In vitro proteolysis of human plasma low density lipoproteins by an elastase released from human blood polymorphonuclear cells

In vitro incubation of human plasma low density lipoproteins (LDL) with human blood polymorphonuclear cells (PMN) for 1 h at 37 degrees C resulted in an increased (2-4-fold) release into the medium of an enzymatic activity which co-eluted with LDL by column chromatography at physiological ionic strength but dissociated from it in high salt media in an ultracentrifugal field. The release of this enzymatic activity increased with increasing concentration of LDL in the medium and caused the hydrolysis of the LDL apoprotein B100 as indicated by the appearance of 7-8 low molecular weight bands (immunoreactive with anti-LDL) which were not present in the electropherogram of control LDL. The proteolytic activity was identified as an elastase by the following criteria: 1) capacity to hydrolyze the synthetic substrate methoxysuccinyl-Ala-Ala-Pro-Val-4-methylcoumaryl-7-amide known to be specific for the PMN elastase, 2) pattern of apo-B proteolysis identical to that exhibited by pure PMN elastase, 3) inhibition of the proteolysis by the elastase inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-CH2Cl, 4) identity in molecular weight (28,000-30,000) of this activity with a pure preparation of PMN elastase labeled with [3H]diisopropylfluorophosphate. Based on thiobarbituric acid analyses and the lack of effect by vitamin E, oxidative events appeared to play no detectable role in apo-B proteolysis. Since we previously reported (Byrne, R. E., Polacek, D., Gordon, J. I., and Scanu, A. M. (1984) J. Biol. Chem. 259, 14531-14543) that high density lipoprotein-3 promotes the in vitro release of PMN elastase which cleaves apo-A-II, it is apparent that in vitro, both LDL and high density lipoprotein, two of the major plasma lipoprotein classes, can affect the export from PMN of an elastase which exhibits proteolytic action on apo-B and apo-A-II.     

The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.     

L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2 - dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor

L-663,536 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2, 2-dimethylpropanoic acid) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human polymorphonuclear leukocytes (PMN) (IC50, 2.5 nM). Similarly, L-663,536 inhibited A23187-induced LTB4 formation by rat peripheral blood and elicited PMN. At concentrations where inhibition of leukotriene biosynthesis occurred in human whole blood (1.1 microM), no effect was seen on cyclooxygenase or 12-lipoxygenase, an effect also observed in washed human platelets. The compound had no effect on rat or porcine 5-lipoxygenase indicating that L-663,536 is not a direct 5-lipoxygenase inhibitor. When administered in vivo L-663,536 was a potent inhibitor of antigen-induced dyspnea in inbred rats pretreated with methysergide (ED50, 0.036 mg/kg p.o.) and of Ascaris-induced bronchoconstriction in squirrel monkeys (1 mg/kg p.o.). The compound inhibited leukotriene biosynthesis in vivo in a rat pleurisy model (ED50, 0.2 mg/kg p.o.), an inflamed rat paw model (ED50, 0.8 mg/kg), a model of leukotriene excretion in rat bile following antigen provocation, and a model in the guinea-pig ear where leukotriene synthesis was induced by topical challenge with ionophore A23187 (ED50, 2.5 mg/kg p.o. and 0.6 micrograms topically). The results indicate that L-663,536 is a potent inhibitor of leukotriene biosynthesis both in vitro and in vivo indicating that the compound is suitable for studying the role of leukotrienes in a variety of pathological situations.     

Neutrophil-mediated activation of epithelial protease-activated receptors-1 and -2 regulates barrier function and transepithelial migration

Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn's disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.     

Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis

Gelatinase A (MMP-2), a matrix metalloproteinase (MMP) involved in tumor invasion and angiogenesis, is secreted as an inactive zymogen (proMMP-2) and activated by proteolytic cleavage. Here we report that polymorphonuclear neutrophil (PMN)-derived elastase, cathepsin G, and proteinase-3 activate proMMP-2 through a mechanism that requires membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Immunoprecipitation of human PMN-conditioned medium with a mixture of antibodies to elastase, cathepsin G, and proteinase-3 abolished proMMP-2 activation, whereas individual antibodies were ineffective. Incubation of HT1080 cells with either purified PMN elastase or cathepsin G or proteinase-3 resulted in dose-and time-dependent proMMP-2 activation. Addition of PMN-conditioned medium to MT1-MMP expressing cells resulted in increased proMMP-2 activation and in vitro invasion of extracellular matrix (ECM), but had no effect with cells that express no MT1-MMP. MMP-2 activation by PMN-conditioned medium or purified elastase was blocked by the elastase inhibitor alpha(1)-antitrypsin but not by Batimastat, an MMP inhibitor, showing that elastase activation of MMP-2 is not mediated by MMP activities. The PMN-conditioned medium-induced increase in cell invasion was blocked by Batimastat as well as by alpha(1)-antitrypsin, showing that PMN serine proteinases trigger a proteinase cascade that entails proMMP-2 activation: this gelatinase is the downstream effector of the proinvasive activity of PMN proteinases. These findings indicate a novel role for PMN-mediated inflammation in a variety of tissue remodeling processes including tumor invasion and angiogenesis.     

