Guinea Pig Polymorphonuclear Leukocytes Research Articles

Characterization of Hyaluronan-Binding Proteins on Guinea Pig Polymorphonuclear Leukocytes: Possible Involvement of Complement Receptor Type 3 (CR3, CD11b/CD18) in the Hyaluronan–Leukocyte Interaction

Hyaluronan (HA), a high-molecular-weight glycosaminoglycan ubiquitously present in the extracellular matrices (ECMs) of animals, plays important roles in ECM organization and cell behavior through binding to hyaluronan-binding proteins (HABPs). We previously reported that HA has anti-inflammatory effects on guinea pig phagocytes, although the nature of guinea pig HABPs was unknown. In this study, we characterized guinea pig HABPs on peritoneal polymorphonuclear leukocytes (PMNs) and blood neutrophils by flow cytometry and affinity chromatography. It was found that PMNs express diverse HABPs with different molecular weights. These HABPs maximally bound with HA over a wide pH range (6-8), and recognized HAs as small as the pentadisaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine. Furthermore, they could be divided into Mg(2+)-dependent and Ca(2+)/Mg(2+)-independent groups. Interestingly, two proteins in the Mg(2+)-dependent group were found to be the two subunits of complement receptor type 3 (CR3, CD11b/CD18). Unlike PMNs, blood neutrophils expressed several functionally inactive HABPs. Among these inactive HABPs, Mg(2+)-dependent proteins including CR3 but not Ca(2+)/Mg(2+)-independent proteins were activated on phorbol ester-stimulation. These results show the existence of diverse HABPs on guinea pig neutrophils and the cell activation-dependent activation of HABPs. It is also suggested that the CR3-HA interaction is possibly involved in the regulation of neutrophil function.

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D

Cytochalasin D (CD) induced production of the superoxide radical ( O 2 − ) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O 2 − production. CD as well as PMA induced the translocation of p47 phox to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCζ antagonist with sequences based on the endogenous PKCζ pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O 2 − production. On the other hand, the production of O 2 − induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.

Participation of cytosolic protein phosphatase in regulation of NADPH oxidase in polymorphonuclear leukocytes.

Calyculin A, a protein phosphatase inhibitor, enhanced phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2-) production and translocation of the cytosolic NADPH oxidase factor, p47phox, to the plasma membrane in guinea pig polymorphonuclear leukocytes (PMNs). When PMNs were treated with t-(5-isoquino-line-sulfonyl)-3-methyl-piperazine (H-7), a protein kinase C (PKC) inhibitor, after exposure to PMA, inhibition of O2- production and of translocation of p47phox to the membrane fraction in PMA-stimulated PMNs were observed. When calyculin A was added to the PMA-stimulated PMNs after the addition of H-7, O2- production was again observed, and translocation of p47phox to the membrane fraction also occurred. The activity of NADPH oxidase, the amount of p47phox and the level of phosphorylation of p47phox in the membrane fraction prepared from PMA-stimulated PMNs, were reduced by the addition of the cytosol fraction from unstimulated PMNs. These reductions were attenuated by calyculin A. These results indicate that the active form of NADPH oxidase in PMNs can be reconstituted after the active complex of the enzyme has disappeared once, and that one of the mechanisms of regulation of this enzyme activity involves the phosphorylation of p47phox in the cyotosol and dephosphorylation of phosphorylated p47phox in the NADPH oxidase complex by protein kinase and protein phosphatase, respectively.

Exogenously added alkylmethylglycerophosphocholine and alkylmethylcarbamylglycerophosphocholine accumulate in plasma membranes more than in intracellular membranes of rabbit platelets

We found that extracellular addition of 2% bovine serum albumin (BSA) to a suspension of rabbit platelets after stimulation with platelet-activating factor resulted in a biphasic extraction of [ 3 H]1-O- alkyl-2-O- methyl (or 2- O-methylcarbamyl)- sn-glycero-3-phosphocholine. A fast phase of extraction of the phospholipid probe by BSA was found to be mainly due to removal of the probe remaining in an outer layer of platelet plasma membrane, whereas a second phase of extraction of the probe by BSA was mostly attributed to redistribution of the probe which had been flipped across the plasma membrane. On the basis of analysis of the biphasic extraction by BSA of 1- O-alkyl-2- O-methyl(or methylcarbamyl)- sn-glycero-3-phosphocholine at various times after its addition, we suggested that the radioactive phospholipid accumulated in plasma membrane more than in intracellular membranes of rabbit platelets. In similar experiments with guinea-pig polymorphonuclear leukocytes, we observed a monophasic extraction of 1- O-alkyl-2- O-methyl, (or methylcarbamyl)- sn-glycero-3-phosphocholine by BSA, indicating its unidirectional movement across the plasma membrane.

