Galvanotaxis of human granulocytes

The galvanotactic response of human granulocytes was investigated theoretically and experimentally. The basic results are: (i) The granulocytes move towards the anode. (ii) The directed movement has been quantified by two different polar order parameters-the McCutcheon index and the average of cos . (iii) The polar order parameters are a function of the applied electric field (= dose-response curve). (iv) The inverse of the galvanotactic constant of migrating cells (analogous to the Michaelis-Menten constant) has a value of-0.2±0.03 V/mm. (v) The galvanotactic response of granulocytes is a non-cooperative process with a cooperativity coefficient of 1±0.2. (vi) The galvanotactic constant is a function of pH. (vii) The protein essential for the galvanotactic response is very likely a G-protein.     

Cytostasis of tumor cell lines by human granulocytes

Purified peripheral blood granulocytes from normal adult donors were tested for cytolytic and cytostatic activity against a variety of tumor-derived, virus-transformed, and normal cell lines. Altogether, 45 donors and 16 cell lines were tested. Although granulocytes mediated antibody-dependent cell-mediated cytolysis, no spontaneous cytolysis, as measured by chromium-51 (⁵¹Cr) or [³H]thymidine ([³H]TdR) release could be detected in assays performed for up to 12 hr, even at an effector:target (E:T) cell ratio of 100:1. In contrast, granulocytes exhibited substantial growth-inhibitory activity (GIA) against most target cells, as measured by uptake of [³H]TdR by the target cells. These results were confirmed by visual counting of target cells. The degree of cytostasis was dependent on the E:T ratio, with a plateau of 80–95% inhibition usually reached at a ratio of 40:1. Inhibition of growth of adherent tumor target cells was accompanied by cell detachment, with both effects apparent by 5 hr and reaching a peak after 15 hr of incubation. With nonadherent targets, the onset and the peak of cytostasis were delayed, being observed after 8 and 24 hr, respectively. Growth of target cells remained inhibited for up to 4 days of culture. A wide variety of target cells were sensitive to granulocyte-mediated cytostasis, including tumor-derived human and mouse cell lines, lymphoblastoid cell lines from normal donors, and embryo fibroblasts. Normal human fibroblasts were inhibited only at high E:T ratios (40:1). PHA-induced lymphoblasts were the only target cells tested that were completely resistant to the cytostatic effects of granulocytes and in fact, their growth was slightly stimulated. There appeared to be two somewhat different mechanisms of growth inhibition by granulocytes, which varied with the target cell. Trypsinization of granulocytes markedly reduced their reactivity against adherent target cells but had little effect on GIA against suspension target cells. Also, the activity against F-265, but not against other target cells, was almost completely abrogated in the presence of catalase, suggesting an important role of hydrogen peroxide in one mechanism of granulocytemediated cytostasis.     

Interactions of human complement with virus particles containing the Nipah virus glycoproteins

Complement is an innate immune response system that most animal viruses encounter during natural infections. We have tested the role of human complement in the neutralization of virus particles harboring the Nipah virus (NiV) glycoproteins. A luciferase-expressing vesicular stomatitis virus (VSV) pseudotype that contained the NiV fusion (F) and attachment (G) glycoproteins (NiVpp) showed dose- and time-dependent activation of human complement through the alternative pathway. In contrast to our findings with other paramyxoviruses, normal human serum (NHS) alone did not neutralize NiVpp infectivity in vitro, and electron microscopy demonstrated no significant deposition of complement component C3 on particles. This lack of NiVpp neutralization by NHS was not due to a global inhibition of complement pathways, since complement was found to significantly enhance neutralization by antibodies specific for the NiV F and G glycoproteins. Complement components C4 and C1q were necessary but not sufficient by themselves for the enhancement of antibody neutralization. Human complement also enhanced NiVpp neutralization by a soluble version of the NiV receptor EphrinB2, and this depended on components in the classical pathway. The ability of complement to enhance neutralization fell into one of two profiles: (i) anti-F monoclonal antibodies showed enhancement only at high and not low antibody concentrations, and (ii) anti-G monoclonal antibodies and EphrinB2 showed enhancement at both high and very low levels of antibody (e.g., 3.1 ng) or EphrinB2 (e.g., 2.5 ng). Together, these data establish the importance of human complement in the neutralization of particles containing the NiV glycoproteins and will help guide the design of more effective therapeutics that harness the potency of complement pathways.     