Human neutrophil elastase releases a ligand-binding fragment from the 75-kDa tumor necrosis factor (TNF) receptor. Comparison with the proteolytic activity responsible for shedding of TNF receptors from stimulated neutrophils.

To localize the protease(s) involved in shedding of tumor necrosis factor receptors (TNF-R) from activated neutrophils (PMN) (Porteu, F., and C. Nathan (1990) J. Exp. Med. 172, 599-607), we tested subcellular fractions from PMN for their ability to cause loss of TNF-R from intact cells. Exposure of PMN to sonicated azurophil granules at 37 degrees C resulted in inhibition of 125I-TNF binding; 50% inhibition ensued when PMN were treated for approximately 1 min with azurophil granules equivalent to 2-3 PMN per indicator cell. The TNF-R-degrading activity in azurophil granules were identified as elastase by its sensitivity to diisopropyl fluorophosphate (DFP), alpha 1-antitrypsin and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (MSAAPV-CK), and by the ability of purified elastase to reproduce the effect of azurophil granules. Elastase preferentially acted on the 75-kDa TNF-R, reducing by 85-96% the binding of 125I-TNF to mononuclear cells expressing predominantly this receptor, while having no effect on endothelial cells expressing almost exclusively the 55-kDa TNF-R. Elastase-treated PMN released a 32-kDa soluble fragment of p75 TNF-R that bound TNF and reacted with anti-TNF-R monoclonal antibodies. In contrast, fMet-Leu-Phe-activated PMN shed a 42-kDa fragment from p75 TNF-R, along with similar amounts of a 28-kDa fragment from p55 TNF-R. Shedding of both TNF-Rs by intact activated PMN was more extensive than shedding caused by elastase and was completely resistant to DFP and MSAAPV-CK. Thus, the TNF-R-releasing activity of azurophil granules is distinct from that operative in intact stimulated PMN and could provide an additional mechanism for the control of cellular responses to TNF at sites of inflammation.     

IL-2-based immunotherapy alters circulating neutrophil Fc receptor expression and chemotaxis

We performed functional assays on polymorphonuclear (PMN) leukocytes from 21 patients with advanced cancers, before, during, and after IL-2 administration. Of these, 19 were treated with high dose bolus IL-2 infusions (10(5) U/kg every 8 h) and 2 patients received low dose continuous infusions of IL-2 (250 U/kg/h). Five of six patients studied after IL-2 therapy had a decrease in their PMN chemotactic response to FMLP after bolus IL-2 (mean 8 doses) or, after the 4th day of continuous infusion IL-2 (pre-IL-2 values of 82% +/- 17% to 45% +/- 1% post-IL-2, p2 less than 0.004) compared with normal control values. In 8 of 10 patients studied, PMN capacity to oxidize intracellular dichlorofluorescein dye, an indirect measurement of O2- production in response to PMA stimulation, decreased after IL-2 administration (pre-IL-2 mean dichlorofluorescein oxidation (by channel number) 243 +/- 128 vs 3-day post-IL-2 87 +/- 86, p2 less than 0.02). Furthermore, a marked decrease in Fc gamma R III (Leu-11, CD16) expression was observed in 12/13 patients' PMN studied after IL-2 therapy (mean percent of PMN population with positive FcR expression was 81.1 +/- 15.4% pre-IL-2 which decreased to 56.0 +/- 30.5% post-IL-2, p2 less than 0.001). Other PMN surface markers (My4, My7, ICAM-1, LFA1, LFA3, Mac1) did not change significantly. PMN-mediated antibody-dependent cellular cytotoxicity did not change after IL-2 therapy (only 4/15 patients demonstrated more than 50% reduction in antibody-dependent cellular cytotoxicity). PMN phagocytosis of Staphylococcus aureus was also not significantly altered by IL-2 administration in six patients studied (pre-IL-2, 99 +/- 17% vs 111 +/- 28% post-IL-2, p2 greater than 0.2). We conclude that the systemic administration of IL-2 by intermittent or continuous administration is associated with marked changes in PMN function and cell surface receptor expression. These alterations may contribute to the apparent increased susceptibility to bacterial infection observed in these patients.     

