Dietary effect of EPA-rich and DHA-rich fish oils on the immune function of Sprague-Dawley rats.
(1/1250)
The dietary effect of fish oils (FOs) rich in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) on the immune function of Sprague-Dawley rats was compared with that of safflower oil. After 3 weeks of feeding at the 10% level of a dietary fat, the IgG and IgM production by splenocytes and IgG production by mesenteric lymph node (MLN) lymphocytes were significantly higher in the FO-fed rats, while no significant difference was found in IgA or IgE productivity by both the spleen and MLN lymphocytes. In the FO-fed rats, peritoneal exudate cells released a lower amount of LTB4, reflecting their lower arachidonic acid level, and a higher amount of LTB5, reflecting their higher EPA level in phospholipids. On these EPA-rich FO exerted a stronger effect than DHA-rich FO immune functions. (+info)
Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells.
(2/1250)
The mouse leukotriene B4 receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4 (LTB4, Kd = 0.24 +/- 0.03 nM). In competition binding experiments, LTB4 was the most potent competitor (Ki = 0.23 +/- 0.05 nM) followed by 20-hydroxy-LTB4 (Ki = 1.1 +/- 0.2 nM) and by 6-trans-12-epi-LTB4 and LTD4 (Ki > 1 microM). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation. (+info)
Differential regulation of beta1 integrins by chemoattractants regulates neutrophil migration through fibrin.
(3/1250)
Chemoattractants differ in their capacity to stimulate neutrophils to adhere to and to migrate through matrices containing fibrin. Formyl methionyl leucyl phenylalanine (fMLP) stimulates neutrophils to adhere closely to, but not to migrate into, fibrin gels. Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We report that alpha5beta1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response to these chemoattractants. fMLP, but not LTB4, activated neutrophil beta1 integrins, as measured by binding of mAb 15/7 to an activation epitope on the beta1 integrins. Antibodies or peptides that block alpha5beta1 integrins prevented fMLP-stimulated neutrophils from forming zones of close apposition on fibrin and reversed fMLP's inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that block beta1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or to migrate through fibrin gels. These results suggest that chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general message that induces neutrophils to crawl and a specific message that prepares neutrophils to stop when they contact appropriate matrix proteins for activated beta1 integrins. (+info)
Effects of petrosaspongiolide M, a novel phospholipase A2 inhibitor, on acute and chronic inflammation.
(4/1250)
The marine product petrosaspongiolide M is a novel inhibitor of phospholipase A2 (PLA2), showing selectivity for secretory PLA2 versus cytosolic PLA2, with a potency on the human synovial enzyme (group II) similar to that of manoalide. This compound was more potent than manoalide on bee venom PLA2 (group III) and had no effect on group I enzymes (Naja naja and porcine pancreatic PLA2). Inhibition of PLA2 was also observed in vivo in the zymosan-injected rat air pouch, on the secretory enzyme accumulated in the pouch exudate. Petrosaspongiolide M decreased carrageenan paw edema in mice after the oral administration of 5, 10, or 20 mg/kg. This marine metabolite (0.01-1.0 micromol/pouch) induced a dose-dependent reduction in the levels of prostaglandin (PG)E2, leukotriene B4, and tumor necrosis factor-alpha in the mouse air pouch injected with zymosan 4 h after the stimulus. It also had a weaker effect on cell migration. The inflammatory response of adjuvant arthritis was reduced by petrosaspongiolide M, which also inhibited leukotriene B4 levels in serum and PGE2 levels in paw homogenates. In contrast with indomethacin, this marine compound did not reduce PGE2 levels in stomach homogenates. Petrosaspongiolide M is a new inhibitor of secretory PLA2 in vitro and in vivo, with anti-inflammatory properties in acute and chronic inflammation. (+info)
The effects of phosphodiesterase type 4 inhibitors on tumour necrosis factor-alpha and leukotriene B4 in a novel human whole blood assay.
(5/1250)
1. The aim of this study was to assess the inhibitory activities of phosphodiesterase type 4 (PDE4) inhibitors on tumour necrosis factor-alpha (TNF-alpha) and leukotriene B4 (LTB4) production in a novel human whole blood assay. 2. Lipopolysaccharide (LPS) stimulation of human whole blood caused a time dependent increase in TNF-alpha and prostaglandin E2 (PGE2) plasma levels. Inhibition of LPS-induced TNF-alpha by the selective PDE4 inhibitor RP73401 was proportionally enhanced with endogenous PGE2 (maximal after 24 h). In contrast, blocking endogenous PGE2 production with indomethacin in blood stimulated with LPS for 24 h decreased the potency of RP73401 to that observed with a 4 h LPS incubation. 3. Non-selective and selective PDE4 inhibitors showed greater inhibition of LPS-induced TNF-alpha after 24 h compared to 4 h. Stereoselectivity was only achieved in the 24 h method. 4. LPS-stimulation of whole blood for either 30 min or 24 h followed by N-formyl-Met-Leu-Phe (fMLP) activation resulted in low plasma LTB4 levels. Combination of both treatments resulted in a greater than 7 fold increase in plasma LTB4 levels. Inhibition of the double LPS and fMLP-activated LTB4 production was observed with non-selective and PDE4-selective inhibitors. Their LTB4 inhibitory potencies were similar to that observed in the 24 h LPS-induced TNF-alpha assay. Thus, stimulation of human whole blood with two LPS stimulations followed by fMLP gives rise to both TNF-alpha and LTB4 and their inhibition by various compounds can be assessed in the same blood sample. 5. Calcium ionophore (A23187) stimulation of whole blood resulted in plasma LTB4 levels similar to the double LPS and fMLP method. Inhibition of A23187-induced LTB4 biosynthesis was also achieved by PDE4-selective inhibitors as well as the direct 5-lipoxygenase (5-LO) inhibitor L-739,010. 6. These results confirm the anti-inflammatory properties of PDE4 inhibitors. Thus, this novel human whole blood can be used to assess the biochemical efficacy of PDE4 inhibitors in human subjects. (+info)
Aminopeptidase B is structurally related to leukotriene-A4 hydrolase but is not a bifunctional enzyme with epoxide hydrolase activity.
