Video analysis of chemotactic locomotion of stored human polymorphonuclear leukocytes

Previous studies of the storage of polymorphonuclear leukocytes (PMNs) have used an empirical approach to define "optimal" conditions. To date, no storage conditions have been described which satisfactorily preserve the chemotactic function of PMNs beyond 24 h. In an effort to define the precise nature of the storage lesion, we studied the chemotactic locomotion of freshly isolated PMNs and PMNs which had been suspended in citrate-phosphate-dextrose-adenine (CPD-A1) plasma and stored in PVC bags, at 20-22 degrees C for 24 h. We used time-lapse video recording and computer image analysis to quantitate the motion of PMNs migrating under agarose. The positions of individual motile cells were traced at 1-min intervals for 5 min. The following parameters were used to quantitate migration: speed (distance/min), persistence of locomotion index (velocity/speed), orientation angle (the angle of the vector describing the next displacement of a cell relative to a direct line toward the chemoattractant), and chemotropic index (cosine of the orientation angle). After 24 h of storage, the following changes were observed: fewer cells migrated, the speed of migrating cells was reduced by 25%, the persistence of locomotion index decreased by 7%, which indicates that migrating cells made slightly more/wider turns, and the chemotropic index was decreased by 30%, which indicates that migrating cells were less accurate in their orientation toward the chemoattractant. Apparently, the storage of PMNs selectively impairs the ability of some cells to orient accurately in a chemotactic gradient and changes the distribution of these locomotor parameters within the population.     

Tumor necrosis factor is chemotactic for monocytes and polymorphonuclear leukocytes

Human recombinant tumor necrosis factor (TNF) induced migration across polycarbonate and nitrocellulose filters of human peripheral blood monocytes and polymorphonuclear leukocytes, TNF was active in inducing migration at concentrations less than 1 U/ml, and maximal responses (observed at greater than 100 U/ml) were comparable to those elicited by standard reference chemoattractants (FMLP, 10 nM; activated human serum, 5%). Checkerboard analysis performed by seeding different concentrations of TNF above and below the filter revealed that maximal induction of migration required a positive concentration gradient between the lower and upper compartments and that TNF elicited an actual chemotactic response in phagocytes. An anti-TNF rabbit antiserum and anti-TNF mouse monoclonal antibody abolished the chemotactic activity of TNF. Recombinant lymphotoxin was also chemotactic for phagocytes, and its activity was blocked by an anti-lymphotoxin antiserum. Human umbilical vein endothelial cells and blood large granular lymphocytes did not respond chemotactically to TNF under conditions in which appropriate reference chemoattractants were active. The chemotactic activity of TNF may serve to recruit phagocytic cells from the blood compartment to amplify resistance against noxious agents.     

Soluble Fas ligand is chemotactic for human neutrophilic polymorphonuclear leukocytes

It has been recently shown that Fas ligand (FasL) expression on islet beta grafts results in neutrophilic infiltration and graft rejection. In this study, we show that human recombinant soluble FasL is endowed with potent chemotactic properties toward human neutrophilic polymorphonuclear leukocytes (neutrophils) at concentrations incapable of inducing cell apoptosis. Furthermore, neutrophils exposed to soluble FasL did not display detectable change of intracellular Ca2+ and did not undergo superoxide production or exocytosis of primary and secondary granules. Our results show that FasL is a potent chemoattractant for human neutrophils without evoking their secretory responses. This finding suggests a novel proinflammatory function for this ligand and may help to clarify the mechanism governing FasL-mediated graft rejection, thereby offering rational bases for controlling and modulating FasL-based immunotherapies.     

