Drug inhibitable ecta-ATPase in leukocytes.

In rat leukocytes, the total ATP-hydrolyzing activity was highest when determined in cells with an intact plasma membrane. The ecto-ATPase, exhibiting a Km for ATP of 0.3 mM, was inhibited 30% by sulfhydryl reagents, whereas dinitrophenol, oligomycin or Ca²⁺ had little or no effect. The ouabain inhibitable, Na, K-activated component amounted to 7% of the total ATPase activity in leukocytes. A major characteristic of the ecto-ATPase was its marked inhibition by micromolar concentrations of both phenothiazine tranquilizers and tricyclic antidepressants.     

Aminopeptidase P from human leukocytes.

Cytosolic aminopeptidase P was obtained in highly purified form from human leukocytes by a four-step procedure. Buffy coats were the starting material. A M(r) of 140,000 was obtained by size-exclusion HPLC for the native enzyme. As shown by SDS/PAGE under reducing and denaturing conditions, the enzyme consisted of likely identical subunits with M(r) of 71,000. Purified aminopeptidase P cleaved off, specifically and efficiently, the N-terminal residues from peptides with N-terminal Xaa-Pro sequences. The penultimate proline was not replaceable by hydroxyproline, alanine and glycine in di-, tri- and tetrapeptides. Polyproline was not hydrolyzed. Dipeptides were cleaved (Arg-Pro, Phe-Pro > Trp-Pro > Pro-Pro) although slower than longer peptides. Cleavage was observed of several biologically active peptides; C-terminal fragment (residues 201-206) of C-reactive protein, oxytocin fragment Tyr-Pro-Leu-Gly, morphiceptin, peptide Gly-Pro-Arg-Pro (inhibitor of fibrin polymerization) and kentsin. In addition, cleavage of a protein, interleukin-6, was also demonstrated. Aminopeptidase P was maximally activated by Mn2+, and to a lesser extent by Co2+. The activity was optimal at pH 8. Ni2+, Zn2+ and especially Cd2+ caused marked inhibition. EDTA, 1,10-phenantroline and dithiothreitol were also inhibitory. Carbobenzoxy-phenylalanine, as well as several N-carbobenzoxy-proline-containing peptides, caused partial inhibition. The observed resistance of Gly-Pro, Pro-Gly, Pro-Phe and Pro-Ile to hydrolysis by the purified enzyme strongly indicates absence of known proline-specific dipeptidases in the aminopeptidase-P preparation.     

Passive mechanical properties of human leukocytes.

Micropipette experiments are used to determine the rheological properties of human leukocytes. Individual cells in EDTA are subjected to a known aspiration pressure via a micropipette, and their surface deformation from the undeformed spherical shape is recorded on a television monitor. The cells are mathematically modeled as homogeneous spheres, and a standard solid viscoelastic model is found to describe accurately the deformation of the cell for small strains. These experimental and theoretical studies provide the basis for further investigations of leukocyte rheology in health and disease.     

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.     

Complement-mediated release of histamine from human leukocytes.

Activation of either the alternative or classical pathway of complement generated a factor which induced release of histamine from both non-allergic and allergic human basophils. This factor probably is derived from the complement system since 1) its formation was associated with loss of C3 activity in human serum, 2) chemotactic factor, probably also a complement product, was generated simultaneously, 3) heat inactivation blocked its formation, 4) anti-C3 and anti-C5 blocked formation of the factor, and finally 5) anti-C5 inhibited the activity of the factor once it had been formed. It appears that both complement-mediated and allergen-mediated release of histamine from basophils are secretory, non-cytolytic pathways since both were maximal at 37 degrees C, required the presence of divalent cations, and were inhibited by theophylline. One consistent difference between these two mechanisms was noted: complement-initiated release of histamine occurred more quickly.     

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.     

The cytochemistry of rabbit peripheral blood leukocytes.

