Rabbit Polymorphonuclear Neutrophils Research Articles

Adenosine inhibition of neutrophil damage during reperfusion does not involve KATP-channel activation

This study tests the hypothesis that cardioprotection exerted by adenosine A2-receptor activation and neutrophil-related events involves stimulation of ATP-sensitive potassium (K(ATP)) channels on neutrophils during reperfusion. The adenosine A2 agonist CGS-21680 (CGS) inhibited superoxide radical generation from isolated rabbit polymorphonuclear neutrophils (PMNs) in a dose-dependent manner from 17.7 +/- 2.1 to 7.4 +/- 1.3 nmol/5 x 10(6) PMNs (P < 0.05). Pinacidil, a K(ATP)-channel opener, partially inhibited superoxide radical production, which was completely reversed by glibenclamide (Glib). Incremental doses of Glib in combination with CGS (1 microM) did not alter CGS-induced inhibition of superoxide radical generation. CGS significantly reduced PMN adherence to the endothelial surface of aortic segments in a dose-dependent manner from 189 +/- 8 to 50 +/- 6 PMNs/mm2 (P < 0.05), which was also not altered by incremental doses of Glib. Infusion of CGS (0.025 mg/kg) before reperfusion reduced infarct size from 29 +/- 2% in the Vehicle group to 15 +/- 1% in rabbits undergoing 30 min of ischemia and 120 min of reperfusion (P < 0.05). Glib (0.3 mg/kg) did not change the infarct size (28 +/- 2%) vs. the Vehicle group and did not attenuate infarct size reduction by CGS (16 +/- 1%). Glib did not change blood glucose levels. Cardiac myeloperoxidase activity was decreased in the ischemic tissue of the CGS group (0.15 +/- 0.03 U/100 mg tissue) compared with the Vehicle group (0.37 +/- 0.05 U/100 mg tissue; P < 0.05). We conclude that adenosine A2 activation before reperfusion partially reduces infarct size by inhibiting neutrophil activity and that this effect does not involve K(ATP)-channel stimulation.

Influence of microvascular adherence on neutrophil leukotriene generation. Evidence for cooperative eicosanoid synthesis.

Profile and quantity of leukotriene (LT) and hydroxyeicosatetraenoic acid (HETE) generation upon selective stimulation of isolated polymorphonuclear neutrophils (PMN) compared with neutrophils in a model of pulmonary leukostasis were investigated. Freshly prepared human PMN (2 x 10(8) were injected into the pulmonary artery of isolated, ventilated, and bloodfree perfused rabbit lungs, resulting in nearly quantitative sticking in the microvasculature. The sequestered neutrophils and, in parallel, aliquots of isolated PMN were stimulated with mAb in the presence of C, known to activate PMN arachidonate metabolism via formation of membrane attack complexes. In the isolated cells, a typical LT profile including LTB4 and its omega-oxidation products, 5-HETE and nonenzymatic hydrolysis products of LTA4 was evoked. The latter indicate secretion of LTA4 in considerable amounts. In the model of pulmonary leukostasis, no nonenzymatic LTA4-derivatives were detected, coincident with a predominance of cysteinyl-LT. This finding gives indirect evidence for an efficient LTA4-transfer between PMN feeder cells and vascular acceptor cells with glutathione-S-transferase activity. Moreover, a threefold increase in the total amount of LTA4-derived products was noted in the model of leukostasis, paralleled by a marked decrease in 5-HETE liberation. This effect was further enhanced by inhibition of lung cyclooxygenase. These findings were corroborated in a homologous system, in which rabbit PMN, sticking in the rabbit lung microvasculature, were stimulated with calcium-ionophore A23187. Collectively, these data suggest a complex interaction between microvascular tissue and adhering neutrophils in LT synthesis, involving transcellular LTA4-shift, modulation of the PMN 5-lipoxygenase pathway, and amplification of LT generation. These findings may be relevant for inflammatory events with neutrophils involved.

Rabbit polymorphonuclear neutrophils elicit endothelium-dependent contraction in vascular smooth muscle.

The present studies were designed to investigate the interaction between activated polymorphonuclear neutrophils (PMNs) and endothelial regulation of vascular smooth muscle function. Rabbit peritoneal PMNs (4 x 10(3)-4 x 10(5) cells/ml) added to muscle bath chambers containing phenylephrine-precontracted rabbit isolated aortic rings produced no effect on vascular tone. However, when PMNs were activated with the chemotactic peptide, f-met-leu-phe (0.1 microM), PMNs produced concentration-dependent vascular contraction, which was dependent on the presence of the endothelium. Aortic rings denuded of endothelium were unaffected by activated PMNs. Superoxide dismutase (100 units/ml) treatment of tissues blocked completely PMN-induced vascular contraction, whereas mannitol (20 mM) had no significant effect on PMN-induced vascular contraction. Pyrogallol (a generator of superoxide anion) produced a response that was similar to that observed with activated PMNs. Superoxide anion production was measured separately, and the time of peak rate of superoxide anion production corresponded to the time of the maximal vascular contractile responses. Activated PMNs added to vascular tissues undergoing endothelium-dependent relaxation mediated by either acetylcholine or A23187 produced a reversal of vascular relaxation. Furthermore, activated PMNs did not have any effect on endothelium-independent vascular relaxation produced by either isoproterenol or nitroglycerin. The present investigation reveals that activated PMNs can release superoxide anion and produce endothelium-dependent contraction. The endothelium-dependent contraction may be the result of superoxide anion inactivation of endothelium-derived relaxing factor.

