Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide

Recombinant human granulocyte colony-stimulating factor (G-CSF) by itself was not an effective stimulus for inducing the release of superoxide (O-2) in human granulocytes. However, G-CSF was able to prime human granulocytes, and enhanced O-2 release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). The preincubation with G-CSF for 5-10 min at 37 degrees C was sufficient for priming the cells. The optimal enhancing effect was obtained at 25 ng/ml of G-CSF. The enhancement of O-2 release by G-CSF was observed over the complete range of effective concentrations of FMLP (10(-8)-10(-6) M). These findings indicate that G-CSF is a potent activator of mature granulocyte functions.     

Nicotine-induced membrane perturbation of intact human granulocytes spin-labeled with 5-doxylstearic acid. Correlation with chemotaxis

The effects of nicotine on intact human granulocytes were examined, using 5-doxylstearic acid as a spin probe. At micromolar concentrations, (-)-nitocine produces a membrane perturbation in granulocytes not observable with oriented lipid bilayers. The effect, which is stereoselective for the (-)-isomer, occurs at concentrations of nicotine that bind to noncholinergic nicotine receptors on granulocytes and which are present in the blood after smoking. At comparable concentrations, (-)-nicotine modulates granulocyte chemotaxis towards a chemotactic peptide in a stereospecific and dose-dependent manner. Cotinine, the major metabolite of nicotine, does not bind to the receptor, does not produce the membrane perturbation observed with nicotine, and has no effect on chemotaxis. These results suggest that (-)-nicotine present in the blood after smoking binds to a receptor on granulocytes, perturbs granulocyte membranes and modulates chemotaxis.     

Isolation of blood granulocytes from golden hamsters for the study of chemotaxis.

Since the number of granulocytes is low in the circulating pool of adult golden hamsters, intraperitoneal injection of oyster glycogen was used to mobilize white blood cells from the non-circulating pool. The percentage of blood granulocytes increased from 7 to 49%. Mobilized granulocytes, purified by a discontinuous Percoll gradient, showed adherence to polycarbonate filters only when coated by collagen. Random migration was high using the modified Boyden chamber technique. While endotoxin-activated serum caused chemotactic response, it was absent when the tripeptide N-formyl-methionyl-leucyl-phenylalanine was applied at concentrations of 10(-7) or 10(-8) M. The true chemotactic response corresponded to the proportion of migrated cells versus the total cell number adherent to both sides of the filter. It is concluded that the golden hamster is a good model for the study of granulocyte sequestration.     

The effects of chemotactic factors on the adhesiveness of rabbit neutrophil granulocytes.

A range of chemotactic factors has been shown to affect the adhesion of rabbit peritoneal neutrophil granulocytes to cultured endothelial cells and to serum-coated glass. At chemotactically optimal concentrations, α_s-casein, β-casein, alkali denatured human serum albumin (HSA) and several synthetic formyl-peptides reduced the number of adherent neutrophils after 30 min to around 50% of control values. These effects were still observed after neutrophils, but not endothelium or serum-coated glass had been exposed to chemotactic factors and washed before use in assays. Two non-chemotactic analogues, native HSA and a non-formyl-peptide were ineffective. The dose responses for adhesion after 30 min in the presence of α_s-casein and formyl-methionyl-leucyl-phenylalanine (FMLP) were found to be inversely related to those for migration towards these substances. After incubation for 60 min in high (10^?8–10^?7 M) concentrations of FMLP, neutrophil adhesion was found to be enhanced. Neutrophil aggregation was also affected by the presence of chemotactic factors in a similar time- and dose-dependent manner to the adhesion to substratum assays. Using FMLP, it was also shown that the timing of the adhesive changes depended on the concentration of chemotactic factor present.     