Surface thermodynamics of phagocytic ingestion of non-opsonized bacteria by granulocytes in liquids of different surface tensions

The free energy of engulfment of four bacterial species by human granulocytes is calculated from contact angle data as a function of the surface tension γ_LV of the suspending liquid. The resulting curves predict that at low liquid surface tensions γ_LV, the phagocytic ingestion increases with decreasing hydrophobicity of the bacteria while at high surface tensions γ_LV, it increases with increasing hydrophobicity. Furthermore, these curves reach a minimum at values of γ_LV equal to the surface tension γ_LV of the bacteria. The experimental results support these predictions. Thus, the determination of the surface tension of the suspending medium at which phagocytic ingestion becomes minimum represents a novel technique to establish the surface tension of ingested particles, such as bacteria. The results obtained in this fashion for the four bacterial species are in good agreement with those obtained from contact angles, as well as those derived from bacterial adhesion experiments.     

Phagocytosis of enteric Campylobacter by human and murine granulocytes

The phagocytosis of enteric Campylobacter strains by murine and human granulocytes was studied in vitro. The number of fluorescein isothiocyanate-labelled bacteria per granulocyte was determined microscopically. The phagocytic index is strain-dependent, ranging from 0.05 to 0.4 bacteria per granulocyte. Human granulocytes phagocytose Campylobacter sp. with a twofold higher effectivity than murine cells. Opsonization with immune sera increased phagocytosis 11.6-fold; flagella-defective mutants were phagocytosed without opsonization with 3.3-fold higher effectivity than the isogenic mother strain. Stimulation and phagocytosis of granulocytes by 14 clinical isolates of Campylobacter sp. was monitored by measuring the oxidative burst and phagocytosis. Stimulation of granulocytes varied from 0.4 to 1.8 (relative units) and phagocytosis ranged from 0.03 to 0.68 bacteria per granulocyte. No statistically significant correlation among bio- or serovars and the degree of stimulation and phagocytosis was observed.     

Synthesis and uptake of taurine by isolated human granulocytes

The synthesis and uptake of taurine were studied in isolated human granulocytes. It was found that granulocytes were capable to metabolize L-cysteine to taurine, and to take up taurine from the incubation medium. The taurine uptake appeared to be temperature dependent, sodium dependent and competitively inhibited by beta--alanine.     

Cryopreservation of human granulocytes.

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to ?80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol.Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.     

Intracellular freezing of human granulocytes

Human granulocyte suspensions were exposed to controlled freezing regimens on a cryomicroscope, and the incidence of intracellular freezing was measured as a function of cooling rate and extracellular nucleation temperature. The presence of intracellular ice was assessed by analysis of serially recorded images of the freeze-thaw process and by correlation with measured patterns of change in the cell volume. For granulocytes suspended in autologous plasma, a threshold was described for intracellular freezing as an empirical function of cooling rate (B) and extracellular nucleation temperature (Tn): B (degrees C/min) = 1.1 Tn (degrees C) + 12.3.     

Humate-induced activation of human granulocytes

Naturally occurring humic substances are particular chemical compounds which are found in humus. They bind to carbohydrates, amino acids and steroids by means of hydrogen bonds, covalent bonds and epsilon donor-acceptor complexes. Three specimens of low-molecular humic substances were tested (two naturally occurring humates and one synthetically prepared humate). They were all capable of stimulating certain functions of human neutrophils (PMN), such as the respiratory burst which results in the production of toxic oxygen compounds. This PMN stimulation can be demonstrated with the help of chemiluminescence, as well as by cytochemistry and with the electron microscope. The main product of the humate-induced PMN response is H2O2. There was no activation of neutrophilic chemokinesis or chemotaxis. It is suggested that the low-molecular humic substances originating from decaying organic material contain chemical structures which can act as signals to change dormant PMN into activated cells.     