Chemotactic peptide receptor modulation in polymorphonuclear leukocytes

The binding of the chemotactic peptide N- formylnorleucylleucylphenylalanine (FNLLP) to its receptor on rabbit polymorphonuclear leukocytes (PMNs) modulates the number of available peptide receptors. Incubation with FNLLP decreases subsequent binding capacity, a phenomenon that has been termed receptor down regulation. Down regulation of the chemotactic peptide receptor is concentration dependent in both the rate and extent of receptor loss. The dose response parallels that of FNLLP binding to the recptor. The time- course is rapid; even at concentrations of FNLLP as low as 3 x 10(-9) M, the new equilibrium concentration of receptors is reached within 15 min. Down regulation is temperature dependent, but does occur even at 4 degrees C. Concomitant with down regulation, some of the peptide becomes irreversibly cell associated. At 4 degrees C, there is a small accumulation of nondissociable peptide that rapidly reaches a plateau. At higher temperatures, accumulation of nondissociable peptide continues after the rceptor number has reached equilibrium, and the amount accumulated can exceed the initial number of receptors by as much as 300%. The dose response of peptide uptake at 37 degrees C reflects that of binding, suggesting that it is receptor mediated. This uptake may occur via a pinocytosis mechanism. Although PMNs have not been considered to be pinocytic, the addition of FNLLP causes a fourfold stimulation of the rate of pinocytosis as measured by the uptake of [3H]sucrose.     

Specific binding sites for the phagocytosis stimulating peptide tuftsin on human polymorphonuclear leukocytes and monocytes

Highly purified and biologically active [³H]tuftsin (specific activity 9 Ci/mmol) was synthesized and its binding to several types of human circulating blood cells was studied at 22°C. The binding to polymorphonuclear leukocytes and to monocytes was found to be specific, fast, saturable and reversible. Values for the dissociation constants (K_D) were derived from equilibrium experiments and are 130 and 125 nM, respectively. The number of binding sites is approximately 50,000 and 100,000 per cell, respectively. Under the same experimental conditions lymphocytes exhibited only a threshold binding capacity for [³H]tuftsin whereas erythrocytes revealed no detectable binding.     

Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo

The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.     

Effects of microcystins on human polymorphonuclear leukocytes

Microcystins (MCs) are cyclic heptapeptides produced by cyanobacteria present in water contaminated reservoirs. Reported toxic effects for microcystins are liver injury and tumour promotion. In this study, we evaluated the effects of two MCs, MC-LR and [Asp(3)]-MC-LR, on human neutrophil (PMN). We observed that even at concentrations lower than that recommended by World Health Organization for chronic exposure (0.1 nM), MCs affect human PMN. Both MCs have chemotactic activity, induce the production of reactive oxygen species, and increase phagocytosis of Candida albicans. MC-LR also increased C. albicans killing. The effect of MCs on PMN provides support for a damage process mediated by PMN and oxidative stress, and may explain liver injury and tumour promotion associated to long-term MCs exposures.     

Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes

Leukotriene B4 (5(S),12(R)-di-hydroxy-eicosa-6,14-cis-8,10-trans-tetraenoic acid [LTB4]) is a product of the 5-lipoxygenation of arachidonic acid, which elicits human PMN leukocyte chemotactic responses in vitro that are 50% of the maximal level at concentrations of 3 X 10(-9) M to 10(-8) M and are maximal at 2 X 10(-8) M to 10(-7) M. The specific binding of highly purified [3H]LTB4 to human PMN leukocytes was assessed both by extracting the unbound and weakly bound [3H]LTB4 with acetone at -78 degrees C and by centrifuging the PMN leukocytes through cushions of phthalate oil to separate the unbound from bound [3H]LTB4. The levels of total binding of [3H]LTB4 and of nonspecific binding of [3H]LTB4, in the presence of a 1500-fold molar excess of nonradioactive LTB4, were approximately two times higher with the phthalate oil method. Scatchard plots of the concentration dependence of the specific binding (total - nonspecific binding) of [3H]LTB4 to PMN leukocytes were linear for the acetone extraction and phthalate oil methods and revealed dissociation constants of 10.8 X 10(-9) M and 13.9 X 10(-9) M, respectively, and mean of 2.6 X 10(4) and 4.0 X 10(4) receptors per PMN leukocyte. The 5(S),12(S)-all-trans-di-HETE analog of LTB4 and 5-HETE competitively inhibited by 50% the binding of [3H]LTB4 to PMN leukocytes at respective concentrations that evoked half-maximal chemotactic responses, whereas neither N-formyl-methionyl-leucyl-phenylalanine nor chemotactic fragments of C5 inhibited the binding. Human erythrocytes exhibited no specific binding sites for [3H]LTB4. Human PMN leukocytes possess a subset of receptors for LTB4 that are distinct from those specific for peptide chemotactic factors.     