A possible competition between 5'-monodeiodination of thyroxine and the respiratory burst in human granulocytes

Thyroxine (T4) pretreatment of A 23187-stimulated human granulocytes in 10(-5)-10(-6) M concentration range inhibited the superoxide anion production of these cells. T4 increased the level of oxidized form of glutathione, whereas the intracellular level of the reduced form decreased. A similar alteration in the ratio of the oxidized to reduced forms of glutathione was detected in granulocytes during yeast cell phagocytosis. In addition, conversion of T4 to triiodothyronine (T3) was also inhibited during phagocytosis. A possible competition between 5'-monodeiodination of T4 and the oxidative burst of human granulocytes is discussed.     

Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems

Although salicylates have been used for centuries as treatment of inflammatory diseases, the mechanism of action of these drugs is still not clear. Aspirin (acetylsalicylic acid) and other nonsteroidal anti-inflammatory drugs (NSAID) inhibit prostaglandin biosynthesis, a property that appears to explain part of their anti-inflammatory activity. However, this mechanism does not appear to explain the anti-inflammatory properties of salicylic acid, which is a major metabolite of ASA in vivo. Results of prior studies in our laboratory have established that benzoic acid, the parent compound of the salicylate group of drugs, is decarboxylated and hydroxylated by the hydroxyl free radical (OH.) produced by stimulated granulocytes. These observations suggested that salicylates might be similarly metabolized by granulocytes. If so, the capacity of salicylates to rapidly react with OH. might relate directly to their known anti-inflammatory properties. Preliminary experiments established that salicylic acid and aspirin were decarboxylated by the hydroxyl free radical generated by the enzyme system xanthine-xanthine oxidase. We then studied the metabolism of salicylates by human granulocytes. Unstimulated granulocyte suspensions did not oxidize ASA or salicylic acid. However, suspensions stimulated by opsonized zymosan particles rapidly oxidized both substrates in pharmacological concentrations. The rate of oxidation of salicylic acid was 16-fold higher than benzoic acid, whereas the rate of oxidation of ASA was four-fold higher. The reaction was oxygen dependent and could be inhibited by known hydroxyl scavengers, particularly dimethylthiourea. The reaction could also be inhibited by superoxide dismutase and azide, indicating that O-2 and heme or an iron-dependent enzyme were required for the reaction. The reaction was not impaired by compounds known to react with the HOCL and the chloramines generated by stimulated PMN. Furthermore, salicylic acid in high concentrations did not impair the HMPS pathway, the production of O-2 or the production of H2O2 by granulocytes. These data provide evidence that salicylates are rapidly oxidized by the hydroxyl free radical produced by granulocytes and not O-2, H2O2, or HOCL. This capacity of salicylates to react rapidly and selectively react with OH. may directly relate to their anti-inflammatory properties. In addition, results of our experiments indicate that stimulated granulocytes acquire the capacity to metabolize these drugs. Therefore, several metabolites of salicylates may be produced at a site of inflammation, all of which may have altered biological activity compared with the parent compound.     

Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes

Experiments were performed to probe the role of exocytotic and endocytotic processes in the regulation of the human granulocyte O-2-generating system. Analytical subcellular fractionation studies indicated that 25-30% of the total cellular b-cytochrome and 8-10% of the flavin co-sedimented with plasma membrane markers, irrespective of stimulation of the cells by the chemoattractants N-formyl-Met-Leu-Phe (FMLP) or C5a. Phorbol myristate acetate stimulation resulted in significant translocation of b-cytochrome but not flavin from the specific granule/Golgi to the plasma membrane-enriched fractions. These results indicated that approximately 3.1 X 10(5) flavin and 0.8-1 X 10(6) b-cytochrome molecules are present in the plasma membrane of an isolated unstimulated human granulocyte and that these levels are invariant upon stimulation with chemoattractants. Maximal instantaneous rates of O-2 generation by cells in these preparations, however, were equivalent for all the stimuli. Since stimulation of granulocytes by phorbol myristate acetate, FMLP, or C5a results in exocytosis and/or endocytosis, then the role of these processes in regulating stimulated O-2 production by controlling the content of plasma membrane redox enzymes is questionable. This conclusion was supported by observations made with cytoplasts, which do not have an intracellular reserve of granules. Cytoplasts prepared from granulocytes produced O-2 at equivalent rates as their parent cells on a per unit surface area basis. These results suggest: 1) that stimulation of granulocytes with chemotactic peptides leads to full generation of O-2 at the cell surface without exocytotic recruitment of additional b-cytochrome and flavoprotein from the cytoplasmic compartment; 2) that these redox enzymes are not internalized along with chemoattractant receptors; and 3) that traffic of these redox enzymes between endo- and plasma membranes is not involved in the regulation of O-2 production in suspensions of human granulocytes stimulated by chemoattractants.     