Activation of progelatinase B by membranes of human polymorphonuclear granulocytes

Isolated human granulocyte plasma membranes contain progelatinase B. The binding of progelatinase B to the membrane, however, is relatively weak, and a considerable part of progelatinase B can be removed by simply washing the membrane with buffer. This detachment does not depend on the ionic strength of the buffer, indicating that electrostatic forces do not play an important role in the binding of progelatinase B to the membrane. A complete removal of progelatinase B is achieved by chromatography of neutrophil membranes on gelatin-agarose. The plasma membrane of human granulocytes activates added progelatinase B. This activation is inhibited by soybean trypsin inhibitor and is thus performed by membrane bound serine proteinases. In contrast to other reports that claimed an important role of elastase in activating progelatinase B, we found that this activation is mostly inhibited by chymostatin and not by elastatinal and is thus primarily due to cathepsin G. Proteinase 3 was shown to activate progelatinase B as efficient as neutrophil elastase, i. e. much weaker than cathepsin G. Binding of cathepsin G and elastase to the neutrophil membrane does not change their ability to activate progelatinase B. However, cathepsin G, the most potent activator of the three neutrophil serine proteinases, is only a weak activator, when compared to stromelysin-1. This, as well as only a weak binding of progelatinase B, make it doubtful that activation of membrane-bound progelatinase B by membrane-bound serine proteinases is of significant physiological importance.     

Activation of the alternative complement pathway and generation of stimulating factors for granulocytes by glass fibers

Continuous-filament glass fibers coated with organic agents, candidate asbestos substitutes, were assessed for their ability to elicit from normal human serum complement-derived cleavage products which are able to stimulate the chemotaxis and the respiratory burst of polymorphonuclear leukocytes. Glass fibers generated chemoattracting and respiratory stimulating factors for polymorphonuclears from human serum. The effect was dose related for chemotaxis from the serum fiber concentration of 75 g/ml to 1,250 g/ml. The serum chemoattracting activity, as well the respiratory stimulation, were dramatically impaired when serum had been preliminarily absorbed with antiC5 antiserum. Since the impairment of chemotactic activity occurred also in the presence of EDTA, but not in the presence of EGTA, we assumed an activation of the alternative complement pathway.Glass fibers were studied in comparison to a UICC sample of Canadian chrysotile asbestos, which is able to activate in vitro the alternative complement pathway.Glass fibers exhibited less ability than asbestos fibers to generate complement cleavage products with chemotactic activity for polymorphonuclears; however, they produced an activity about equal to 80% of a chemotactic standard stimulus such as zymosan-activated plasma.Abbreviations AF asbestos fibers - antiCS-abs-S NHS absorbed with antiserum against C5 - EDTA-CH-S NHS treated with EDTA - EGTA-Ch-S NHS treated with EGTA - GF continuous filament glass fibers coated with a binder of organic substances - NHP normal human plasma - NHS normal human serum - PMN polymorphonuclear luekocytes - ZAP zymosan-activated plasma     

Cationic proteins of human granulocytes. VI. Effects on the complement system and mediation of chemotactic activity.

The chymotrypsin-like cationic proteins of human granulocytes are shown to possess the ability to produce conversion of the complement components C1s, C4, C3, and C5 as detected by crossed immuno-electrophoresis. This ability seems to be a direct proteolytic effect. Incubation of cationic proteins with serum or functionally pure preparations of C3 and C5 is shown to generate the formation of chemotactic activity which is abolished by prolonged incubation. Also, the chemotactic activity of porcine C5a or spontaneously activated C5 is abolished by incubation with cationic proteins. It is suggested that the chymotrypsin-like cationic proteins of human granulocytes after extrusion from the phagocytic cell play an important role for generation of inflammatory mediators.