Inhibition of polymorphonuclear leukocyte functions by fluorinated nitrobenzenes

The bifunctional fluorinated nitrobenzenes, 1,5-difluoro-2,4-dinitrobenzene (DFDNB) and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (DFDNDPS), and the monofunctional 1-fluoro-2,4-dinitrobenzene (FDNB) inhibit chemotaxis, phagocytosis, exocytosis and the respiratory burst of rabbit polymorphonuclear leukocytes. Inhibition occurs in the micromolar concentration range; the bifunctional compounds are stronger inhibitory than the monofunctional one. The inhibitory effect can be counteracted by sulfhydryl compounds and not with amino-group containing compounds. The results suggest that an interaction with vulnerable sulfhydryl groups, located in a hydrophobic surrounding, is the basis of the inhibitory effect of the fluorinated nitrobenzenes.     

Inhibition of polymorphonuclear leukocyte capping by a chemotactic factor.

Incubation of human polymorphonuclear leukocytes with colchicine and fluorescein-concanavalin A leads to the formation of a polarized cap of fluorescence not seen if cells are incubated with fluorescein-Con A along. When cells are preincubated with a chemotactic factor before colchicine treatment, the capping is inhibited in a dose-related manner. Studies with alpha-methylmannoside indicate that the caps represent extracellular fluorescein-Con A and are not areas of Con A internalization. Experiments utilizing an irreversible inhibitor of serine esterases suggest that a chemotactic factor-activated enzyme is involved in the inhibition of cap formation in the human neutrophil.     

Inhibition of phosphoinositide metabolism in human polymorphonuclear leukocytes by S-adenosylhomocysteine

Transmembrane signaling by chemoattractants in leukocytes appears to require activation of phosphoinositide metabolism with subsequent generation of the second messenger substances, inositol(1,4,5)trisphosphate and diacylglycerol. In addition, previous studies have shown that conditions which lead to an intracellular increase in S-adenosylhomocysteine (AdoHcy), a by-product and competitive inhibitor of S-adenosylmethionine-mediated methylation reactions, inhibit all chemoattractant-mediated functions of leukocytes, suggesting that AdoHcy also interferes with chemoattractant transmembrane signaling. In the present study, we determined whether AdoHcy altered the metabolism of phosphoinositides in human polymorphonuclear leukocytes. Treatment of 32P-labeled polymorphonuclear leukocytes with the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, plus exogenous adenosine and L-homocysteine thiolactone, conditions which cause an increase in AdoHcy, produced as much as a 37% decrease in the amount of [32P]phosphatidylinositol 4-monophosphate associated with the cells. The formation of inositol bisphosphate was inhibited by as much as 45% by erythro-9-(2-hydroxy-3-nonyl)adenine, adenosine, and L-homocysteine thiolactone suggesting decreased availability of phosphatidylinositol 4-monophosphate. In support of this, AdoHcy, in concentrations ranging from 0.01 to 0.1 mM, inhibited the transfer of gamma-32P from gamma-[32P] ATP to phosphatidylinositol (PtdIns). The inhibition of PtdIns kinase was competitive with an apparent Ki for AdoHcy of 43 microM. Increased intracellular AdoHcy reduced chemoattractant-mediated increases in inositol(1,4,5)trisphosphate formation suggesting abrogation of transmembrane signaling. These findings for the first time demonstrate that AdoHcy is a competitive inhibitor of PtdIns kinase and thus a regulator of the phosphoinositide pathway.     

Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.     

Inhibition of TASK-1 potassium channel by phospholipase C

Thetwo-pore-domain K⁺ channel, TASK-1, was recently shown tobe a target of receptor-mediated regulation in neurons and in adrenalglomerulosa cells. Here, we demonstrate that TASK-1 expressed inXenopus laevis oocytes is inhibited by differentCa²⁺-mobilizing agonists. Lysophosphatidic acid, via itsendogenous receptor, and ANG II and carbachol, via their heterologouslyexpressed ANG II type 1a and M₁ muscarinic receptors,respectively, inhibit TASK-1. This effect can be mimicked by guanosine5'-O-(3-thiotriphosphate), indicating the involvementof GTP-binding protein(s). The phospholipase C inhibitor U-73122reduced the receptor-mediated inhibition of TASK-1. Downstream signalsof phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmicCa²⁺ concentration, and diacylglycerol) do not mediate theinhibition. Unlike the G_q-coupled receptors, stimulation ofthe G_i-activating M₂ muscarinic receptorcoexpressed with TASK-1 results in an only minimal decrease of theTASK-1 current. However, additional coexpression of phospholipaseC-₂ (which is responsive also to G_i-subunits) renders M₂ receptor activation effective.This indicates the significance of phospholipase C activity in thereceptor-mediated inhibition of TASK-1.

