Interplay between shear stress and adhesion on neutrophil locomotion

Leukocyte locomotion over the lumen of inflamed endothelial cells is a critical step, following firm adhesion, in the inflammatory response. Once firmly adherent, the cell will spread and will either undergo diapedesis through individual vascular endothelial cells or will migrate to tight junctions before extravasating to the site of injury or infection. Little is known about the mechanisms of neutrophil spreading or locomotion, or how motility is affected by the physical environment. We performed a systematic study to investigate the effect of the type of adhesive ligand and shear stress on neutrophil motility by employing a parallel-plate flow chamber with reconstituted protein surfaces of E-selectin, E-selectin/PECAM-1, and E-selectin/ICAM-1. We find that the level and type of adhesive ligand and the shear rate are intertwined in affecting several metrics of migration, such as the migration velocity, random motility, index of migration, and the percentage of cells moving in the direction of flow. On surfaces with high levels of PECAM-1, there is a near doubling in random motility at a shear rate of 180 s(-1) compared to the motility in the absence of flow. On surfaces with ICAM-1, neutrophil random motility exhibits a weaker response to shear rate, decreasing slightly when shear rate is increased from static conditions to 180 s(-1), and is only slightly higher at 1000 s(-1) than in the absence of flow. The random motility increases with increasing surface concentrations of E-selectin and PECAM-1 under static and flow conditions. Our findings illustrate that the endothelium may regulate neutrophil migration in postcapillary venules through the presentation of various adhesion ligands at sites of inflammation.     

Effects of culture filtrates of Blastomyces dermatitidis on neutrophil locomotion

A potent chemotactic activity for neutrophils is detectable in liquid culture filtrates of Blastomyces dermatitidis. The production of this activity is medium-dependent and culture age-dependent. The highest levels of cytotaxin were produced in filtrates of B. dermatitidis grown in tissue culture medium 199 for three or more weeks. This factor(s) stimulates directed as well as random migration. It functions directly and independently of serum. It is stable at -20 degrees C and 56 degrees C, and has a molecular weight greater than 10000 daltons. These properties define a new microbial chemotactic factor.     

Effects of leukocyte random motility and chemotaxis in tissue inflammatory response.

A rapidly growing body of experimental evidence indicates that defects in leukocyte motility and chemotactic response correlate with increased susceptibility to and severity of bacterial infection in tissue. While this is understandable in qualitative terms, the sensitivity of the correlation is remarkable.In the present study, a theoretical analysis has been developed to relate the dynamics of bacterial growth to the growth and transport parameters of bacteria and leukocytes in tissue. The model considers a local tissue region in the vicinity of a venule and applies continuum unsteady state species conservation equations to the bacterial population, the phagocytic leukocytes, and a chemotactically active chemical mediator assumed to be produced by the bacteria. The analysis quantifies the effects of key parameters, such as leukocyte random motility and chemotactic coefficients, phagocytic and growth rate constants, and leukocyte vessel wall permeability, upon host ability to eliminate the bacteria.As an example, the model's predictions are compared to experimental results correlating inhibition of leukocyte chemotaxis by hemoglobin with its adjuvant action in experimental peritoneal infection by E. coli.     

The effects and comparative differences of neutrophil specific chemokines on neutrophil chemotaxis of the neonate

Neutrophil specific chemokines are potent chemoattractants for neutrophils. IL-8/CXCL8 is the most extensively studied member of this group, and its concentrations increase during inflammatory conditions of the newborn infant including sepsis and chronic lung disease. A significant amount of information exists on the effects of IL-8/CXCL8 on neutrophil chemotaxis of neonates, but little is known about the other neutrophil specific chemokines. The aim of this study was to determine the relative potency of the neutrophil specific chemokines on chemotaxis of neonatal neutrophils and to compare this effect with the effect on adult neutrophils. Neutrophils were isolated from cord blood or healthy adult donors and incubated in a Neuroprobe chemotaxis chamber. Chemokine concentrations ranging from 1-1000 ng/mL were used. Differences in chemotactic potency existed among the seven neutrophil specific chemokines. Specifically, at 100 ng/mL, the order was IL-8/CXCL8>GRO-alpha/CXCL1>GCP-2/CXCL6>NAP-2/CXCL7>ENA-78/CXCL5>GRO-gamma/CXCL2>GRO-beta/CXCL3. This pattern was observed for adult and neonatal neutrophils. We conclude that (1) neutrophils from cord blood exhibit the same pattern of potency for each ELR chemokine as neutrophils from adults, and (2) migration of neonatal neutrophils is significantly less than that of adults at every concentration examined except the lowest (1 ng/mL).     