Nicotine-induced membrane perturbation of intact human granulocytes spin-labeled with 5-doxylstearic acid Correlation with chemotaxis

The effects of nicotine on intact human granulocytes were examined, using 5-doxylstearic acid as a spin probe. At micromolar concentrations, (−)-nitocine produces a membrane perturbation in granulocytes not observable with oriented lipid bilayers. The effect, which is stereoselective for the (−)-isomer, occurs at concentrations of nicotine that bind to noncholinergic nicotine receptors on granulocytes and which are present in the blood after smoking. At comparable concentrations, (−)-nicotine modulates granulocyte chemotaxis towards a chemotactic peptide in a stereospecific and dose-dependent manner. Cotinine, the major metabolite of nicotine, does not bind to the receptor, does not produce the membrane perturbation observed with nicotine, and has no effect on chemotaxis. These results suggest that (−)-nicotine present in the blood after smoking binds to a receptor on granulocytes, perturbs granulocyte membranes and modulates chemotaxis.     

Fibronectin is stored but not synthesized in mature human peripheral blood granulocytes

To investigate whether peripheral blood granulocytes can synthesize the adhesive glycoprotein, fibronectin, we sought to demonstrate the presence of messenger RNA coding for fibronectin within mature circulating granulocytes. Polyadenylated-enriched RNA was isolated from human peripheral blood granulocytes, human skin fibroblasts (synthesize fibronectin) and HeLa cells (lack fibronectin) and probed with a cDNA clone coding for the cell attachment domain of fibronectin. Hybridization of a fibronectin cDNA fragment occurred with fibroblast RNA but did not occur with granulocyte RNA despite a 100 fold excess granulocyte RNA. Incubation of granulocytes with n-formyl methionyl leucyl phenylalanine, a chemotactic peptide known to augment the release of fibronectin from granulocytes, failed to induce detectable levels of mRNA for fibronectin in granulocytes. There was no difference in the quantity of fibronectin released from chemotactic peptide-stimulated granulocytes pre-incubated in the presence or absence of the protein synthesis inhibitor, cycloheximide, suggesting that fibronectin exists in a stored form in granulocytes. These data suggest that fibronectin in mature granulocytes is the product of synthesis during early myeloid maturation.     

Phospholipase A2 activation in chemotactic peptide-stimulated HL60 granulocytes: synergism between diacylglycerol and Ca2+ in a protein kinase C-independent mechanism

In dimethylsulfoxide-differentiated HL60 granulocytes, the chemotactic peptide N-formyl-Met-Leu-Phe (FMLP) augments arachidonic acid (AA) release via phospholipase A2 activity induced by the Ca2+-ionophore, A23187. Evidence indicates that this augmentation is mediated by diacylglycerols formed endogenously during FMLP receptor activation: The augmentation is mimicked by the synthetic diglyceride 1-oleoyl-2-acetyl-glycerol (OAG) and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate; Pertussis toxin inhibits FMLP-induced augmentation but not OAG-induced augmentation: At suboptimal concentrations FMLP and OAG act cooperatively to augment ionophore A23187-induced AA release but not at optimal concentrations. These data indicate that phospholipase A2 activation in FMLP-stimulated HL60 granulocytes involves cooperative interactions between diacylglycerol formed endogenously and Ca2+. Interestingly, this effect of diacylglycerol appears not to be mediated by protein kinase C, since a specific protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) does not inhibit receptor-mediated release of AA by stimulated HL60 granulocytes.     