Synthesis, absolute configuration and pharmacological evaluation of tiaprofenic acid antipodes

Summary Dextrorotatory and levorotatory antipodes (RU 40518, RU 40519) of the non-steroidal antiinflammatory drug (NSAID) racemic tiaprofenic acid (RU 15060) have been prepared by means of a new patented stereoconversion process. Unlike other arylpropionic acids, their absolute configurations, determined by circular dichroism, were R for the dextrorotatory enantiomer and S for the levorotatory enantiomer. Pharmacological evaluation showed that R-(+)-RU 40518 displays a potent in vitro inhibition of guinea-pig polymorphonuclear leukocytes (PMNL) cyclooxygenase and in vivo a better analgesic activity than its racemate or S-(-)-RU 40519 on the writhing test in the rat. Racemate and enantiomers showed similar antiinflammatory activity in carrageenan-induced paw oedema in the rat. These findings indicate that unlike other NSAIDs, the dextrorotatory enantiomer R is the active antipode for tiaprofenic acid.

Tyrosine phosphorylation and translocation of phospholipase C-γ 2 in polymorphonuclear leukocytes treated with pervanadate

We examined in detail the tyrosine phosphorylation of proteins, especially inositol phospholipid-specific phospholipase C (PLC) γ 2, during activation of respiratory burst of guinea pig polymorphonuclear leukocytes (PMNs) by pervanadate. The pervanadate, generated from a combination of H 2O 2 and orthovanadate, induced concomitantly tyrosine phosphorylation of 145, 120, 104, 76, 68, 60, 53, 42, 37, 28, and 25 kDa proteins and superoxide anion (O 2 −) production of PMNs. The pretreatment of PMNs with genistein caused an inhibition of tyrosine phosphorylation of these proteins, and also markedly depressed O 2 − production. Among the above proteins, a 145 kDa protein was found to be identical with the protein recognized by the anti-PLCγ 2 antibody on Western blots. PLCγ 2 was detected in the cytosol fraction but not in the membrane fraction of resting PMNs, whereas it was detected in both cytosol and membrane fractions of pervanadate treated PMNs. PLC activity of pervanadate treated PMNs was higher than that of resting cells. In addition, the enzyme activity of the cytosol fraction from the former cells was significantly lower than that from the latter cells, whereas the enzyme activity of membrane fraction from the former cells was significantly higher than that from the latter cells. These findings suggest that the tyrosine residue(s) of PLCγ 2 is phosphorylated and the enzyme is translocated from the cytosol to membrane fractions in PMNs by pervanadate treatment.

Inhibitory effect of acetylshikonin on the activation of NADPH oxidase in polymorphonuclear leukocytes in both whole cell and cell-free systems.

The effects of acetylshikonin (AS) on the activation of NADPH oxidase (EC 1.6.99.6) in guinea pig polymorphonuclear leukocytes (PMNs) in both whole cell and cell-free activation systems were investigated. When PMNs were treated with AS before exposure to phorbol myristate acetate (PMA), superoxide (O2-) generation in these cells was significantly reduced, but after exposure of PMNs to PMA, inhibition of O2- generation by AS did not occur. Thiol compounds completely abolished the inhibitory effect of AS on the O2- generating activity of PMNs. In the cell-free system, AS inhibited the activation of NADPH oxidase induced by myristate in a combination of cytosol and membrane fractions obtained from intact PMNs, but did not inhibit the activity of NADPH oxidase already induced. These results suggest that AS inhibits the generation of NADPH oxidase complex in the activation of respiratory burst of PMNs, but does not directly inhibit the activity of NADPH oxidase already generated.

Derivatives of 2-[[N-(Aminocarbonyl)-N-hydroxyamino]methyl]-1,4- benzodioxan as orally active 5-lipoxygenase inhibitors.