Resistance of some capsular serotype D strains of Pasteurella multocida to rabbit polymorphonuclear neutrophil phagocytosis

The mechanism of resistance of Capsular Type D strains of Pasteurella multocida to killing by rabbit polymorphonuclear neutrophils (PMN) was studied using an in vitro assay that differentiates intra- from extracellular bacteria. Two Capsular Type D strains (3761 and 3766), resistant to killing by rabbit PMN, and one Type A strain (R1), susceptible to PMN destruction, were compared. After combining opsonized bacteria and PMN, the Capsular Type D Strains 3761 and 3766 remained extracellular while the Capsular Type A Strain R1 was internalized by PMN. Thus, both Type D strains were resistant to phagocytosis by rabbit PMN.

Polymorphonuclear neutrophil chemotaxis modulated by Bacteroides fragilis peptidoglycan.

Peptidoglycan was isolated from Bacteroides fragilis with boiling sodium dodecyl sulfate, and some was treated with pronase to eliminate contaminating protein. This peptidoglycan was chemotactic for rabbit polymorphonuclear neutrophils and had even greater chemotactic activity along with some chemokinetic activity after it was partially hydrolyzed with lysozyme. Significant chemotaxis-inhibitory activity was observed for an acid-precipitable component of the lysozyme-treated crude peptidoglycan of B. fragilis.

Effect of acute nonimmune inflammation on locomotion of exudate and blood rabbit neutrophils.

The random and directed locomotion of rabbit polymorphonuclear neutrophils (PMNs) were investigated in vitro by the agarose technique. PMNs were either recruited in serum-elicited pleural exudates or isolated from blood collected before or after pleurisy development. Directed PMN migration was assessed in the presence of N-formyl-methionyl-leucyl-phenylalanine (FMLP), isologous rabbit serum (IRS) or cell-free exudates as chemotactic stimuli. Neutrophils collected from blood before pleurisy induction or 4 h afterwards displayed similar random migration, and similar migration oriented by FMLP, IRS, or cell-free exudates. However, both random and FMLP-induced migration were greater for exudate than blood PMNs. In the presence of IRS or cell-free exudate as chemoattractants, exudate and blood PMNs migrated over similar distances. These results suggest that exudation does not deactivate elicited PMNs but alters their locomotion responses according to the stimulus applied.

Inhibition of in vitro neutrophil responses to chemotactic factors by piroxicam.

The effect of piroxicam on polymorphonuclear neutrophils (PMN) functions induced by several stimuli was evaluated in vitro. Preincubation of rabbit or human PMN with piroxicam inhibited the cellular responses elicited by N-formyl-methionyl-leucyl-phenylalanine (FMLP) such as superoxide anion (O2-) generation, granule enzyme release and chemotaxis. The effectiveness of piroxicam on each response was superior to those of indomethacin and ibuprofen. Also when either concanavalin A, zymosan-treated serum or ionophore A23187 was used as stimuli, piroxicam inhibited O2- generation of PMN. The inhibitory effect of piroxicam on FMLP-induced O2- generation was dependent on the concentration of stimuli and was reversed by increasing the extracellular calcium concentration. In addition, piroxicam had no effect on the activity of a chymotrypsin-like esterase, N-acetyl-phenylalanine-beta-naphthyl esterase, isolated from rabbit PMN. These results suggest that at least some of the anti-inflammatory effects of piroxicam may be mediated by affecting PMN functions, and the inhibition of O2- generation of PMN by piroxicam may be related to its capacity to modulate the association of calcium with these cells.

Inhibition of in Vitro Neutrophil Responses to Chemotactic Factors by Piroxicam

The effect of piroxicam on polymorphonuclear neutrophils (PMN) functions induced by several stimuli was evaluated in vitro. Preincubation of rabbit or human PMN with piroxicam inhibited the cellular responses elicited by N-formyl-methionyl-leucyl-phenylalanine (FMLP) such as superoxide anion (O2−) generation, granule enzyme release and chemotaxis. The effectiveness of piroxicam on each response was superior to those of indomethacin and ibuprofen. Also when either concanavalin A, zymosan-treated serum or ionophore A23187 was used as stimuli, piroxicam inhibited O2− generation of PMN. The inhibitory effect of piroxicam on FMLP-induced O2− generation was dependent on the concentration of stimuli and was reversed by increasing the extracellular calcium concentration. In addition, piroxicam had no effect on the activity of a chymotrypsin-like esterase, N-acetyl-phenylalanine-β-naphthyl esterase, isolated from rabbit PMN. These results suggest that at least some of the anti-inflammatory effects of piroxicam may be mediated by affecting PMN functions, and the inhibition of O2− generation of PMN by piroxicam may be related to its capacity to modulate the association of calcium with these cells.