Paradoxical effects of adenosine on neutrophil chemotaxis

Chemotaxis of rabbit neutrophils is most sensitive to inhibition by 3-deazaadenosine, followed by 3-deaza-(+/-)aristeromycin, 3-deaza-(+/-)aristeromycinylhomocysteine, 3-deazaadenosylhomocysteine, and adenosylhomocysteine, in that order. Although adenosine by itself had no effect on the chemotaxis of neutrophils, it essentially abolished the inhibitory effects of 3-deaza-adenosine on chemotaxis and the reduction of nitroblue tetrazolium. Paradoxically, adenosine enhanced the inhibition of chemotaxis by 3-deazaadenosylhomocysteine slightly and that of 3-deaza-(+/-)aristeromycin significantly. Adenosine alone unexpectedly inhibited phospholipid methylation to the same extent as 3-deazaadenosine, and reduced protein carboxymethylation to a lesser degree. The inhibition of these two methylation reactions by 3-deazaadenosine was, however, not substantially altered in the presence of adenosine. Drastic changes in the ratio of adenosylmethionine/nucleosidylhomocysteine were observed in the presence of adenosine, 3-deazaadenosine, 3-deaza-(+/-)aristeromycin, or of adenosine in combination with each of the latter compounds. There was no significant effect on the binding of chemotactic peptide to receptors, or on the ratio of ATP/ADP in cells treated by the analogs. These results suggest that the inhibition of methylation reactions per se is not enough to account for the inhibition of both chemotaxis and the reduction of nitroblue tetrazolium by neutrophils.     

T lymphocytes and neutrophil granulocytes differ in regulatory signaling and migratory dynamics with regard to spontaneous locomotion and chemotaxis

Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1-induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis.     

The influence of contact guidance on chemotaxis of human neutrophil leukocytes

Chemotaxis of human neutrophil leukocytes moving on or in aligned 3D fibrin gels is more efficient if the cells are moving along the axis of fibre alignment than if they have to cross the fibres. This was shown by using two assays, one in which the cells were responding to a distant (600 micrometers) gradient source diffusing from a filter paper impregnated with formyl-Met-Leu-Phe and incorporated into the gel, the other in which the cells were responding to nearby (20--30 micrometers) Candida albicans spores in serum. In the former assay, impairment of chemotaxis across the axis of fibre alignment was highly significant. In the latter, cells showed efficient chemotaxis to the spores, but took more irregular paths when crossing the aligned fibres than when running along them. Neutrophils show contact guidance in aligned collagen or fibrin gels (Wilkinson et al., Exp cell res 140 (1982) 55) [1], thus the cells were subjected simultaneously to two directional cues in these experiments, one the chemotactic gradient and the other a contact guidance field. These cues may reinforce or interfere with each other depending on their relative orientation. Since many tissues in vivo show alignment or more complex forms of patterning, tissue architecture is likely to be an important determinant of the efficiency of cellular mobilization in inflamed or infected sites.     

Oscillatory pericellular proteolysis and oxidant deposition during neutrophil locomotion.

To better understand the mechanism of leukocyte migration in complex environments, model extracellular matrices were prepared using gelatin, Hanks' solution, Bodipy-BSA (fluorescent upon proteolysis), and dihydrotetramethylrosamine or hydroethidine (fluorescent upon oxidation). Using quantitative microfluorometry, neutrophil-mediated extracellular pulses of reactive oxygen metabolites (ROMs) and pericellular proteolysis were periodically observed showing that these functions occur as quantal bursts. However, chronic granulomatous disease neutrophils, which do not produce ROMs, did not display ROM deposition. Matrices show an alternating pattern of green (proteolytic) and red (oxidative) fluorescence, indicating these functions are out of phase. Electric fields phase-matched with metabolic oscillations, which increase the amplitude of intracellular NAD(P)H oscillations, increase ROM deposition and pericellular proteolysis; this further supports the link between intracellular chemical oscillators and extracellular functions. This phase relationship may allow ROMs to inactivate protease inhibitors, followed by protease activation.     

A visual analysis of chemotactic and chemokinetic locomotion of human neutrophil leucocytes. Use of a new chemotaxis assay with Candida albicans as gradient source.

