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J M Lackie

Publications and source records attributed to J M Lackie.

At least 37 records · Page 2Linked to original sources
PubMed1987

A critique of the methods used to assess leucocyte behaviour.

Methods for assessing the adhesive and locomotory properties of leucocytes are reviewed critically and a suggestion made as to the best strategy for testing an unknown compound on leucocyte behaviour. In particular the distinction is made between reductionist assays, where a single property is being investigated, and realistic assays where an attempt is made to mimic the situation in vivo. The realistic assays are often difficult (or impossible) to interpret in behavioural terms, because more than one cell activity is involved, but such assays (e.g. simple Boyden chambers), used with caution, may be preferable for initial screening, If effects are obvious in an assay of this type then more complex reductionist assays, to determine the cause of the altered behaviour, can be tried. In discussing adhesion, data from a flow-chamber system is presented to show the very rapid changes in adhesiveness when cells encounter an immune complex-coated surface. A fully automated tracking system for obtaining speed and persistence parameters for neutrophils is described, and some of the problems involved in estimating these parameters are illustrated. Movement of neutrophils in collagen gels provides a more realistic model of the environment in which they must operate in vivo, and the effects of incorporating immune complexes into such gels are reported.

Animals↗
PubMed1986

Neutrophil leucocyte chemotaxis is not induced by a spatial gradient of chemoattractant.

Chemotaxis and directed locomotion of neutrophil leucocytes are generally thought to be determined by the directed response of the cell to stable, spatial gradients of chemoattractants. In most cases, however, cells are also exposed to characteristic temporal changes in the attractant concentration during the lifetime of the gradient, especially as it develops. We have attempted to test whether neutrophils can respond to a spatial gradient in which these temporal changes are essentially absent. Gradients of formyl-peptides were made across a narrow barrier of agarose gel that separated two fluid reservoirs, and the cells were observed cinematographically as they moved between gel and glass. In gradients predeveloped at low temperature, at which cell motion and responses to attractant were inhibited, neutrophils showed no tendency to accumulate up-gradient when warmed to 37 degrees C. Yet their speed and turning behaviour was related to the local concentration of formyl-peptide. However, gradients that developed at 37 degrees C, whilst the cells were responsive, elicited directed locomotion. We also tested populations that were either spreading into or already evenly distributed across micropore filters to see how cells might sense directional cues. We reasoned that evenly distributed populations could accumulate in a spatial gradient only if cells were able to 'read' it. However, no redistribution occurred without an applied impulse of attractant. It seems that the oriented, temporal component of an attractant signal is essential if a directed response (i.e. non-random turning) is to occur; a spatial gradient of soluble attractant alone does not induce neutrophil accumulation or taxis. This finding has implications for the termination of the acute inflammatory response, for clinical tests of leucocyte behaviour and for morphogen signal interpretation by cells in developing tissues.

Animals↗
PubMed1986

Cellular proliferation in the vitreous: the use of vitreous explants as a model system.

An in vitro model of cellular proliferation in the vitreous has been developed using explants of bovine vitreous gel. Various cell types, including chick embryo pigmented retinal epithelium, choroidal fibroblasts, retinal glial cells, bovine retinal capillary endothelial cells, and peritoneal mouse macrophages, were cultured at 37 degrees C on vitreous gels for periods up to 8 days and compared for their effects on the structure of the gel. Choroidal fibroblasts caused a very marked reduction in the size of the gel and produced the strongest traction on the gel fibril structure; in contrast, peritoneal macrophages caused virtually no reduction in vitreous volume and little or no visible traction on the gel as detected by phase-contrast microscopy. The remaining cell types usually formed sheets on the surface of the gel; this was associated with an initial reduction in vitreous volume of approximately 50%, but little change thereafter. The cell mediated traction events on the structure of the vitreous gel were followed by time lapse video microscopy and by electron microscopy at various times after seeding of cells on the gels. The appearances corresponded closely with reported clinico-pathological studies of cellular proliferation in the vitreous. We believe that this in vitro model has several advantages over in vivo models of cellular proliferation in the vitreous, in that it permits analysis of individual cell behaviour and it is eminently suitable for pharmacologic manipulation.

Animals↗
PubMed1985

Studies on the locomotory behaviour and adhesive properties of mononuclear phagocytes from blood.

Mononuclear phagocytes isolated from peripheral blood move slowly on albumin-, collagen- and fibronectin-coated glass, as judged by analysis of time-lapse film, although their movement is stimulated somewhat by serum. When a differential-adhesion method is used to purify monocytes from a crude mononuclear cell fraction it appears to select a particularly slow-moving sub-group. Despite their slow rate of movement (especially in comparison with neutrophils) monocytes move over and penetrate monolayers of endothelial cells, apparently without difficulty; they are, however, restricted to the upper surface of a fibroblast monolayer. Penetration of reconstituted collagen gels by freshly isolated monocytes was not observed but cultured monocytes, which spontaneously detached from the culture substratum over a 48 h period, did invade collagen gels. The adhesive properties of these cultured cells, measured in a flow-chamber assay, did not differ from freshly isolated monocytes purified on serum-coated dishes and detached with EDTA, and their invasive capacity does not seem to be a consequence of altered adhesive properties. The behavioural differences between monocytes and neutrophils are considerable, although both cell types have to leave the circulation and penetrate tissues in vivo.

Animals↗
PubMed1984

Chemokinetic accumulation of human neutrophils on immune complex-coated substrata: analysis at a boundary.