Differential activation by fMet-Leu-Phe and phorbol ester of a plasma membrane phosphatidylcholine-specific phospholipase D in human neutrophil

Signal transduction involving phosphatidylcholine hydrolysis has been investigated in human neutrophils (PMN) after in situ generation of [3H]alkylacyl-sn-glycero-3-phosphocholine ([3H]alkylacyl-GPC) by cell incubation with [3H]alkylacetyl-GPC. When PMN were stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe(fMLP) or phorbol myristate acetate (PMA) in the presence of cytochalasin B, both 1-O-alkyl-2-acyl-sn-glycero-3-phosphate (PA) and 1-O-alkyl-2-acyl-sn-glycerol (AAG) were generated. On addition of the agonists in the presence of ethanol, phosphatidylethanol (PEt) [corrected] was formed with a concomitant decrease in PA and AAG. These results indicate the presence of a phospholipase D (PLD) acting on phosphatidylcholine in human PMN. The kinetics of hydrolysis were quite different according to the stimulus. Whereas fMLP induced a maximum rise in PA and AAG at 30-45 s, these products began to appear only after 1 min upon cell incubation with PMA. Similar amounts of products were formed at 1 min with fMLP and only at 5 min with PMA. Although similar time courses of PA generation were obtained in the absence of cytochalasin B, AAG were no longer involved and therefore cannot account for intracellular second messenger under physiological conditions. Subcellular distribution studies demonstrated the exclusive location of PA and PEt [corrected] in the plasma membrane. The possible involvement of PA in respiratory burst activation is discussed.     

Continuous subcutaneous injection reduces polymorphonuclear leukocyte activation by granulocyte colony-stimulating factor

The use of granulocyte colony stimulating factor (G-CSF) for recovery from neutropenia has been established; however, acute lung injury due to G-CSF-induced polymorphonuclear leukocyte (PMN) activation is a serious complication. This study was designed to compare the activation of PMN with single bolus administration and continuous administration of G-CSF. Healthy volunteers (age 33.8 +/- 1.4 yr; n = 6) received a single bolus injection of 50 microm/m2 of G-CSF (SI; n = 6) or continuous subcutaneous injection of 50 microm/m2 of G-CSF for 24 h (CI; n = 6) and were followed for 48 h. Circulating leukocyte counts, markers of activation on PMN, and circulating levels of G-CSF, IL-6, and PMN elastase were measured. SI rapidly increased serum G-CSF levels, which peaked at 4 h, whereas CI gradually increased G-CSF levels, which remained at a steady level from 8 to 24 h. SI caused a rapid decrease in PMN counts at 0.5 h followed by sustained increase to peak at 12 h. CI gradually increased PMN counts, which peaked at 24 h, but the peak values were not significantly different between the groups. SI-induced activation of PMN, which was characterized by increased expression of CD11b, decreased expression of L-selectin, and increased F-actin content, led to increases in serum IL-6 and PMN elastase level. Such changes were all attenuated with CI (P < 0.05). We conclude that continuous subcutaneous injection of G-CSF resulted in a marrow response similar to that to a single injection but yielded reduced PMN activation.     