N-Hydroxyureas based on the 1,4-benzodioxan template were prepared from appropriately substituted 1,4-benzodioxan-2-methanols as the key intermediates and evaluated in the in vitro guinea pig polymorphonuclear leukocyte 5-lipoxygenase (5-LO) assay for their 5-LO inhibitory activity. Placement of a 7-phenoxy or 7-p-fluorophenoxy substituent resulted in a dramatic increase in in vitro potency. Selected compounds were subsequently assayed in an ex vivo dog model of LTB4 synthesis at a dose of 1.0 mg/kg. The 7-phenoxy derivatives 16 and 17 showed modest duration of action (DA) in this dog model. The 6-regioisomers 21 and 22 were less potent. Replacement of the 7-phenoxy group of 16 with the p-fluorophenoxy moiety enhanced the DA dramatically. Compound 18 (CGS 25667), which had an IC50 value of 100 nM in the in vitro guinea pig 5-LO assay, had a DA of 8.5 h (zileuton, DA = 8.5 h) at the oral dose of 1.0 mg/kg. Optical antipodes (24, 26) of 18 were independently synthesized in high (> 95%) enantiomeric purity from commercially available optically active glycidyl tosylates and evaluated. In the in vitro assay, the 2S-(-)-enantiomer (24, CGS 25997, IC50 = 85 nM) was found to be twice as active as the 2R-(+)-counterpart (26, CGS 25998, IC50 = 180 nM). In the ex vivo experiment, 24, which dose dependently inhibited plasma 5-LO activity, was shown to be significantly longer acting than 26, with a DA of 8.4 h when dosed orally at 1.0 mg/kg.

Differences in the effects of two hexachlorobiphenyls on superoxide generation by polymorphonuclear leucocytes stimulated by N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate.

The effects of hexachlorobiphenyls (HCBs) on superoxide (O2-) generation by guinea pig polymorphonuclear leucocytes were examined. 2,3,6,2',3',6'-HCB by itself had only a weak inductive effect on O2- generation. This compound, however, enhanced O2- generation stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) about twofold, but not the generation induced by phorbol myristate acetate (PMA). On the other hand, 3,4,5,3',4',5'-HCB suppressed O2- generation stimulated by both FMLP and PMA. The inhibitory potency of this compound was far greater with PMA (ID50, 5 microM) than with FMLP (ID50, 40 microM).

Piperazinealkanol ester derivatives of indomethacin as dual inhibitors of 5-lipoxygenase and cyclooxygenase.

Piperazinealkanol ester derivatives of indomethacin were prepared and tested for inhibitory activities against 5-lipoxygenase (5-LO) and cyclooxygenase (CO). They inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) formation by the cytosol of guinea pig polymorphonuclear leukocytes and thromboxane B2 (TXB2) formation by washed rabbit platelet suspension. Of the test compounds, 2-[4-(2-hydroxyethyl)-1-piperazinyl]-1-phenylethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indolylacetate dimaleate (II-8) was found to be the most active dual inhibitor of 5-LO and CO, and its inhibitory potency was higher than that of 2-[4-(3-hydroxypropyl)-1-piperazinyl]-ethyl [1-(4-chlorobenzoyl)-5-methoxy-2-methyl]-3-indolyacetate (CR-1015) (I), the lead compound.

Heterogeneity in lysosomal fusion with phagocytic vesicles and cell membrane in non-phagocytosing guinea-pig polymorphonuclear leukocyte.

The fusion of lysosomes and other granules with phagocytic vesicles (endo-fusion) and cell membrane (exocytosis) was simultaneously examined in non-phagocytosing guinea-pig polymorphonuclear leukocytes (PMNs). beta-Glucuronidase as a typical lysosomal enzyme, acid phosphatase as another lysosomal enzyme, and alkaline phosphatase as a specific granule enzyme were assayed. PMNs released these three enzymes in the presence of serum but the extents of exocytosis differed considerably: release of beta-glucuronidase, alkaline phosphatase and acid phosphatase was 6.9, 4.3 and 3.3%, respectively. Acid phosphatase was released even in the absence of serum, whereas the other two enzymes were not. These three enzymes showed also different responses in endofusion: the preformed phagocytic vesicles fused with the granules containing beta-glucuronidase and acid phosphatase, but scarcely fused with those containing alkaline phosphatase, although all these enzymes were recovered in increasing amounts in the phagolysosome fraction when the cells were allowed to phagocytose continuously. These results suggest that fusion of these three types of granules with phagocytic vesicles (endo-fusion) and plasma membrane (exocytosis) is heterogeneous and regulated by different mechanisms in guinea-pig PMNs.

