Robert C. Murphy

Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils

The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils.

Cysteinyl-leukotrienes receptor activation in brain inflammatory reactions and cerebral edema formation: a role for transcellular biosynthesis of cysteinyl-leukotrienes

We studied the effect of intravascular activation of human neutrophils on the synthesis of cysteinyl leukotrienes (cysLT) and the formation of cerebral edema in guinea‐pig brains. Challenge with the chemotactic formylated tripeptide fMLP (0.1 µM) of neutrophil‐perfused brain in vitro resulted in blood‐brain barrier disruption associated with a significant increase of cysLT. Both events were completely prevented by neutrophil pretreatment with a specific 5‐lipoxygenase (5‐LO) inhibitor. Perfusion with the 5‐LO metabolite leukotriene B4 (10 nM), together with neutrophils treated with the 5‐LO inhibitor, did not restore the alteration in permeability observed upon perfusion with untreated and activated neutrophils. The dual cysLT1‐cysLT2 receptor antagonist BAYu9773 was more potent and more effective than a selective cysLT1 antagonist in preventing the brain permeability alteration induced by neutrophil activation. RT‐PCR showed significant expression of cysLT2 receptor mRNA in human umbilical vein endothelial cells. Intravital microscopy in mice showed that inhibition of leukotriene synthesis significantly reduced firm adhesion of neutrophils to cerebral vessels without affecting rolling. These data support the hypothesis that neutrophil and endothelial cells cooperate toward the local synthesis of cysLT within the brain vasculature and, acting via the cysLT2 receptor on endothelial cells, may represent a contributing pathogenic mechanism in the development of cerebral inflammation and edema.

Transcellular biosynthesis of cysteinyl leukotrienes in vivo during mouse peritoneal inflammation

Leukotrienes (LTs) are lipid mediators of inflammation formed by enzymatic oxidation of arachidonic acid. One intriguing aspect of LT production is transcellular biosynthesis: cells expressing 5-lipoxygenase (5LO) form LTA4 and transfer it to cells expressing LTA4 hydrolase (LTA4H) or LTC4 synthase (LTC4S) to produce LTB4 or LTC4. This process has been demonstrated in vivo for LTB4, but not for cysteinyl LTs (cysLTs). We examined transcellular cysLT synthesis during zymosan-induced peritonitis, using bone marrow transplants with transgenic mice deficient in key enzymes of LT synthesis and analyzing all eicosanoids by liquid chromatography/tandem mass spectrometry. WT mice time-dependently produced LTB4 and cysLTs (LTC4, LTD4, and LTE4). 5LO−/− mice were incapable of producing LTs. WT bone marrow cells restored this biosynthetic ability, but 5LO−/− bone marrow did not rescue LT synthesis in irradiated WT mice, demonstrating that bone marrow-derived cells are the ultimate source of all LTs in this model. Total levels of 5LO-derived products were comparable in LTA4H−/− and WT mice, but were reduced in LTC4S−/− animals. No differences in prostaglandin production were observed between these transgenic or chimeric mice. Bone marrow cells from LTA4H−/− or LTC4S−/− mice injected into 5LO−/− mice restored the ability to synthesize LTB4 and cysLTs, providing unequivocal evidence of efficient transcellular biosynthesis of cysLTs. These results highlight the potential relevance of transcellular exchange of LTA4 for the synthesis of LTs mediating biological activities during inflammatory events in vivo.

Differential Metabolism of Exogenous and Endogenous Arachidonic Acid in Human Neutrophils

Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A4(LTA4) by another and this can then be exported for conversion to LTB4 or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC4 synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA4 products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA4 that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA4products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB4by the LTA4 hydrolase. This result was not because of saturation of the LTA4 hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC4 synthase), the LTA4 synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA4 for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.

Dietary linoleate preserves cardiolipin and attenuates mitochondrial dysfunction in the failing rat heart

AIMS Cardiolipin (CL) is a tetra-acyl phospholipid that provides structural and functional support to several proteins in the inner mitochondrial membrane. The majority of CL in the healthy mammalian heart contains four linoleic acid acyl chains (L(4)CL). A selective loss of L(4)CL is associated with mitochondrial dysfunction and heart failure in humans and animal models. We examined whether supplementing the diet with linoleic acid would preserve cardiac L(4)CL and attenuate mitochondrial dysfunction and contractile failure in rats with hypertensive heart failure. METHODS AND RESULTS Male spontaneously hypertensive heart failure rats (21 months of age) were administered diets supplemented with high-linoleate safflower oil (HLSO) or lard (10% w/w; 28% kilocalorie fat) or without supplemental fat (control) for 4 weeks. HLSO preserved L(4)CL and total CL to 90% of non-failing levels (vs. 61-75% in control and lard groups), and attenuated 17-22% decreases in state 3 mitochondrial respiration observed in the control and lard groups (P < 0.05). Left ventricular fractional shortening was significantly higher in HLSO vs. control (33 ± 2 vs. 29 ± 2%, P < 0.05), while plasma insulin levels were lower (5.4 ± 1.1 vs. 9.1 ± 2.3 ng/mL; P < 0.05), with no significant effect of lard supplementation. HLSO also increased serum concentrations of several eicosanoid species compared with control and lard diets, but had no effect on plasma glucose or blood pressure. CONCLUSION Moderate consumption of HLSO preserves CL and mitochondrial function in the failing heart and may be a useful adjuvant therapy for this condition.

Biosynthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid from 5-hydroperoxyeicosatetraenoic acid in the murine macrophage

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of arachidonic acid shown to possess important biological activities within different cell types. In the neutrophil, a specific NADP+-dependent dehydrogenase utilizes 5-lipoxygenase-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) as the required substrate. In the present study, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HpETE), rather than 5-HETE, was found to be the biosynthetic precursor of 5-oxo-ETE in the murine macrophage. The macrophage was not able to convert 5-HETE into 5-oxo-ETE even when preincubated with phorbol ester or with other lipid hydroperoxides. The factor responsible for the conversion of 5-HpETE into 5-oxo-ETE was found predominantly in the cytosolic fraction of the macrophage, with an approximate molecular weight of 50,000–60,000, as assessed by size exclusion chromatography. Formation of 5-oxo-ETE was rapid and the catalytic protein was found to have an apparent K m of 5.3 μm for the eicosanoid. Furthermore, the protein could efficiently utilize 5(R,S)-HpETE as substrate and was heat and protease labile. This novel pathway of 5-oxo-ETE biosynthesis in the murine macrophage was consistent with reduction of a 5-hydroperoxy group to an intermediate alkoxy radical that could be subsequently oxidized to the 5-oxo product. Such a mechanism would enable racemic 5-HpETE, derived from free radical oxidation of arachidonic acid, to be efficiently converted into this potent chemotactic eicosanoid.

Pulmonary Surfactant Phosphatidylglycerol Inhibits Mycoplasma pneumoniae-stimulated Eicosanoid Production from Human and Mouse Macrophages

Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases. Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38MAPK and p44/42ERK1/2 MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD2 and PGE2 from RAW264.7 cells and thromboxane B2 (TXB2) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation of p44/42ERK1/2 or inactivation of cytosolic phospholipase A2α (cPLA2α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response.

Leukocytes regulate retinal capillary degeneration in the diabetic mouse via generation of leukotrienes

Understanding the early pathogenesis of DR may uncover new therapeutic targets to prevent or slow the progression of this sight‐threatening disorder. We investigated the role of leukocyte‐mediated generation of LTs in regulation of retinal capillary degeneration and inflammation in the diabetic mouse. We generated (1) chimeric mice that lacked the ability to generate LTs by transplanting 5LO−/− bone marrow cells into ND.WT mice and into SD.WT mice and (2) “control” chimeric mice by transplanting WT bone marrow cells into 5LO−/− mice or into WT mice. Retinas from diabetic chimeric mice with WT marrow demonstrated capillary degeneration to the same extent as retinas from diabetic, nonchimeric WT mice. In contrast, retinas from diabetic chimeric mice with 5LO−/− marrow developed significantly less capillary degeneration and pericyte loss (P<0.05). In the retinas from chimeric mice with WT marrow, diabetes induced a rise in leukocyte adherence to the microvasculature, expression of the NF‐κB p65 subunit, and ICAM1, superoxide generation, and retinal microvascular permeability, yet these characteristic responses were blunted by >50% in diabetic chimeras containing 5LO−/− leukocytes (P<0.05). Our data suggest the critical involvement of leukocytes and LTs in the regulation of inflammation and capillary degeneration in DR.

Lysophospholipid acyltransferases and eicosanoid biosynthesis in zebrafish myeloid cells.

Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation-reacylation cycle from a well-established, versatile vertebrate model species.

The Endocannabinoid Metabolite Prostaglandin E2 (PGE2)-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP Receptors

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine mediate an array of pro- and anti-inflammatory effects. These effects are related, in part, to their metabolism by eicosanoid biosynthetic enzymes. For example, N-arachidonoyl-ethanolamine and 2-arachidonoyl-glycerol can be metabolized by cyclooxygenase-2 into PG-ethanolamide (PG-EA) and PG-glycerol (PG-G), respectively. Although PGE2 is a recognized suppressor of neutrophil functions, the impact of cyclooxygenase-derived endocannabinoids such as PGE2-EA or PGE2-G on neutrophils is unknown. This study’s aim was to define the effects of these mediators on neutrophil functions and the underlying cellular mechanisms involved. We show that PGE2-G, but not PGE2-EA, inhibits leukotriene B4 biosynthesis, superoxide production, migration, and antimicrobial peptide release. The effects of PGE2-G were prevented by EP1/EP2 receptor antagonist AH-6809 but not the EP4 antagonist ONO-AE2-227. The effects of PGE2-G required its hydrolysis into PGE2, were not observed with the non-hydrolyzable PGE2-serinol amide, and were completely prevented by methyl-arachidonoyl-fluorophosphate and palmostatin B, and partially prevented by JZL184 and WWL113. Although we could detect six of the documented PG-G hydrolases in neutrophils by quantitative PCR, only ABHD12 and ABHD16A were detected by immunoblot. Our pharmacological data, combined with our protein expression data, did not allow us to pinpoint one PGE2-G lipase, and rather support the involvement of an uncharacterized lipase and/or of multiple hydrolases. In conclusion, we show that PGE2-G inhibits human neutrophil functions through its hydrolysis into PGE2, and by activating the EP2 receptor. This also indicates that neutrophils could regulate inflammation by altering the balance between PG-G and PG levels in vivo.