Modification of low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway

In previous studies we reported that polymorphonuclear cell (PMN) elastase cleaves apoB-100 of human plasma low density lipoprotein (LDL) into seven or eight large Mr fragments (1, Polacek, D., R.E. Byrne, G.M. Fless, and A.M. Scanu. 1986. J. Biol. Chem. 261: 2057-2063). In the present studies we examined the interaction of native and elastase-digested LDL (ED-LDL) with primary cultures of human monocyte-derived macrophages (HMD-M). For this purpose LDL was digested with purified PMN elastase, re-isolated by ultracentrifugation at d 1.063 g/ml to remove the enzyme, and radiolabeled with 125I. At all LDL concentrations in the medium, the degradation of 125I-labeled ED-LDL was 1.5- to 2.5-fold greater than that of 125I-labeled native LDL, and for both lipoproteins species it was further enhanced by prior incubation of the cells in autologous lipoprotein-deficient serum (ALPDS). ED-LDL incubated with HMD-M in a medium containing [14C]oleate stimulated cholesteryl [14C]oleate formation 2- to 3-fold more than native LDL. In competitive degradation experiments, unlabeled ED-LDL did not inhibit the degradation of 125I-labeled acetylated LDL, whereas it caused a 90% inhibition of the degradation of 125I-labeled native LDL. At 4 degrees C, the binding of both 125I-labeled native and 125I-labeled ED-LDL was specific and of a high affinity. At saturation (Bmax), the binding of 125I-labeled ED-LDL was 2-fold higher (68 ng/mg cell protein) than that of 125I-labeled native LDL (31 ng/mg), with Kd values of 6.5 x 10(-8) M and 2.1 x 10(-8) M, respectively. A possible explanation of the binding data was provided by electrophoretic analyses suggesting that ED-LDL was twice the size of native LDL and thus potentially capable of delivering proportionately more cholesterol to the cells. Taken together, the results indicate that 1) digestion of LDL by purified PMN elastase results in a greater mass of ED-LDL (relative to native LDL) being degraded per unit time by HMD-M; 2) uptake of ED-LDL occurs via the LDL receptor; and 3) LDL digested by PMN elastase undergoes a physical change that may be responsible for its unique interactions with HMD-M. We speculate that if this process were to occur in vivo during an inflammatory process, macrophages could acquire excess cholesterol and be transformed into foam cells which are considered to be precursors of the atherosclerotic process.     

Calcium changes in immune complex-stimulated human neutrophils. Simultaneous measurement of receptor occupancy and activation reveals full population stimulus binding but subpopulation activation

Immune complexes (ICs) induce an initial transient increase in cytosolic intracellular calcium [( Ca2+]in) levels in human neutrophils (PMN). Changes in PMN [Ca2+]in were measured with the fluorescent calcium indicator Indo-1 ( [1-[2-amino-5-(6-carboxylindol-2-yl]-phenoxyl]-2-(2'-amino-5 '- methylphenoxy]ethane-N,N,N'N'-tetraacetic acid), at the level of individual cells by flow cytometry. Two kinds of immune complexes (ICs) were used in this study: an insoluble (IIC) and a more soluble less valent immune complex (SIC) with fewer available Fc receptor binding ends per molecule of SIC than IIC. Simultaneous binding and activation studies performed on the flow cytometer with fluoresceinated IIC or SIC demonstrated that a majority of the cells bound each stimulus uniformly. However, only an IC dose-dependent proportion of those IC-bound cells responded with an increase in [Ca2+]in. Analysis of Indo-1 fluorescence signals from neutrophils exposed to IIC, corrected for the contribution of the nonresponding population, indicated that every dose of IIC elicited a similar maximum [Ca+2]in within the responding population. In contrast, the magnitude of the increase in [Ca2+]in elicited by low doses of SIC did become dependent on dose. Cells treated with pertussis toxin and exposed to IIC exhibited a normal [Ca2+]in response both in magnitude and expression. Therefore, [Ca2+]in responses induced by immune complexes are expressed by subpopulations of PMN, in a response which is dependent on the valency of the stimulus. In addition, pertussis toxin sensitive G protein(s) appear not to have a major role in IIC-induced [Ca2+]in changes, membrane potential changes, production of superoxide anions, and elastase release.     

Transforming growth factor alpha in arterioles: cell surface processing of its precursor by elastases.

Analysis of the transforming growth factor alpha (TGF alpha) cDNA predicts that the mature TGF alpha polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGF alpha to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGF alpha, NR6 cells were transfected with the rat TGF alpha cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGF alpha precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGF alpha polypeptide. The released TGF alpha was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGF alpha but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic alpha 1-chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGF alpha precursor from the fixed cells. The location of TGF alpha in the arterioles and ability of PMN leukocytic elastase to process the membrane-bound TGF alpha precursor suggests a novel role for this elastase at the wound site.     

Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils

Stimulation of human polymorphonuclear leukocytes (PMN) may result in the metabolism of phospholipids other than phosphoinositides to generate second-messenger intermediary metabolites. We investigated agonist-induced breakdown of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC), which constitutes almost half the diradyl-GPC fraction in human PMN (Mueller, H. W., O'Flaherty, J. T., Green, D. G., Samuel, M. P., and Wykle, R. L. (1984) J. Lipid Res. 25: 383-388), in cells prelabeled with 1-O-[3H] alkyl-2-acyl-GPC. We also utilized normal-phase high pressure liquid chromatography to quantitate the accumulation of diradylglycerols (1-O-alkyl-2-acylglycerols and diacylglycerols) in stimulated PMN. Phorbol-12-myristate-13-acetate (PMA), 1-oleoyl-2-acetyl-sn-glycerol-, calcium ionophore A23187-, and f-methionyl-leucyl-phenylalanine (fMLP) stimulation of PMN resulted in a time- and concentration-dependent hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA) and 1-O-[3H]alkyl-2-acylglycerol. In all cases formation of 1-O-[3H]alkyl-2-acyl-PA preceded that of 1-O-[3H]alkyl-2-acylglycerol. The times between addition of stimulus and appearance of 1-O-[3H] alkyl-2-acylglycerol varied for PMA (40 s at 1.6 microM), A23187 (5 min at 5 microM), and fMLP (30 sec at 1 microM). Preincubation of cells with 1 microgram/ml pertussis toxin (PT) inhibited the breakdown of 1-O-[3H]alkyl-2-acyl-GPC in cells stimulated with 1 microM fMLP, indicating a role for a PT-sensitive G protein with this stimulus. Quantitation of diglycerides as diradylglycerobenzoates in PMN stimulated with PMA (10 min), A23187 (10 min), or fMLP demonstrated marked accumulation of both 1-O-alkyl-2-acylglycerols and diacylglycerols. The highest increases over controls were observed for fMLP (33-fold for 1-O-alkyl-2-acylglycerols and 17-fold for diacylglycerols). In stimulated PMN prelabeled with 1-O-[3H]hexadecyl-2-acyl-GPC and 1-O-alkyl-2-acyl-sn-glycero-3-[32P]phosphocholine, the ratio of 3H to 32P in 1-O-alkyl-2-acyl-PA compared to 1-O-alkyl-2-acyl-GPC suggested the involvement of a phospholipase D in the hydrolysis of 1-O-[3H]-alkyl-2-acyl-GPC. Thus, stimulation of human PMN results in the hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC to yield 1-O-[3H] alkyl-2-acyl-PA and 1-O-[3H]alkyl-2-acylglycerol possibly initiated by activation of a phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation and priming of protein kinase C translocation by a Ca2+ transient-independent mechanism. Studies in human neutrophils challenged with platelet-activating factor and other receptor agonists

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 stimulate human polymorphonuclear neutrophils (PMN) to translocate protein kinase C from the cytosol to plasmalemma as judged by their abilities to increase PMN binding of and receptor numbers for [3H]phorbol dibutyrate [( 3H]PDB) (O'Flaherty, J.T., Jacobson, D.P., Redman, J.F., and Rossi, A.G. (1990) J. Biol. Chem. 265, 9146-9152). Platelet-activating factor (PAF) had these same effects. Moreover, two potent PAF analogs (but not an inactive analog) increased [3H]PDB binding; a PAF antagonist blocked responses to PAF without altering those to fMLP; and PMN treated with PAF became desensitized to PAF while retaining sensitivity to fMLP. Indeed, PMN incubated with 1-100 nM PAF for 5-40 min had markedly enhanced [3H]PDB binding responses to fMLP. PAF thus acted through its receptors to stimulate and prime protein kinase C translocation. Its effects, however, did not necessarily proceed by a standard mechanism: Ca2(+)-depleted PMN failed to raise Fura-2-monitored cytosolic Ca2+ concentrations [( Ca2+]i), yet increased [3H]PDB binding and receptor numbers almost normally after PAF challenge. PAF also primed Ca2(+)-depleted PMN to fMLP. Nevertheless, [3H]PDB binding responses to PAF were blocked in PMN loaded with Ca2+ chelators, viz. Quin 2, Fura-2, or 5,5'-dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Exogenous Ca2+ reversed Quin 2 inhibition, and a weak chelator 4,4'-difluoro-BAPTA, lacked inhibitory actions. The chelators similarly influenced fMLP and leukotriene B4. Thus, PMN can by-pass [Ca2+]i to translocate protein kinase C. They may achieve this using a regulatable pool of Ca2+ that evades conventional [Ca2+]i monitors or a signal that needs cell Ca2+ to form and/or act. This signal may mediate function in Ca2(+)-depleted cells, the actions of [Ca2+]i-independent stimuli, cell priming, and protein kinase C movements that otherwise seem [Ca2+]i-induced.     