Histamine release by chemotactic, formyl methionine-containing peptides.

Certain formyl dipeptides and tripeptides containing methionine released histamine from human basophils at concentrations of 10(-4) to 10(-7) M. However, N-formyl amino acids did not release histamine. Tripeptides, in general, were more active than dipeptides. An acyl group was required for histamine release although an N-terminal position for Met was not essential. Histamine release from human basophils by these peptides correlated well with their chemotactic activity for rabbit leukocytes.     

Effect of storage conditions on function of granulocytes

The effect of collection technique, anticoagulant, pH, glucose, and temperature on in vitro granulocyte function were studied after 24 hr of storage in the liquid state. Collection by CL did not adversely affect granulocyte function, however, cells collected by FL had accelerated loss of bactericidal activity and chemotactic response. Citrate anticoagulants provided better maintenance of bacteridical activity, NBT reduction, and chemotactic response than heparin, EDTA, and ion-exchange anticoagulants. Chemiluminescence was well maintained when the initial pH of the preservative solution (CPD plasma) was between 6.5 and 8.0 but maintenance of chemotaxis required pH of 7.0–7.5. Glucose concentrations of 80–1000 mg/dl provided adequate maintenance of chemiluminescence and chemotaxis. Bacterial killing was well maintained by storage at either 1–6 or 20–24 °C. Storage at 1–6 °C caused decreased chemotaxis, decreased ability of granulocytes to adhere and spread on a foreign surface, and a decreased intravascular recovery and shortened half-life after transfusion. Although short-term liquid storage may be practical, at present, granulocytes should be transfused as soon as possible after collection.     

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.     

Investigations on the role of Golgi-mediated, ligand-receptor processing in the activation of granulocytes by chemoattractants: differential effects of monensin

Human granulocytes were exposed to different concentrations of the ionophore monensin for 20 min at 37 degrees C. Subsequent exposure to 50 nM of the chemoattractant fMet-Leu-[3H]Phe for up to 30 min at 37 degrees C resulted in a receptor-mediated uptake that was inhibited 80% at a monensin concentration of 30 microM. 50% inhibition was observed at 1-10 microM monensin with no significant change in fMet-Leu-Phe dose dependency. Subcellular fractionation of cells treated with monensin, indicated that the low density UDP-galactosyltransferase activity associated with internalized receptor-fMet-Leu-Phe complexes in untreated cells was absent. The high density galactosyltransferase activity cosedimenting with specific granule markers, however, was unaffected. Monensin also inhibited chemotaxis toward fMet-Leu-Phe as measured by migration of granulocytes through millipore filters and fMet-Leu-Phe induction of polarized morphology. Incubation of cell suspensions with up to 30 microM monensin, both before and during measurement of fMet-Leu-Phe stimulated superoxide production, did not affect the magnitude, kinetics, or transiency of the radical generation. Monensin did, however, shift the dose dependency of superoxide production of fMet-Leu-Phe to higher concentrations. These differential effects of monensin suggest that endocytosis of complexes of the chemoattractant and receptor is not involved in the activation or termination of the fMet-Leu-Phe stimulated superoxide production. They also are consistent with a role for receptor modulation and processing in the chemotactic response.     

Stimulation of Locomotion of Peripheral Blood Monocytes by Human Plasma Fibronectin

The motility of human peripheral blood granulocytes and monocytes in response to human plasma fibronectin was quantified by an in vitro assay using blind-well chemotaxis chambers. Purified fibronectin under nondenaturing conditions produced increased migration of granulocytes only at concentrations higher than 100 nm, and induced increased chemotactic and random locomotion of monocytes at concentrations higher than 0.1 nm. The monocyte migration-inducing activity of fibronectin was concentration dependent, and was strongly inhibited by low concentrations of colchicine (100 nm–100 μm). These findings suggest the possibility that plasma fibronectin serves as a chemotactic stimulus for monocytes in vivo and attracts these cells to sites of microscopic tissue injury where plasma fibronectin is deposited.     