Cytochalasin binding in lymphocytes and polymorphonuclear leukocytes

The binding of [³H]cytochalasin B (CB) to intact cells was compared in lymphocytes, polymorphonuclear leukocytes (PMNLs) and erythrocytes over a broad range of cytochalasin concentrations. Binding curves consistent with the presence of high and low affinity binding sites were demonstrated in all three cell types. However, in contrast to observations in erythrocytes, in lymphocytes and PMNLs CB binding was unaffected by d-glucose. p-Hydroxymercuribenzoate and p-hydroxymercurisulfonate were only partially inhibitory and unlabeled cytochalasins E, D and A (CE, CD, CA) inhibited [³H]CB binding more effectively than unlabeled CB. While attempts to demonstrate that plasma membrane-rich subcellular fractions from lymphocytes selectively bind [³H]CB were inconclusive, radioautographic studies on unbroken cells indicated that most or all of the high affinity CB-binding sites in lymphocytes and PMNLs were in close proximity to the cell surface.     

An actin-nucleating activity in polymorphonuclear leukocytes is modulated by chemotactic peptides

We examined the actin-nucleating activity in polymorphonuclear leukocyte lysates prepared at various times after chemotactic peptide addition. The actin nucleation increases two- to threefold within 15 s after peptide addition, decays to basal levels within 90 s, and is largely independent of cytoplasmic calcium fluxes. The peptide-induced nucleation sites behave as free barbed ends and therefore may increase the level of polymerized actin in vivo. The new nucleation sites may also determine the cellular sites of actin polymerization. This localization of actin polymerization could be important for the directional extension of lamellipodia during chemotaxis.     

The decreasedin vitro chemotactic activity of polymorphonuclear leukocytes of newborns and infants

Using Boyden's technique, a statistically significant decrease in the chemotactic activity of polymorphonuclear (PMN) leukocytes was found during the early postnatal period, i.e. in the cord blood and in blood of newborns within the first 10-15 d of life after stimulation of cells with both zymosan-activated adult serum (ZAS) and with an abacterial filtrate of Escherichia coli broth culture (ECF). After this period, the responsiveness of leukocytes to both chemotactic agents increased and remained at the same level during the whole observation period, i.e. up to the age of 6 months. Nevertheless even then it did not reach fully the responsiveness of the leukocytes of mothers and pregnant women. Zymosan-activated serum was shown to be a more potent chemotactic stimulus to leukocytes of infants as compared to the E. coli filtrate.     

High affinity binding of immunoglobulin G fragments to human polymorphonuclear leukocytes

Human neutrophil elastase splits IgG into Fc, Fabc, and Fab fragments. The Fc and Fabc fragments bind with high affinity (KD 2.1 and 2.5 nM respectively) to a small number of binding sites (1175 and 1370 sites/cell respectively) on untreated human polymorphonuclear leukocytes. Molecular mass determination of the binding site by crosslinking of Fc fragments to the neutrophils followed by SDS electrophoresis yields one band corresponding to a molecular mass of 67 kDa for the binding site. Incubation of neutrophils with rIFN-gamma (50 ng/ml, 18 h, 37 degrees C) enhances the expression of binding sites by about 6 fold to about 14,500 sites/cell, while the binding affinity and the molecular mass of the ligand receptor complex remain constant. By comparison with known affinities of leukocyte Fc receptors it is concluded that IgG fragments bind to the high affinity FcRI receptor of human neutrophils.     