Effects of methylprednisolone on exercise-induced increases of plasma levels of polymorphonuclear elastase and myeloperoxidase in man. Preliminary results

The aim of the present study was to verify whether a single oral dose of methylprednisolone could modulate the exercise-induced release of polymorphonuclear neutrophil (PMN) elastase and myeloperoxidase. Four healthy, male subjects were submitted to a 20 min downhill run (-20%) at 60% VO(2) max, 3 h after oral absorption of a placebo or a single dose of 32 mg methylprednisolone. A marked neutrophilia (+103% of basal PMN count; p < 0.02) was observed 3 h after methylprednisolone ingestion. During both exercise trials, placebo and methylprednisolone, PMN counts were increased by 46% and 19% (p < 0.05), respectively. The running test caused marked and significant (p < 0.05) increases in plasma myeloperoxidase concentration (MPO). The magnitude of MPO changes was the same in the two trials (+110%). Exercise also resulted in significant changes in plasma elastase concentration (EL) in both experimental conditions (placebo: +104%, p < 0.05; methylprednisolone: +338%, p < 0.005). Plasma elastase levels reached at the end of exercise on methylprednisolone were significantly higher than after placebo (p < 0.05). A significant relationship was found between EL and PMN in methylprednisolone trial only (r = 0.72; l0 < 0.005). These results showed that the transient exercise-induced release of elastase and myeloperoxidase were not decreased by methylprednisolone.     

Functional differences between the 40 kDa and 50 to 70 kDa IgG Fc receptors on human neutrophils revealed by elastase treatment and antireceptor antibodies

Most previous studies of IgG FcR on neutrophils (PMN) have focused on a single FcR of Mr = 50 to 70 kDa, which is recognized by mAb 3G8 and anti-Leu-11a. In the course of studying the effects of extracellular proteases on PMN receptor expression and function, we found that treatment with human leukocyte elastase reduced the expression of this FcR on the PMN surface by as much as 85% in flow cytometric studies, but did not inhibit ingestion of IgG-coated particles or O2- production induced by multivalent IgG complexes, and caused only a 35% decrease in IC binding to PMN. Since a second FcR with Mr = 40 kDa recognized by mAb IV-3, recently has been identified on PMN, we sought to determine if this FcR was resistant to elastase and thus accounted for the elastase stability of IgG-mediated PMN functions. Elastase treatment that reduced 3G8 binding by 85% caused no decrease in binding of mAb IV-3. For non-elastase-treated PMN, mAb IV-3 against the 40 kDa FcR caused as much as 79 +/- 7% inhibition of IgG-induced O2- production, whereas mAb 3G8 against the 50 to 70 kDa FcR caused only 32 +/- 5% inhibition. In contrast, for IC-binding, mAb IV-3 caused only 15 +/- 6% inhibition, whereas mAb 3G8 caused as much as 80 +/- 9% inhibition, a reversal of their relative effects on O2- production. In parallel studies with elastase-treated PMN, mAb IV-3 actually blocked more IC binding than did mAb 3G8, 55 +/- 4% vs 40 +/- 6%, respectively, presumably because most of the 50 to 70 kDa FcR molecules had been cleaved. The effect of the two mAb together in blocking IC binding was additive, whereas for blocking of O2- production, mAb 3G8 added little or nothing to the effect of mAb IV-3 alone. Direct 125I-labeled Ab binding studies with intact PMN revealed seven times as many 50 to 70 kDa as 40kDa FcR, 110,200 +/- 9600 and 15,100 +/- 700 sites/cell, respectively. Our findings suggest that the elastase-resistant 40 kDa FcR is primarily responsible for IgG-mediated activation of human PMN, whereas the elastase-sensitive 50 to 70 kDa FcR predominates in IC binding, by virtue of its numerical superiority, but does not directly activate the cell. The latter may serve to hold IgG-coated microorganisms or other multivalent IC in place at the PMN surface, enhancing contact with the 40 kDa FcR and thus facilitating cell activation in a cooperative manner.     