Patent Evaluation: Hydroxylamine derivatives as lipoxygenase inhibitors

SummarySummaryNovelty: Novel substituted heteroaryl hydroxylamine derivatives are claimed to be lipoxygenase (LPO) inhibitors. They are said to be particularly effective against 5-LPO. They are potentially useful for the treatment of inflammatory and allergic disorders, asthma, dermatitis, ischaemia and endotoxin shock.Biology: 5-LPO inhibition was determined by measuring the inhibition of the synthesis of 5-HETE and LTB4 in guinea-pig polymorphonuclear leukocytes. Methods to measure antiinflammatory and anti-asthmatic activity are also described. However, no specific biological data are presented.Chemistry: A total of eighty-six compounds are disclosed. Syntheses are given in thirteen examples. Nine compounds are specifically claimed including 2-[3-(4-fluorophenyl)propyl]-6-[2-(N-aminocarbonyl-N-hydroxyamino)ethoxy]-1-oxo-1,2,3,4-tetrahydroisoquinoline.

Effects of high-molecular-weight hyaluronates on the functions of guinea pig polymorphonuclear leukocytes

The effect of hyaluronate (HA) with a molecular weight of 1,900,000 (HA190) purified from products of Streptococcus equi on various functions of guinea pig peritoneal polymorphonuclear leukocytes (PMNs) was examined. HA190 effectively inhibited both the chemotactic activity of PMNs for a chemotactic peptide, formyl-Met-Leu-Phe, and the phagocytic activity of the cells for serum-opsonized zymosan (SOZ) in a dose-dependent manner. When the inhibitory activity of the HAs of different molecular weights, including HA190, HA of molecular weight 800,000 (HA80), and HA of molecular weight 300,000 (HA30), was assessed, the HA190 showed the strongest inhibitory activity and HA30 the lowest activity. In contrast, significant inhibition of the superoxide generation by PMNs upon stimulation with SOZ was not observed even in the presence of HA190 and HA80 at the highest concentration used (2.5 mg/mL). This finding indicated that the HAs studied did not prevent the interaction of PMNs with stimuli. From these data it is concluded that the binding of high-molecular-weight HAs to the HA receptors on PMNs may produce intracellular signals that are responsible for suppression of phagocytosis and chemotaxis but not for superoxide generation.

Structure of Magnolianin, a novel trilignan possessing potent 5-lipoxygenase inhibitory activity

Magnolianin, a novel trilignan with a 1, 4-benzodioxane ring, isolated from the bark of Magnolia obovata, has been assigned structure on the basis of extensive analysis of 2D 1H and 13C NMR of its triacetate derivative. Magnolianin has been found to inhibit strongly (IC 50 = 0.45 μM) 5-HETE formation by die cytosol of guinea pig polymorphonuclear leukocytes.

Phagocytosis of monosaccharide-binding latex particles by guinea-pig polymorphonuclear leucocytes

Phagocytosis of monosaccharide-binding latex particles by guinea-pig polymorphonuclear leucocytes (PMNs) was investigated. When neuraminic acid or glucuronic acid was chemically bound to the surface of latex particles the degree of phagocytosis was somewhat lower than that for bare, glucose-, mannose- or rhamnose-binding latex particles. These experimental findings were interpreted in terms of the kind of monosaccharide and the surface potential and surface hydrophobicity of latex particles. As a result, it was concluded that the inhibitory action of neuraminic acid or glucuronic acid on phagocytosis arises from an increase in the surface hydrophilicity of latex particles brought about by the hydroxyl groups and negative charges on the chemically bound neuraminic acid or glucuronic acid molecules. Physically adsorbed glucuronic acid molecules were also found to decrease the degree of phagocytosis of latex particles by PMNs, though not so remarkable as the chemically bound monosaccharide molecules.