Studies of leukotriene B4-specific binding and function in rat polymorphonuclear leukocytes: absence of a chemotactic response

Rat PMN isolated from peripheral blood show a small amount of high-affinity (specific) binding of [3H]-LTB4 at nanomolar concentrations. This binding is reversible and has a stereospecificity similar to rat PMN aggregation in response to several LTB4 analogs. This population of binding sites shares many characteristics with a population of high-affinity binding sites in human PMN; however, human PMN bind a significantly greater amount of [3H]-LTB4 to a second population of specific binding sites that is not present in rat PMN. The aggregation responses of human and rat peripheral blood PMN to LTB4 are similar in magnitude and specificity, but unlike human PMN, LTB4 fails to elicit a chemotactic response in rat PMN at concentrations from 10(-10) M to 10(-6) M. Rat PMN also fail to metabolize exogenous LTB4 when compared with human PMN. These data suggest that different PMN functions, such as chemotaxis and aggregation, may involve different classes of specific receptors. The finding that rat PMN do not exhibit chemotaxis to LTB4 calls for a reevaluation of the relevance to inflammation in humans of studies of inflammation performed in rat models.     

Nerve growth factor: a chemotactic factor for polymorphonuclear leukocytes in vivo

Previous studies have shown that mouse submandibular gland nerve growth factor (NGF) stimulates chemotactic migration of human polymorphonuclear leukocytes in vitro. The results of the present study demonstrate that subdermal injection of NGF in mice also stimulates rapid and marked chemotactic recruitment of leukocytes. This property of NGF is manifest in the nanomolar range of concentrations, it requires the known serine class protease activity of the growth factor, and it does not require participation of the fifth component of complement. Another, as yet unrecognized, C5-independent pathway must be involved. Chemotactic stimulation of cells involved in the early inflammatory response to injury may help to explain earlier observations that NGF can accelerate the rate of contraction of experimentally induced wounds in mice.     

Fucose-binding Lotus tetragonolobus lectin binds to human polymorphonuclear leukocytes and induces a chemotactic response.

Fucose-binding L. tetragonolobus lectin to the surface of human polymorphonuclear leukocytes (PMN) and induces a chemotactic response. Both surface binding and chemotaxis are inhibited by free fucose but not by fructose, mannose, or galactose. The lectin-binding sites on PMN are unrelated to the A, B, or O blood group antigen. Utilization of this lectin should be a useful tool in isolating PMN membrane components and in analyzing the mechanism of neutrophil chemotaxis.     

Effects of endotoxin on oxidative metabolism of polymorphonuclear leukocytes

The influence of endotoxin on rat polymorphonuclear leucocytes (PMN) ability to generate oxygen free radicals (OFR) has been studied by chemiluminescence method. PMNs derived from intact animals were used as a control. PMNs derived from animals with 1.5 h endotoxemia increased OFR production after stimulation by latex. In contrast, PMNs derived from intact animals and preincubated with endotoxin for 1.5 h decreased OFR production after stimulation bw latex. It was proposed that stimulating effect of endotoxin on PMNs in vivo was mediated by plasma components.     

Studies on the sodium and potassium transport in rabbit polymorphonuclear leukocytes

Rabbit polymorphonuclear leukocytes obtained from peritoneal exudates, incubated at 37°C. following exposure to 4°C., actively reaccumulate potassium while little or no net extrusion of sodium takes place. Preventing the utilization of oxidative metabolism with potassium cyanide, 2,4-dinitrophenol, or a nitrogen atmosphere does not inhibit the recovery process. Inhibitors blocking anaerobic glycolysis (sodium iodoacetate and sodium fluoride in low concentrations) completely abolish the capacity to reaccumulate potassium and cause a further dissipation of the sodium and potassium gradients. Water movements have been shown to be secondary to cation shifts. It is postulated that separate transport mechanisms exist for sodium and potassium and that the process of potassium reaccumulation relies on anaerobic glycolysis as a source of energy.