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.     

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.     

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.     

Taurine uptake in chicken leukocytes and erythrocytes.

1. The intracellular taurine concentration and rate of taurine uptake of chicken erythrocytes and two leukocyte populations were determined from one to six weeks of age. 2. Plasma taurine concentrations increased significantly from the time of hatching to week 2 and remained constant thereafter. 3. Intracellular taurine concentrations in both leukocyte populations increased significantly with age without any significant change in the erythrocytes. 4. Taurine uptake rate for erythrocytes was significantly higher at weeks 1-3 while both leukocyte populations showed no significant change during the six week period studied.     

N-acetyl-beta-glucosaminidase activity in normal and malignant leukocytes.

An improved cytochemical method demonstrating N-acetyl-beta-glucosaminidase in peripheral blood and bone marrow leukocytes is described. A significant elevation in enzyme activity in circulating monocytes from patients with solid tumor malignancies was observed. In a large series of cases of acute nonlymphocytic leukemia, elevated levels were found in the vast majority of those leukemias that had a predominant monocytic component identified either morphologically or by standard cytochemical methods. This reaction would appear to be useful as a monocyte marker.     

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.     

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.     

Adrenergic desensitization in leukocytes of normal and asthmatic subjects.

Cyclic AMP was measured in leukocytes of normal and asthmatic subjects before and after one week of treatment with equal amounts of ephedrine. During the control and placebo periods, the measurements of cyclic AMP in leukocytes of asthmatic subjects were similar to those of normal individuals. After one week of treatment with ephedrine, both groups exhibited suppression of the leukocyte cyclic AMP response to adrenergic stimulation in vitro: however, the suppression of response was significantly greater in asthmatic subjects (p less than .u1). Subcutaneous administration of epinephrine was followed by further suppression of the leukocyte cyclic AMP response to in vitro stimulation which was similar in both groups during all treatment periods. The results indicate that in vivo exposure to adrenergic medications is followed by desensitization of the leukocyte responses to subsequent adrenergic stimulation in vitro. After administration of small doses of medication, the severity and/or duration of desensitization is significantly greater in asthmatic leukocytes.