Effect of ibudilast, a novel antiasthmatic agent, on anaphylactic bronchoconstriction: predominant involvement of endogenous slow reacting substance of anaphylaxis.

The effect of ibudilast on anaphylactic bronchoconstriction was studied in guinea pigs sensitized actively with ovalbumin (OA). Animals were treated with indomethacin, tripelennamine and propranolol prior to the antigen challenge. Anaphylactic bronchoconstriction was prevented by ibudilast (1-4 mg/kg i.v. and 5-20 mg/kg p.o.) dose-dependently. FPL55712 and phenidone were also effective. Even when administered at the maximum development of bronchoconstriction, ibudilast (0.5 and 2 mg/kg i.v.) and FPL 55712 caused significant reduction of the increased airway tone, while phenidone did not. Ibudilast (1-4 mg/kg i.v.) and FPL55712 inhibited leukotriene D4-induced airway responses in nonsensitized guinea pigs pretreated with indomethacin and propranolol. Ibudilast (1.6 and 4 mg/kg i.v.) inhibited platelet-activating-factor (PAF)-induced airway responses in nonsensitized guinea pigs pretreated with indomethacin and propranolol, however, FPL 55712 inhibited PAF-induced airway responses only at a high dose such as 10 mg/kg i.v. Ibudilast (4 mg/kg i.v.) did not inhibit acetylcholine-induced airway response. Ibudilast showed inhibition of the release of slow-reacting substance of anaphylaxis (SRS-A) from guinea pig chopped lung sensitized with OA, which was significantly diminished by indomethacin. The drug little affected the activity of phospholipase A2 and 5-lipoxygenase in guinea pig polymorphonuclear leukocytes. These results indicate that ibudilast inhibits anaphylactic bronchoconstriction which is considered to be largely mediated by endogenously released SRS-A. The inhibitory effect of ibudilast on anaphylactic bronchoconstriction in the presence of indomethacin is considered to be exerted through its antagonism to SRS-A.

Phagocytosis of monosaccharide-binding latex particles by guinea-pig polymorphonuclear leucocytes

Phagocytosis of monosaccharide-binding latex particles by guinea-pig polymorphonuclear leucocytes

Surface electric characteristics of guinea-pig polymorphonuclear leucocytes

The charge distribution in the surface region and the isoelectric point of guinea-pig polymorphonuclear leucocytes were investigated on the basis of their electrophoretic mobility data at different pH values and ionic strengths of the medium. As a result, it was shown that the distribution of the negative charges arising from the acidic groups is fairly uniform while the positive charges arising from the basic groups increase with increasing inward distance from the boundary of the surface region and the medium, and that the isoelectric point is dependent on the ionic strength of the medium. The dependence of the isoelectric point on the ionic strength was found to decrease after the cells were treated with neuraminidase.

Effects of itraconazole on phagocytosis and killing of Candida glabrata by polymorphonuclear leucocytes from guinea pigs.

Itraconazole, a systemically active antifungal, was tested for its effects on microscopically assessed phagocytosis and killing of Candida glabrata 233 in vitro. Yeast cells were exposed to itraconazole in culture and guinea-pig peritoneal polymorphonuclear leucocytes were exposed to the drug injected intraperitoneally in vivo. At a concentration of 10(-7) M and with exposure times of 1 h, itraconazole pre-treatment of the leucocytes had no effect on the ability of PMNL to ingest or kill C. glabrata. However, pre-treatment of the growing C. glabrata cells under the same conditions significantly increased their vulnerability to both phagocytosis and intracellular killing. Longer exposures of the yeasts to itraconazole further increased their susceptibility to leucocyte phagocytosis, and it also rendered the cells vulnerable to killing merely by immersion in sodium deoxycholate solution. These findings indicate that short exposures of C. glabrata to low itraconazole concentrations damages the cells sublethally and renders them highly susceptible to leucocyte killing. Itraconazole had no direct effects on leucocyte function itself.

Stimulation of superoxide anion production in guinea pig polymorphonuclear leukocytes by hypotonic conditions in combination with protein kinase C activators

Conditions for Superoxide anion (O − 2) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O − 2 production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O − 2 production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O − 2 production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in parallel with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 μ m OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O − 2 production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.