Chemotropism indices for polymorphonuclear leukocytes.

Trajectories of polymorphonuclear leukocytes which are responding to a chemical gradient are analyzed in order to deduce probability distributions of the angles between successive path segments. The turn angle probability distributions thus obtained are seen to be strongly dependent on the direction of locomotion prior to a turn, in that cells usually turn to maintain alignment along an axis directed towards the chemoattractant source. A mathematical model based on these observations is developed in order to understand the relationship between net chemotactic response and parameters characterizing stochastic movements of individual cells. In particular, the manner in which the chemotropism index depends on details of the turn-angle distributions is examined. When bias in the direction of turn is induced by a chemotactic field, transition from random motion to directed response occurs most abruptly if the turn-angle distribution is narrow. "Accommodation," viz., a dependence of the mean angle of turn upon prior orientation, is found to have relatively little effect on the magnitude of the response.     

Effect of mechanical deformation on structure and function of polymorphonuclear leukocytes

Kitagawa, Yuko, Stephan F. Van Eeden, Darlene M. Redenbach,Maleki Daya, Blair A. M. Walker, Maria E. Klut, Barry R. Wiggs, andJames C. Hogg. Effect of mechanical deformation on structure andfunction of polymorphonuclear leukocytes. J. Appl.Physiol. 82(5): 1397-1405, 1997.The presentstudies were designed to test the hypothesis that mechanicaldeformation of polymorphonuclear leukocytes (PMN) leads to functionalchanges that might influence their transit in the pulmonarycapillaries. Human leukocytes were passed through 5- or 3-µm-porepolycarbonate filters under controlled conditions. Morphometricanalysis showed that the majority of PMN were deformed and that thisdeformation persisted longer after filtration through 3-µm filtersthan through 5-µm filters (P < 0.05) but did not result in the cytoskeletal polarizationcharacteristic of migrating cells. Flow cytometric studies of thefiltered PMN showed that there was a transient increase in thecytosolic free Ca²⁺ concentrationafter both 3- and 5-µm filtration (P < 0.01) with an increase in F-actin content after 3-µm filtration(P < 0.05). AlthoughL-selectin expression on PMN wasnot changed by either 5- or 3-µm filtration, CD18 and CD11b wereincreased by 3-µm filtration (P < 0.05). Priming of the PMN withN-formyl-methionyl-leucyl-phenylalanine (0.5 nM) before filtration resulted in an increase of CD11b by both 5 (P < 0.05)- and 3-µm(P < 0.01) filtration. Neither 5- nor 3-µm filtration induced hydrogen peroxide production. We conclude that mechanical deformation of PMN, similar to what occurs in thepulmonary microvessels, induces both structural and functional changesin the cells, which might influence their passage through the pulmonarycapillary bed.

     

Phosphorylase kinase from human polymorphonuclear leukocytes.

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.     

Human polymorphonuclear leukocytes aminopeptidases

In human polymorphonuclear leukocytes a methionine, leucine, arginine, phenylalanine and alanine aminopeptidase activities were detected, both in cytosol and secondary granules. All activities were EDTA sensitive and their pH optima were in the range of pH 6.5 to 8.6. In the cytosol two enzymes could be distinguished, broad substrate specificity aminopeptidase of pH 4.7-4.9 and a chloride dependent arginine aminopeptidase of pI 5.3-5.5. The granules contain aminopeptidase of pI 4.0-4.6 and of pI 9.8-10.2, different from those in the cytosol. Among them broad specificity aminopeptidases and possibly specific methionine and leucine aminopeptidases could be discerned.     

Monokines released during short-term Fc gamma receptor phagocytosis up-regulate polymorphonuclear leukocytes and monocyte-phagocytic function.

Freshly explanted monocytes phagocytosing IgG antibody-coated erythrocyte targets (EIgG) release a factor(s) that stimulates phagocytosis by neighboring monocytes and polymorphonuclear leukocytes (PMN). Culture supernatants obtained after 30-min incubation of adherent monocytes with EIgG, but not unopsonized sheep erythrocytes, markedly up-regulated the extent of PMN phagocytosis and enhanced the rate at which monocytes ingested EIgG. The presence of this factor(s) was first evident in phagocytic studies in which monocytes were prepared by a colloidal silica-based continuous gradient technique (Sepracell-Mn). After introduction of erythrocyte targets, there was a 20- to 30-min delay before initiation of phagocytosis that was not observed with monocytes prepared by the standard Percoll-gradient technique. Experiments suggest that, when compared with monocytes prepared by the Percoll-gradient method, Sepracell-Mn monocytes are closer to a base line state of activation with regard to the expression of Fc gamma RI and the ability to ingest EIgG. The mechanism of PMN upregulation by the monocyte factor(s) was explored. Monocyte supernatants did not induce an increase in the surface expression of PMN Fc gamma RI, II, or III. Neither anti-TNF, anti-IL-2, nor anti-GM-CSF had any significant effect on monocyte supernatant activity. Neutrophil activating protein-1 was not detected by ELISA. In contrast, anti-IL-1 completely blocked the effect of the supernatant on subsequent monocyte phagocytosis, and partially inhibited its effect on PMN phagocytosis. Furthermore, it was shown that RIL-1 as well as TNF markedly enhanced monocyte and PMN ingestion of EIgG. These results suggest that monocytes, after Fc gamma R-mediated phagocytosis, release monokines, including at least IL-1, which enhance the phagocytic function of neighboring PMN and monocytes to augment the host defense process.     