The locomotion of human blood neutrophil leucocytes was observed and analysed by time-lapse cinematography (1) under conditions where chemokinetic locomotion was stimulated, i.e. in a uniform concentration of casein; (2) in response to chemotactic gradients generated at a point-like source, namely blastospores of the pathogenic yeast Candida albicans in normal human plasma, and (3) in response to soluble chemotactic factors diffusing from Sephadex beads. Neutrophils moving in purely chemokinetic conditions tended to persist in straight paths and showed a preference for narrow angles of turn suggesting a “persistent random walk” type of locomotion rather than a pure random walk. Cells responding to Candida spores showed near straight-line locomotion to the gradient source over short distances (ca 50 μm) and brief time periods. They phagocytosed the spores on arrival and were usually immediately able to respond to a new gradient. Colchicine treatment caused the cells to turn through wider angles, but they were still able to home onto and phagocytose the spores. Colchicine-treated cells showed bizarre and fluctuating shapes but were nonetheless usually polarized towards the gradient source. Gradients from large sources, such as Sephadex beads containing soluble chemotactic factors, were more easily disturbed than those from Candida spores and directional locomotion of cells towards the beads was only seen in certain sectors. The angles of turn made by moving cells under these conditions were an important determinant of chemotaxis since paths of those cells reaching beads showed longer straight segments and narrower angles of turn than those which failed to show a directional response.     

The effect of Helicobacter pylori on neutrophil chemotaxis is independent of cagA

Sixteen Helicobacter pylori strains were studied in order to determine their neutrophil chemotactic activity and the association with the presence cagA gene. Neutrophil chemotactic activity was detected by a modified Boyden chamber method and the results were expressed in terms of chemotactic index (CI). The presence of cagA was determined by PCR. Of the 16 strains, eight were cagA+ and eight were cagA-. All of the isolated strains showed chemotactic activity. The mean value of CI of the patient group was significantly higher than the negative control (P < 0.01). The mean value of CI of zymosan-activated serum (P < 0.05) and the reference strain H. pylori NCTC 11637 (HP11637) (P < 0.01) was significantly higher than the patient group's mean value of CI. There were no statistical significance in the CI between cagA+ and cagA- strains (P > 0.05). It is concluded that H. pylori attracts neutrophils by chemotaxis, however, there is no association with cagA.     

Leucocyte locomotion and chemotaxis : The influence of divalent cations and cation ionophores

Human blood neutrophil leucocytes and monocytes incubated in the absence of Ca²⁺ and Mg²⁺ showed reduced, but still substantial migration into micropore filters towards chemotactic agents, compared with cells migrating in a divalent cation-rich medium. This reduction in migration could be reversed by adding low doses of divalent cation ionophores (X537A or A23187) to the Ca²⁺- and Mg²⁺-free medium which suggests that migrating leucocytes in media depleted of extracellular divalent cations can make use of intracellular divalent cations and that the intracellular cation exchange necessary for locomotion is facilitated by the ionophores. At higher doses, the ionophores inhibited locomotion, as did procaine which reduces membrane permeability to cations. Little effect of K⁺ depletion or of ouabain on leucocyte locomotion was noted.     

The equine neck and its function during movement and locomotion

During both locomotion and body movements at stance, the head and neck of the horse are a major craniocaudal and lateral balancing mechanism employing input from the visual, vestibular and proprioceptive systems. The function of the equine neck has recently become the focus of several research groups; this is probably also feeding on an increase of interest in the equine neck in equestrian sports, with a controversial discussion of specific neck positions such as maximum head and neck flexion. The aim of this review is to offer an overview of new findings on the structures and functions of the equine neck, illustrating their interplay. The movement of the neck is based on intervertebral motion, but it is also an integral part of locomotion; this is illustrated by the different neck conformations in the breeds of horses used for various types of work. The considerable effect of the neck movement and posture onto the whole trunk and even the limbs is transmitted via bony, ligamentous and muscular structures. Also, the fact that the neck position can easily be influenced by the rider and/or by the employment of training aids makes it an important avenue for training of new movements of the neck as well as the whole horse. Additionally, the neck position also affects the cervical spinal cord as well as the roots of the spinal nerves; besides the commonly encountered long-term neurological effects of cervical vertebral disorders, short-term changes of neural and muscular function have also been identified in the maximum flexion of the cranial neck and head position. During locomotion, the neck stores elastic energy within the passive tissues such as ligaments, joint capsules and fasciae. For adequate stabilisation, additional muscle activity is necessary; this is learned and requires constant muscle training as it is essential to prevent excessive wear and tear on the vertebral joints and also repetitive or single trauma to the spinal nerves and the spinal cord. The capability for this stabilisation decreases with age in the majority of horses due to changes in muscle tissue, muscle coordination and consequently muscle strength.     