     

Inhibition of phospholipase A2 and phospholipase C by polyamines

The polyamines spermine, spermidine, and putrescine inhibit the activity of phospholipase A₂ (Naja naja) and phospholipase C (Clostridium welchii) on phospholipid vesicles and mitochondrial membranes as sources of substrate phospholipids. The inhibitory effect is highest for spermine and lowest for putrescine. With both enzymes, inhibition is stronger when phospholipid vesicles rather than mitochondrial membranes are used as the substrate. No clear competition of polyamines with Ca²⁺, which is required for the activity of both enzymes, has been observed. The inhibition appears to be due to steric hindrance of enzyme-substrate interaction due to the binding of the organic polycations to the phospholipid bilayer.     

Cytochalasin E-induced oxidative metabolism in polymorphonuclear leukocytes.

The extent of the cyanide-resistent oxidative burst of polymorpho-nuclear leukocytes after stimulation with cytochalasin E was shown to depend markedly on the osmolarity of the cell-suspension medium. With granulocyte concentrations up to 2 X 10(6) cells/ml, optimal oxygen consumption and releases of H2O2 and superoxide anions were reached at 180 mOsmol and 2 X 10(-5) M cytochalasin E. After removal of unbound activator, the cellular oxidative activity remained unaltered and continued to depend on the used osmotic conditions. It is proposed that binding of cytochalasin to the plasma membrane induces an irreversible activation of the oxidative system, whereas the resulting metabolic activity depends on conformational changes in the plasma membrane.     

Identification of the polymorphonuclear leukocyte C5a receptor

The peptide C5a is thought to play an important role in the inflammatory response primarily through its action on the polymorphonuclear leukocyte (PMN). The receptor for C5a on human PMN has now been identified by affinity labeling. Cross-linking 125I-C5a to intact PMN with disuccinimidyl suberate produced a species that had a molecular mass on sodium dodecyl sulfate gels of 5.2 X 10(4) daltons. We believe this species represents a complex between C5a and its receptor for the following reasons. The band is eliminated if the cross-linking experiment is performed in the presence of a large excess of unlabeled C5a, but is unaffected by the presence of nonspecific protein or the chemotactic factors N-formyl-Met-Leu-Phe and leukotriene B4. The 5.2 X 10(4)-dalton species is not observed if the cross-linker is omitted. Finally, the dose-response curves for the inhibition of binding of 125I-C5a by unlabeled C5a and the inhibition of cross-linking are similar. Subtraction of the molecular mass of C5a from that of the complex gives a molecular mass for the binding moiety of the C5a receptor of 4.0 X 10(4) daltons.     

Effects of endotoxin on oxidative metabolism of polymorphonuclear leukocytes

The influence of endotoxin on rat polymorphonuclear leucocytes (PMN) ability to generate oxygen free radicals (OFR) has been studied by chemiluminescence method. PMNs derived from intact animals were used as a control. PMNs derived from animals with 1.5 h endotoxemia increased OFR production after stimulation by latex. In contrast, PMNs derived from intact animals and preincubated with endotoxin for 1.5 h decreased OFR production after stimulation bw latex. It was proposed that stimulating effect of endotoxin on PMNs in vivo was mediated by plasma components.     

Inhibition of Bacillus cereus phospholipase C by univalent anions.

The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.     

Phospholipid metabolism by phagocytic cells. Phospholipases A2 associated with rabbit polymorphonuclear leukocyte granules

Polymorphonuclear leukocytes obtained from sterile peritoneal exudates in rabbits contain two phospholipid-splitting activities (phosphatidylacylhydrolases EC 3.1.1.4), one most active at pH 5.5 and the other between pH 7.2 and 9.0. Hydrolysis of phospholipid was demonstrated using Escherichia coli labeled during growth with [1-(14)C]oleate and then autoclaved to inactivate E. coli phospholipases and to increase the accessibility of the microbial phospholipid substrates. The acid and alkaline phospholipase activities are both membrane bound, calcium dependent, and heat stable, and they appear to be specific for the 2-acyl position of phospholipids. Evidence was also obtained suggesting that the E. coli envelope phospholipids with oleate in position 2 are more readily degraded than those with palmitate. The two activities are associated with azurophilic as well as specific granules (obtained by zonal centrifugation) and with phagosomes (isolated after ingestion of paraffin particles by the granulocytes). Phospholipase A activities at pH 5.5 and pH 7.5 degrade the two major phospholipids of E. coli, phosphatidylethanolamine and phosphatidylglycerol, to the same extent, but the phospholipase activity at acid pH does not hydrolyze micellar dispersions of phosphatidylethanolamine. By contrast, phospholipase A(2) activity at pH 7.5 degrades both types of phosphatidylethanolamine substrates. Heparin and chondroitin sulfate inhibit phospholipase activity at pH 5.5 but have little effect on activity at pH 7.5. All detergents tested inhibited phospholipase activity, and both activities are inhibited by reaction products, free fatty acid and lysophosphatidylethanolamine. This product inhibition is only partially prevented by addition of albumin. Supernatant fractions of granulocyte homogenates contain a heat-labile inhibitor of granule phospholipase activity at pH 7.5. Boiling the fraction not only removes the inhibition but actually results in stimulation of hydrolysis at pH 7.5 as well as pH 5.5. These granule-associated phospholipase A activities of polymorphonuclear leukocytes differ in several of their properties from granule or lysosomal phospholipases of other phagocytic cells.     