Egestion of degraded meningococci by polymorphonuclear leukocytes.

Quantitative studies were carried out on the in vitro phagocytosis of 14C-labeled Neisseria meningitidis by mouse polymorphonuclear leukocytes. Intact, "loaded" leukocytes were found to excrete radioactive bacterial products back into supernatant fluids. Morphological events associated with the exocytosis events revealed a fusion between the phagocytic vacuole and plasma membranes of the leukocyte followed by an emptying of the vacuole contents. Egested materials were free from whole meningococci and consisted mainly of membranous vesicles.     

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.     

Transport of 1,5-anhydro-D-glucitol into human polymorphonuclear leukocytes.

1,5-Anhydro-D-glucitol (AG) is one of the main polyols and its structure resembles glucose. It has been proposed that decreased serum AG concentrations in diabetic patients are a novel indicator of diabetic metabolic derangement. However, the pathway of AG metabolism still remains to be clarified. In this study we investigated the transport of AG into human polymorphonuclear leukocytes (PMNLs) isolated from healthy volunteers and found that 0.1 mM 3-O-methy-D-glucose (3OMG) was equilibrated with a half saturation time of 10 s, while the uptake rate of AG was much slower. The concentration dependence of AG uptake revealed that the AG transport velocity reached a plateau, with a Km of about 50 mM and Vmax of about 25 nmol/min/10(7) cells. Transport of 14C-labeled 3OMG was inhibited by unlabeled D-glucose or AG in a dose-dependent manner. The mean inhibition constant (Ki) for D-glucose and for AG were 1.06 and 4.93 mM, respectively. Cytochalasin B (20 microM) inhibited 3OMG transport by 90% but AG transport by only 50%. S/V for 14C-labeled AG transport plotted against the concentration of unlabeled 3OMG showed a non-linear and biphasic pattern. These results suggest that AG influx into PMNLs is mediated not only by the cytochalasin B-sensitive glucose transport system but also via another facilitated transport system.     

Alterations in membrane fluidity of diabetic polymorphonuclear leukocytes.

Plasma membrane fluidity of polymorphonuclear leukocytes was investigated in 28 patients with insulin dependent diabetes mellitus and 30 healthy controls. Membrane fluidity was measured by steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated into the plasma membrane. The fluorescence anisotropy values in resting (unstimulated) polymorphonuclear leukocytes from diabetic subjects were significantly higher than those of controls (0.318 +/- 0.003 vs 0.287 +/- 0.003, P less than 0.001). The addition of the respiratory burst stimulus phorbol myristate acetate induced a stable increase in fluorescence anisotropy values in both groups. Fluorescence anisotropy values of stimulated polymorphonuclear leukocytes from the diabetic and control groups were not significantly different (P greater than 0.05). These data demonstrate a decrease in plasma membrane fluidity of resting polymorphonuclear leukocytes obtained from diabetic subjects. This finding could be in part explained by an increase in their basal respiratory burst activity.     

Superoxide production by polymorphonuclear leukocytes

Summary Phagocytosis by polymorphonuclear leukocytes triggers a burst of oxidative metabolism resulting in hydrogen peroxide and superoxide production, and these active oxygen species function in the killing of microorganisms. A new cytochemical technique, based on a manganese dependent diaminobenzidine oxidation, has been developed to detect superoxide in these cells. It has been shown that superoxide generation is associated with the plasma membrane in cells activated by particulate (zymosan) and nonparticulate (phorbol myristate acetate) stimuli. This membraned activity is maintained during invagination such that reduced oxygen is generated within the endocytic vacuoles. Reaction product is absent from unstimulated cells; additionally, formation of precipitate is blocked by omission of Mn⁺⁺, low temperature, glutaraldehyde prefixation, and the presence of superoxide dismutase in the incubation medium.In honour of Prof. P. van Duijn     

Interaction of polymorphonuclear leukocytes, bradykinin and carragenin. Transference reaction in the rat

1. Injected in the paw of the rat, polymorphonuclear leucocytes do not increase the oedematogen action of bradykinin, but increase the action of lambda carrageenan. 2. This potentiation of carrageenan action is not modified when PG biosynthesis in leucocytes is inhibited by indomethacin or aspirin. It does not appear in rats previously treated by indomethacin or aspirin. 3. Our results suggest that rat polymorphonuclear leucocytes increase the inflammatory reaction, when they are stimulated by carrageenan, by the release of a phospholipase A2 activity which induces PG biosynthesis in rat paw tissues.     

Cytoplasmic strains and strain rates in motile polymorphonuclear leukocytes.

A new method is presented to measure local cytoplasmic deformation and rate of deformation in motile active neutrophils. The deformation is expressed in terms of biomechanical strains and strain rates. For this purpose small phagocytosed latex microspheres were used as intracellular markers. Planar Lagrangian and Eulerian strains and the rate of strain were estimated from the positions of a triad of internalized markers. Principal strains, stretch ratios, and principal directions were computed. The intracellular strains were found to be large relative to the overall cell shape change. Principal cytoplasmic stretch ratios showed large extension in the direction of pseudopod formation and cell locomotion and contraction in perpendicular directions. Regional strain analysis showed contractile strains to predominate in the vicinity of the pseudopod or leading edge of motion. The transitional region between the pseudopod and the main cell body exhibited large shear strains. The posterior region, where the uropod is located, also revealed large extensions but small contractile strains. The rate of strains are relatively small, nonuniform in time, and largely independent of the strain. The method we propose to measure cytoplasmic strain can be applied to a variety of problems in cell mechanics.