(6/1250)
Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contains the consensus sequence HEXXHX18E (324-347), conserved among the M1 family of metallopeptidases. To determine if these putative zinc-binding residues (His324, His328 and Glu347) and the active-site Glu325 are essential for the enzyme activity, we replaced the histidines with tyrosines and the glutamic acid residues with alanines using site-directed mutagenesis. The cDNAs were expressed in Escherichia coli, and the resulting recombinant proteins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogeneity. None of the expressed mutated proteins showed aminopeptidase activity. The E325A enzyme contained 1 mol of zinc per mol of protein, and the other three mutants, H324Y, H328Y and E347A, did not contain significant amounts of zinc, as determined by atomic absorption spectrometry. From sequence-homology searches, Ap B is known to be closely related to leukotriene (LT)-A4 hydrolase (EC 3.3.2.6). We examined human placental Ap B and recombinant rat Ap B, both of which had been purified previously [Fukasawa, Fukasawa, Kanai, Fujii and Harada (1996) J. Biol. Chem. 271, 30731-30735], to determine whether or not they had epoxide hydrolase activities. However, neither enzyme hydrolysed LTA4 into LTB4. We then replaced some amino acids in the domain of the rat enzyme similar to the LTA4-binding site of LTA4 hydrolase. However, these mutants, Y408F, N409S and NE409-410SS also did not possess any epoxide hydrolase activity. We concluded that Ap B is an M1-family zinc metallopeptidase without epoxide hydrolase activity. (+info)
Polyisoprenyl phosphate (PIPP) signaling regulates phospholipase D activity: a 'stop' signaling switch for aspirin-triggered lipoxin A4.
(7/1250)
It is of wide interest to understand how opposing extracellular signals (positive or negative) are translated into intracellular signaling events. Receptor-ligand interactions initiate the generation of bioactive lipids by human neutrophils (PMN), which serve as signals to orchestrate cellular responses important in host defense and inflammation. We recently identified a novel polyisoprenyl phosphate (PIPP) signaling pathway and found that one of its components, presqualene diphosphate (PSDP), is a potent negative intracellular signal in PMN that regulates superoxide anion generation by several stimuli, including phosphatidic acid. We determined intracellular PIPP signaling by autocoids with opposing actions on PMN: leukotriene B4 (LTB4), a potent chemoattractant, and lipoxin A4 (LXA4), a 'stop signal' for recruitment. LTB4 receptor activation initiated a rapid decrease in PSDP levels concurrent with activation of PLD and cellular responses. In sharp contrast, activation of the LXA4 receptor reversed LTB4-initiated PSDP remodeling, leading to an accumulation of PSDP and potent inhibition of both PLD and superoxide anion generation. Thus, an inverse relationship was established for PSDP levels and PLD activity with two PMN ligands that evoke opposing responses. In addition, PSDP directly inhibited both isolated human recombinant (Ki = 6 nM) and plant (Ki = 20 nM) PLD. Together, these findings link PIPP remodeling to intracellular regulation of PMN function and suggest a role for PIPPs as lipid repressors in signal transduction, a novel mechanism that may also explain aspirin's suppressive actions in vivo in cell signaling. (+info)
Fish macrophages express a cyclo-oxygenase-2 homologue after activation.
(8/1250)
In mammals, the increased generation of prostaglandins (PG) during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of a novel cyclo-oxygenase (COX) isoform, termed COX-2, which is distinct from the well-characterized constitutive activity (COX-1). Goldfish (Carassius auratus) macrophages exposed to bacterial lipopolysaccharide and leucocyte-derived macrophage-activating factor(s) showed a significant increase in the generation of the major COX product, PGE2, within the first 6 h of stimulation. The selective COX-2 inhibitor, NS398, inhibited this elevated generation of PGE, whereas the basal level of this product synthesized by unstimulated macrophages was unaffected by such exposure. PGE generation by goldfish macrophages was similarly inhibited by the glucocorticoid, dexamethasone, and an inhibitor of protein synthesis, cycloheximide, suggesting that this stimulation may be due to an inducible enzyme equivalent to mammalian COX-2. The complete coding sequence of rainbow trout (Oncorhynchus mykiss) COX-2 was obtained by PCR. The gene contains a 61 bp 5'-untranslated region (UTR), a 1821 bp open reading frame and a 771 bp 3'UTR containing multiple copies of an mRNA instability motif (ATTTA). The predicted translation product had high homology to known mammalian and chicken COX-2 (83-84%) and COX-1 (77%) sequences. Reverse-transcriptase PCR with cDNA from control and bacterially challenged fish revealed that trout COX-2 expression was not constitutive but could be induced. Overall, these studies show for the first time that the inducible isoform of COX has a long evolutionary history, probably dating back to the evolution of fish over 500 million years ago. (+info)