Protection of crystal-induced polymorphonuclear leukocyte membranolysis by phosphocitrate

The protection afforded by phosphocitrate, a phosphorylated polycarboxylic acid, against crystal-induced membrane damage to polymorphonuclear leukocytes was studied in vitro. Membranolysis was assessed by nitro blue tetrazolium salt reduction, lactate dehydrogenase release, and scanning electron microscopy. Phosphocitrate protected strongly against hydroxyapatite crystal-induced damage, an action attributable to crystal surface binding of phosphocitrate rather than to the membrane. The ability of phosphocitrate to prevent hydroxyapatite crystallization, together with its membrane protective effect against preformed crystals, would suggest that the compound might have a useful future role against crystal-induced arthropathies.     

Induction of polymorphonuclear leukocyte response by human cytomegalovirus

Neutrophils are important in the defense against bacterial infections, by ingesting and killing invading microorganisms. Because of the higher incidence of bacterial infections in patients with active human cytomegalovirus (HCMV) infections, we hypothesized that HCMV-infected neutrophils were inefficient in eliminating the bacteria. Therefore, we mock infected or infected neutrophils with HCMV by contact with HCMV-infected human pulmonary artery endothelial cells. We found that HCMV infection without N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation increased the surface expression of CD11b to the same extent as fMLP stimulation of mock infected cells. Also, HCMV-infected neutrophils became more efficient in phagocytosing serum opsonized yeast particles than mock infected cells. Furthermore, we observed an increase in intracellular free calcium and chemiluminescence in HCMV-infected cells, in response to fMLP compared to fMLP-treated mock cells. We also found that apoptosis was significantly inhibited in HCMV-infected neutrophils. In conclusion, our results suggest that neutrophils become more effective in performing their effector functions when infected with HCMV. Thus, the higher incidence of bacterial infections in HCMV patients might not be due directly to a dysfunction in the neutrophils. Instead, the fact that apoptosis is inhibited may cause over-reactive neutrophils to remain in the tissues, where they will start leaking their contents, damaging the tissues and contributing to inflammatory processes.     

Modulation of polymorphonuclear leukocyte locomotion by synthetic amphiphiles

The capacity of synthetic amphiphiles, poly(ethyleneglycol) 6000 (PEG) esterified with saturated fatty acids (C₂–C₁₈), to modify polymorphonuclear leukocyte (PMNL) locomotion has been investigated. It was noticed that PEG-myristate (M-PEG; C₁₄) stimulated the random locomotion of PMNL populations in concentrations up to about 1 g/L. The esters with shorter aliphatic chains had negligible effects, whereas those with longer chains, PEG-palmitate (P-PEG; C₁₆) and PEG-stearate (S-PEG; C₁₈) reduced the locomotion, irrespectively of concentration. The ability of the PMNL to be stimulated by an attractant liberated from normal human serum was slightly impaired by M-PEG, but not by P-PEG. The response to M-PEG of individual PMNL was heterogeneous in that some cells were stimulated and others were inhibited. However, the average result was a reduction of the motility. This indicates that methods used for the study of the locomotion of cell populations may not always reflect the average behavior of the whole population. It was also concluded that the different effects of M-PEG and P-PEG owed to dissimilar effects on the membrane structure of the PMNL since (1) M-PEG perturbated the PMNL membrane more than P-PEG, as assayed by the release of superoxide anion (0 ₂ ⁻ , although the binding was smaller, and (2) M-PEG and P-PEG increased and decreased the membrane fluidity, respectively, as measured with fluorescent bleaching and recovery after bleaching of labeled PMNL. The results indicate a subtle coupling between membrane structure and PMNL locomotion.     

Locomotion forces generated by a polymorphonuclear leukocyte.

There have been very few studies which have measured the physical forces generated by cells during active movements. A special micropipette system has been designed to make it possible to observe cell motion within the pipette and to apply a pressure to counter the chemotactic migration of the cell. This provides a direct measure of the locomotion force generated by the cell. The average velocity of forward motion is 0.33 microns/s in the absence of counter-pressure. The application of a positive counter-pressure (C-P) causes a decrease in the velocity of the forward motion of the cell. At 17 cm H2O of C-P, the cell velocity drops to zero and even moves backward with a higher C-P. The results show that the decrement of velocity is linearly related to the magnitude of the C-P with a complete stoppage at a pressure of 17 cm H2O which corresponds to a force of 0.003 dyn. The maximum work rate of the cell is approximately 2.5 x 10(-8) erg/s.