Biosynthesis of platelet activating factor in rabbit polymorphonuclear neutrophils.

The synthesis of platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was studied in rabbit peritoneal polymorphonuclear neutrophils. Upon stimulation with ionophore A23187 and Ca2+, these cells are able to incorporate [3H]acetate or 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine into platelet activating factor. Under the same incubation conditions, however, the cells do not synthesize platelet activating factor from [14C]hexadecanol, which is an immediate precursor of O-alkyl chains in the de novo pathway. In the absence of ionophore, [14C] hexadecanol is incorporated into 1-O-alkyl-2-acyl-sn-glycerol-3-phosphate and subsequently into the 1-O-alkyl-linked choline and ethanolamine phosphoglyceride pools. However, in the presence of ionophore, [14C] hexadecanol incorporation is limited to phosphatidic acid, perhaps due to the inhibition of choline phosphotransferase. These findings provide strong evidence that platelet activating factor is synthesized by a deacylation-reacylation mechanism. Upon stimulation, these cells can utilize both plausible substrates of this pathway to make the final product, while under the same conditions it appears that a key step of the de novo pathway is inhibited.

Effects of a phagocytosis-stimulating factor on the phagocytic process of polymorphonuclear neutrophils.

The effect of phagocytosis-stimulating factor (PSF) on phagocytosis was studied in detail. PSF did not affect the Fc receptor-mediated phagocytosis, whereas PSF enhanced the ingestion step, but not attachment step, of C3b receptor-mediated phagocytosis by polymorphonuclear neutrophils (PMNs), suggesting that PSF may specifically modulate the C3b receptor function of PMNs. PSF generated from guinea pig PMNs enhanced phagocytosis by rabbit PMNs, and rabbit PMN-produced PSF accelerated the phagocytosis by guinea pig PMNs, indicating that PSF is not specific for animal species. The effects of PSF on some PMN functions, such as O2- generation, chemotaxis, adherence, and enzyme release, were also studied. Only O2- generation from PMNs was significantly increased in the initial phase of phagocytosis, and this stimulation of O2- generation was completely parallel with the stimulation of phagocytosis by PSF. Resting PMNs hardly generated the superoxide anions by PSF treatment, suggesting that PSF does not affect the O2- forming system directly.

Idiopathic Neutropenia with Normocellular Bone Marrow: an Immune‐Complex Disease

The presence of circulating immune-complexes (IC) and their in vivo interaction with polymorphonuclear neutrophils (PMN) have been detected in two cases of idiopathic neutropenia with normocellular bone marrow. The injection of patients' sera into New Zealand White rabbits caused a striking neutropenia due to sequestration of PMN in the vascular bed of kidneys and lungs. The kinetics of PMN disappearance from peripheral blood and the pattern of sequestration overlapped that induced by the injection of pre-formed soluble IC. Treatment with plasmapheresis caused an early and lasting increase of PMN; rabbit PMN were almost unaffected by the injection of patient serum after the course of plasmapheresis. These data are consistent with the possibility that idiopathic neutropenia with normocellular bone marrow may be caused by persistent in vivo interaction between IC and circulating PMN.

Lipid peroxidation and morphological changes in mammalian cells treated with the glutathione oxidant, diamide.

Chinese hamster ovary cells treated with the glutathione oxidant diamide formed large amounts of lipid peroxide. This effect was greater at 18 °C than at 0 °C and was apparently not a direct consequence of glutathione oxidation because it occurred at concentrations well above those needed to oxidize cellular glutathione. The reagent was toxic at 18 °C but not at 0 °C and caused extensive blebbing in 50% of the treated cells at this temperature. Electron microscopic examination of rabbit polymorphonuclear neutrophils disclosed that diamide caused formation of a large, organelle-free bleb and a band of fibrogranular material. It also inhibited phagocytosis of yeast particles by these cells. These effects were reversed when the cells were incubated at 37 °C in the absence of diamide. The results indicate that, although diamide is relatively specific for glutathione under some circumstances, effects observed with intact cells under most experimental conditions may reflect processes other than oxidation of endogenous glutathione.

Phagocytosis ofCandida albicansby rabbit neutrophils

Phagocytosis and killing of ingested Candida albicans by rabbit polymorphonuclear neutrophils were studied in vitro using cells and sera from normal and immunized animals. Optimal phagocytosis was observed when neutrophils and sera obtained from immunized animals were used. The phagocytic index reached its peak at the end of 1 h. The fate of ingested Candida was studied by supravital staining. The highest killing activity was shown by the combination of neutrophils and serum from immunized animals at the end of 1 h. There was no enhancement of this activity on further incubation. Neutrophils derived from non-immunized animals in the presence of normal serum were least effective in candidacidal activity. The candidacidal activity and the inhibitory effect on the germtube formation by the ingested Candida were most marked in the neutrophils obtained from immunized animals.