Effect of single oral administrations of non steroidal antiinflammatory drugs to healthy volunteers on arachidonic acid metabolism in peripheral polymorphonuclear and mononuclear leukocytes

The effects of a single oral administration of acetylsalicylic acid (500 mg), indomethacin (50 mg) and piroxicam (40 mg) to healthy volunteers on functional and biochemical parameters of platelets, polymorphonuclear (PMN) and mononuclear (MNL) leukocytes were evaluated. Blood was collected before and two hours after the drug intake and blood cells separated according to conventional techniques. The considered drugs almost completely suppressed the aggregation of platelets, whereas they did not affect either PMN and MNL aggregation. Superoxide anion generation by leukocytes was (PMN), or no effect (MNL) was observed after piroxicam and indomethacin respectively. The formation of arachidonate metabolites via the 5-lipoxygenase pathway by PMN and MNL challenged with 10 microM A23187 was unchanged following aspirin and indomethacin. In this respect a selective increase of 5-HETE and LTC4 synthesis by MNL only was detected after piroxicam administration. The three drugs similarly reduced TXB2 synthesis by platelets and PMN (-80% for aspirin and indomethacin, and -40% for piroxicam). As far as MNL is concerned, aspirin inhibited this metabolite by 80%, while indomethacin reduced it by 40% only. In contrast piroxicam increased TXB2 synthesis by stimulated MNL. It can be concluded that the considered antiinflammatory drugs 1) differently affect the cyclooxygenase enzyme in platelets and leukocytes; 2) at variance with the situation in platelets, the inhibition of thromboxane formation by leukocytes is not related to modifications of cellular function; 3) the formation of arachidonate metabolites via the 5-lipoxygenase pathway is affected by piroxicam only.     

Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver

Tracer quantities (in 0.2 ml) of 13N-labeled glutamate, alanine, or glutamine(amide) were administered rapidly (less than or equal to 2 s) via the portal vein of anesthetized adult male rats. Liver content of tracer at 5 s was 57 +/- 6 (n = 6), 24 +/- 1 (n = 3), and 69 +/- 7 (n = 3)% of the injected dose, respectively. Portal-hepatic vein differences for the corresponding amino acids were 17 +/- 6, 26 +/- 8, and 19 +/- 9% (n = 4), respectively, suggesting some export of glutamate and glutamine, but not of alanine, to the hepatic vein. Following L-[13N]glutamate administration, label rapidly appeared in liver alanine and aspartate (within seconds). The data emphasize the rapidity of nitrogen exchange via linked transaminases. By 30 s following administration of either L-[13N]glutamate or L-[13N]alanine, label in liver glutamate was comparable; yet, by 1 min greater than or equal to 9 times as much label was present in liver glutamine(amine) following L-[13N]glutamate administration than following L-[13N]alanine administration. Conversely, label in liver urea at 1 min was more pronounced in the latter case despite: (a) comparable total pool sizes of glutamate and alanine in liver; and (b) label incorporation from alanine into urea must occur via prior transfer of alanine nitrogen to glutamate. The data provide evidence for zonal differences in uptake of alanine and glutamate from the portal vein in vivo. The rate of turnover of L-[amide-13N]glutamine was considerably slower than that of L-[13N]alanine or of L-[13N]glutamate, presumably due in part to the higher concentration of glutamine in that organ. Nevertheless, it was possible to show that despite occasional suggestions to the contrary, glutamine(amide) is a source of urea nitrogen in vivo. The present findings continue to emphasize the rapidity of nitrogen exchange reactions in vivo.     

Functional relevance of carnitine transporter OCTN2 to brain distribution of l-carnitine and acetyl-l-carnitine across the blood–brain barrier

Transport of L-[3H]carnitine and acetyl-L-[3H]carnitine at the blood-brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl-L-[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl-L-carnitine and in the absence of sodium ions. Similar transport properties for L-[3H]carnitine and/or acetyl-L-[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of L-[3H]carnitine and acetyl-L-[3H]carnitine by RBEC1 were sodium ion-dependent, saturable with K(m) values of 33.1 +/- 11.4 microM and 31.3 +/- 11.6 microM, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT-PCR method. Furthermore, the uptake of acetyl-L-[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of L-[3H]carnitine and acetyl-L-[3H]carnitine in jvs mice were slightly lower than those of wild-type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of L-carnitine and acetyl-L-carnitine from the circulating blood to the brain across the BBB.     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.