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.     

Chemotactic responses of human peripheral blood monocytes to the complement-derived peptides C5a and C5a des Arg

We examined responses of human peripheral blood polymorphonuclear leukocytes (PMN) and monocytes to the highly purified human complement-derived peptides C5a and C5a des Arg. As reported previously, C5a proved to be approximately 10- to 20-fold more potent than C5a des Arg as a chemoattractant for human PMN. C5a also was more potent than C5a des Arg in causing PMN to acquire a polarized morphology. In contrast, we found that human monocytes do not distinguish between C5a and C5a des Arg when these peptides are used as chemoattractants. In two different assay systems, both peptides acted at identical concentrations to stimulate suboptimal and optimal migration of monocytes. Human monocytes also did not distinguish between C5a and C5a des Arg when these peptides were used as inducers of polarization. Studies performed with functionally active, [125I]-labeled C5a and C5a des Arg, however, demonstrated that binding of C5a des Arg to monocytes differed from binding of C5a. Although [125I]-C5a des Arg appeared to bind to the same receptor as [125I]-C5a, binding of labeled C5a des Arg occurred with an affinity that was approximately 100-fold less than that observed with labeled C5a. These results indicate that leukocyte chemotactic and polarization responses to C5a and C5a des Arg vary, depending on the target cell type.     

Liquid preservation of highly purified human granulocytes

Highly purified human granulocytes isolated from continuous flow centrifugation leukapheresis concentrates by counterflow centrifugation-elutriation were stored at 4 °C in concentrations of 6 × 10⁶ to 1 × 10⁷ granulocytes per milliliter for up to 14 days. The in vitro physiological function assays of phagocytosis, oxygen consumption associated with phagocytosis, bacterial growth inhibition, chemotaxis, and five enzyme analyses indicated good storage survival for up to 4 days. Stored granulocytes separated from other blood cells have greater storage stability than granulocytes stored as leukapheresis concentrates. After 14 days of storage a small percentage of granulocytes still maintained all physiological functions, with the exception of chemotaxis. Of the five enzymes assayed, only the enzyme activity of leucine aminopeptidase decreased significantly by the 14th day of storage. The storage stability of each physiological function assayed decreased as follows: bacterial growth inhibition (most stable), phagocytosis, oxygen consumption, and chemotaxis (least stable).     

Factors affecting the stability of cryogenically preserved granulocytes

Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing.Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4–10 °C) in phosphate-buffered saline (or Ca²⁺-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES.Granulocytes (30 × 10⁶ ? 50 × 10⁶) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to ?80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g.Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, ethidium-reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation.Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality.The present efficiency of preservation of human granulocytes for 3–4 weeks of liquid nitrogen storage is 90–100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.     

Development of a new method of analysing chemotactic deactivation of human neutrophil granulocytes

Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessing the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemotaxin. This is called chemotactic deactivation. We developed a new deactivation chamber to determine chemotaxis and chemotactic deactivation of human PMN. This novel chamber is a modification of the commercially available acrylic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both blocks are separated by a polycarbonate membrane. PMN from the wells in the upper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific light absorbance. The chemotactic activity is quantified as a ratio of stimulated migration and random migration (chemotactic index=CI). For our novel chamber, only the upper blocks of this commercial chamber were connected like a sandwich, including a polyvinylpyrrolidone-free polycarbonate membrane with a pore size of 3 microm. The wells in the upper compartment were filled with 5 x 10(4) PMN and deactivating chemotaxin. The lower block was then filled with the chemotactic stimulus and the chamber was then incubated in humidified air with 5% CO2 atmosphere at 37 degrees C. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, sensitive and fast. The determination of chemotaxis and deactivation are performed in the same chamber, thus avoiding cell loss due to nonspecific adherence in other incubation tubes. The chamber can be used to characterize the chemotactic activity of chemoattractants of unknown structure via known and unknown receptors. This new chamber can be very helpful in detecting unknown chemotactic stimuli, which are not detectable by, for example, antibodies.