexes. In the (NZB X NZW)F1 strain, the observation of decreased 5'-nucleotidase activity and increased percentage of Ia-positive cells at all ages suggests a primary abnormality. Therefore, different genetic or exogenous factors may be responsible for the activation of the hepatic RES in these autoimmune strains.

Abnormal binding of soluble IgG immune complexes to hepatic nonparenchymal cells of autoimmune mice.

Because the liver is the major organ responsible for removal of soluble immune complexes (IC), the surface binding characteristics of preformed model IC to unstimulated mouse liver nonparenchymal cells (NPC) in suspension were studied. NPC of non-autoimmune C3H/FeJ, C3H/HeJ, A/J, DBA/2 and the autoimmune NZB/W F1 and MRL/lpr female mice of various ages were isolated by perfusion of the portal vein with collagenase followed by separation of NPC from hepatocytes with a metrizamide gradient. Thirty-five percent of NPC of all mouse strains were nonspecific esterase-positive and phagocytosed latex beads. Radiolabeled mouse IgG anti-DNP covalently cross-linked stable IC were separated by gel filtration and bound to NPC under various conditions. Marked differences were noted in maximal number of IC bound per cell between the autoimmune and non-autoimmune mouse strains: 3.3 to 4.0 X 10(5) in the non-autoimmune strains vs 0.3 to 1.4 X 10(5) molecules of IC bound per cell in the autoimmune strains at 1 to 6 mo. Insignificant differences were noted in Ka by Scatchard plot analysis (3.5 to 5.0 X 10(8) M-1) and rate of reversibility of binding as determined by dissociation of surface-bound IC with an excess of heat-aggregated gamma-globulin (T 1/2:1.5 to 2 min). These data demonstrate a decreased number of available binding sites for IC in unstimulated NPC from NZB/W F1 and MRL/lpr female mice throughout their life spans. Although the findings are consistent with saturation of binding sites of the NPC with native IC, the abnormality found in the 1-mo-old autoimmune mice (who do not have detectable autoantibodies) suggests a primary defect in FC receptor expression or an altered state of activation of NPC that may contribute to the disease process.