The alterative effect of isobutylmethylxanthine in hypofunctional human neutrophil chemotaxis

The effects of isobutylmethylxanthine on the locomotion of chemotactically normofunctional and hypofunctional human neutrophils were compared. The hypofunctional cells were obtained from the peripheral blood of selected subjects with juvenile periodontitis. The chemically oriented migration (i.e. chemotaxis) of these cells was less than 50% of normal. Isobutylmethylxanthine caused a doubling of the CHO.Met.Leu.Phe.OH-induced chemotactic responsiveness of hypofunctional cells, while no effect of the drug was detected with respect to random migration. Isobutylmethylxanthine had no effect on chemotaxis or random migration in the normofunctional cell populations. Since isobutylmethylxanthine inhibits cAMP catabolism, these data suggest that the cyclic nucleotide plays a role in intracellular transduction mechanisms which affect chemotaxis.     

Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells

AMP-activated protein kinase (AMPK) is a key energy sensor, known to regulate energy metabolism in diverse cell types. Hypoxia is encountered frequently in the microenvironments of inflammatory lesions and is a critical regulator of function in inflammatory cells. Energy deficiency is one of the consequences of hypoxia, but its potential role in modulating leucocyte function has received little attention. Using micropore chemotaxis assays to assess migratory responses of the monocyte-like cell line U937, it was found that the AMPK activators AICAR and phenformin rapidly reduced random migration (chemokinesis) as well as chemotaxis due to stromal cell-derived factor (SDF)1alpha. There was an approximate 50% reduction in both chemokinesis and chemotaxis following 30 min preincubation with both AICAR and phenformin (P < 0.01), and this continued with up to 24 h preincubation. The binding of SDF1alpha to its receptor CXCR4 was unaltered, suggesting AMPK was acting on downstream intracellular signalling pathways important in cell migration. As AMPK and statins are known to inhibit HMG CoA reductase, and both reduce cell migration, the effect of mevastatin on U937 cells was compared with AMPK activators. Mevastatin inhibited cell migration but required 24 h preincubation. As expected, the inhibitory effect of mevastatin was associated with altered subcellular localization of the Rho GTPases, RhoA and cdc42, indicating decreased prenylation of these molecules. Although the effect of AMPK activation was partially reversed by mevalonate, this was not associated with altered subcellular localization of Rho GTPases. The data suggest that activation of AMPK has a general effect on cell movement in U937 cells, and this is not due to inhibition of HMG CoA reductase. These are the first data to show an effect of AMPK on cell movement, and suggest a fundamental role for energy deficiency in regulating cellular behaviour.     

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

mTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis

Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca²⁺ uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases.     

Inhibition of lymphocyte and neutrophil chemotaxis by pertussis toxin

The cells of the mammalian immune system possess special migratory properties within their in vivo environment, a surveillance characteristic that is thought to be important in the protection of the organism from transformants and exogenous pathogens. Pertussis toxin (PT) has been shown to disrupt the intensity of this process by seriously affecting lymphocyte recirculation in vivo. The mechanisms responsible for this inhibition were investigated by using the in vitro model systems of polymorphonuclear leukocyte and lymphocyte chemotaxis. The type of inhibition that was observed in these in vitro assay systems was quite similar to that observed in vivo, because PT could depress chemotaxis in vitro as well as the accumulation of radiolabeled lymphocytes and neutrophils within a peripheral site of inflammation in vivo. The alterations in neutrophil motility were found to be associated with a stimulus-specific inhibition of the triggering of superoxide anion generation and lysosomal secretion. Some inhibition of neutrophil adherence to plastic surfaces was also observed, most notably after augmentation of adherence with the chemoattractant fMLP. The observed alterations in cellular function after PT treatment occurred in the absence of defects in chemoattractant binding to the neutrophil cell surface, or of membrane potential changes stimulated by ligand binding. The effect of PT in this system was found to be associated with an abnormality in the regulation of intracellular free calcium, suggesting that the substrate for PT in neutrophils is involved in the regulation of calcium ion channels.