The authors report on their investigations of the differentiation between neutrophilic and eosinophilic granulocytes of the peripheral blood in healthy rabbits. By varying the pH-value and the incubation time it is possible to achieve a reliable differentiation between neutrophilic and eosinophilic granulocytes of the rabbit by means of 6 different cytochemical methods which could only be made incompletely with the routine methods used up till now (Eosin-, Giemsa-, Wright's-colouring etc.). Moreover, monocytes and lymphocytes can also be identified reliably.     

Staining of rabbit eosinophil and pseudoeosinophil leukocytes.

Air-dried rabbit blood was stained by HE, PAS and a modification of the Undritz II method. Eosin stained granules red in the eosinophil leukocytes. PAS was negative and the modified Undritz method failed to give consistent results. Cells with eosinophilic granules appeared in the corneal stroma 1 h after removing the corneal epithelium. They were stained red consistently by both eosin and the modified Undritz II method. Electron micrographs failed to demonstrate crystalloids in the granules. Because of the staining characteristics and the lack of crystalloids in their granules these cells were classified as pseudoeosinophil leukocytes. The electron micrographs showed some glycogen 12 h after denuding the cornea, however, glycogen was not well stained by PAS until 18 h after denuding.     

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.     

Polymorphonuclear leukocytes in antibody-dependent cellular cytotoxicity.

Human polymorphonuclear leukocytes (PMN) lyse antibody-coated target cells in an immunologically specific fashion--antibody-dependent cellular cytotoxicity (ADCC). PMN-mediated cytolysis is independent of complement, de novo protein synthesis, and DNA replication. Cytolysis is rapid, detectable at low PMN:target cell ratios, and exceeds lymphocyte-mediated ADCC. The interaction appears to be mediated via an Fc receptor and is inhibited by aggregated gamma-globulin. A role for PMN in host tumor cell immunity is discussed.     

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.     

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.     

Macrophage recognition of ICAM-3 on apoptotic leukocytes.

Cells undergoing apoptosis are cleared rapidly by phagocytes, thus preventing tissue damage caused by loss of plasma membrane integrity. In this study, we show that the surface of leukocytes is altered during apoptosis such that the first Ig-like domain of ICAM-3 (CD50) can participate in the recognition and phagocytosis of the apoptotic cells by macrophages. Macrophage recognition of apoptotic cell-associated ICAM-3 was demonstrated both on leukocytes and, following transfection of exogenous ICAM-3, on nonleukocytes. The change in ICAM-3 was a consistent consequence of apoptosis triggered by various stimuli, suggesting that it occurs as part of a final common pathway of apoptosis. Alteration of ICAM-3 on apoptotic cells permitting recognition by macrophages resulted in a switch in ICAM-3-binding preference from the prototypic ICAM-3 counterreceptor, LFA-1, to an alternative macrophage receptor. Using mAbs to block macrophage/apoptotic cell interactions, we were unable to obtain evidence that either the alternative ICAM-3 counterreceptor alpha d beta 2 or the apoptotic cell receptor alpha v beta 3 was involved in the recognition of ICAM-3. By contrast, mAb blockade of macrophage CD14 inhibited ICAM-3-dependent recognition of apoptotic cells. These results show that ICAM-3 can function as a phagocytic marker of apoptotic leukocytes on which it acquires altered macrophage receptor-binding activity.     

Hyperthermia,antipyretics and function of polymorphonuclear leukocytes.

Whether hyperthermia (temperature, 40 degrees C), salicylates, acetaminophen or phenacetin has an adverse effect on polymorphonuclear leukocyte (PMNL) function was examined. Migration experiemnts were carried out in Boyden chambers with bacterial chemotactic factor as the attract, and bactericidal assays were done with Staphylococcus aureus and serum from an AB blood group donor as a source of opsonins. PMNL viability was determined by the trypan blue exclusion method. Neither hyperthermia nor any of the drugs tested affected PMNL viability adversely, but sodium salicylate and phenacetin suppressed PMNL migration. Early staphylococcal killing was greater at 40 degrees C; however, after 2 hours the converse was true. Bactericidal activity was suppressed by acetylsalicylic acid, sodium salicylate and phenacetin. Hence it appears PMNL function is similar at 37 degrees and 40 degrees C but that some commonly used antipyretics have an adverse effect on PMNL activity.