The locomotory behavior of human blood neutrophil leukocytes was studied at a boundary between two surfaces with different chemokinetic properties. This was achieved by time-lapse cinematography of neutrophils moving on coverslips coated with BSA, then part-coated with immune complexes by adding anti-BSA IgG with a straight-line boundary between the BSA and the immune complexes. Cell locomotion was filmed in microscopic fields bisected by the boundary, and kinetic behavior was assessed by comparing speed (orthokinesis), turning behavior (klinokinesis), and the rate of diffusion of the cells on each side of the boundary, using a recently described mathematical analysis of kinesis. In the absence of serum or complement, the proportion of motile cells and their speed and rate of diffusion were greater on BSA than on antiBSA, but there was no consistent difference in turning behavior between cells on the two surfaces. The immune complexes were therefore negatively chemokinetic in comparison with BSA, and this resulted from a negative orthokinesis with little or no contribution from klinokinesis. As would be predicted theoretically, this resulted in gradual accumulation of cells on the immune complexes even in the absence of a chemotactic factor. In further studies, a parallel plate flow chamber was used to show that, under conditions of flow, neutrophils accumulated much more rapidly on a surface coated with BSA-anti-BSA than on BSA alone. Moreover, neutrophils on immune complex-coated surfaces lost their ability to form rosettes with IgG-coated erythrocytes. This suggests that neutrophils on immune complex-coated surfaces redistribute their Fc receptors (RFc gamma) to the under surface, and that the lowered speed of locomotion is due to tethering of neutrophils by substratum-bound IgG-Fc.

Antigen-Antibody Complex↗
PubMed1984

Adhesion of neutrophil leucocytes under conditions of flow.

By passing a suspension of polymorphonuclear leucocytes through a parallel-plate chamber their adhesiveness can be assessed by scoring the number of cells trapped on the lower plate, and the fluid shear stress can be defined for a given flow rate. Since the adhesiveness of the cell at the instant of collision must exceed the distractive shear if the cell is to stop, the kinetics of cell accumulation provide a measure of the adhesiveness of the leucocytes and the adhesive interaction can be quantified. This measure of adhesion does not suffer from the complication that the force required to remove the cells from the surface will be greater if the cells have the opportunity to spread before the distractive force is applied. The assay is described in detail and the results of modifying the surface of the flow chamber and altering the composition of the suspension medium are used to illustrate the method. Plasma proteins generally seemed to reduce the adhesiveness of neutrophil leucocytes, whether they were present as a coat of adsorbed protein or in the suspension medium during perfusion. Neutrophil leucocytes, unless suspended in relatively high concentrations of plasma, were considerably more adhesive than other cells that have been tested in this assay system.

Blood Flow Velocity↗
PubMed1983

Leucocyte locomotion: comparison of random and directed paths using a modified time-lapse film analysis.

A semi-automated method, using a graphic digitizing tablet, for analysing the the tracks of cells taken from time-lapse film is described and illustrated with sample data. Three film sequences of neutrophil leucocytes moving on protein-coated substrata are analysed, and in one of these the cells are moving in a non-random fashion. The randomness of the tracks is tested by an objective method. Cells in the non-random (directed) sequence move faster and make smaller angled turns. No correlation between step length and turning angle could be shown for the random sequences. The method described here should make it possible to undertake visual analyses of cell locomotion with considerably less effort.

Animals↗
PubMed1983

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.

Candida albicans↗
PubMed1983

Neutrophil behaviour in the presence of protease inhibitors.

Neutrophil leucocytes circulate in plasma, which contains broad-spectrum antiproteases such as alpha-2-macroglobulin and alpha-1-antitrypsin, and move into environments in which antiproteases are present. Plasma antiproteases significantly reduce the adhesiveness of leucocytes in both static and dynamic adhesion assays, although their action does not seem to depend upon their anti-protease capacity. Contrary to expectation, leucocyte movement over surfaces and through both rigid and deformable three-dimensional matrices (micropore filters and collagen gels) is unaffected by antiproteases. This renders the idea that forward movement depends upon the proteolytic cleavage of redundant adhesion sites less probable. Various non-physiological antiproteases (chloromethyl ketones, soya bean trypsin inhibitor, and epsilon-aminocaproic acid are also ineffective in reducing locomotion, although they do cause minor changes in adhesiveness.

Cell Adhesion↗
PubMed1983

Effect of modified alpha 2macroglobulin on leucocyte locomotion and chemotaxis.

The effects of human alpha 2macroglobulin (alpha 2M) on locomotion of human neutrophils and monocytes in micropore filter assays in vitro were studied. Native alpha 2M had no effect on cell locomotion. In contrast, alpha 2M which had been modified by interaction with proteases (trypsin, collagenase), or with ammonium sulphate, stimulated locomotion of both cell types. The degree of locomotory response induced in the cells by alpha 2M correlated closely with changes in molecular conformation of alpha 2M as estimated by measurements of binding of the fluorescent probe, 1-anilinonaphthalene-8-sulphonic acid (ANS). However neutrophil locomotion stimulated by modified alpha 2M appeared to be solely chemokinetic, whereas monocytes showed both chemokinetic and chemotactic responses to modified alpha 2M. The reason for this difference in response between the two cell types is unclear, but it is reflected in the lack of specific binding of alpha 2M-protease complexes by neutrophils. Earlier workers had shown specific binding of such complexes by mononuclear phagocytes.

Ammonium Sulfate↗
PubMed1981

Effect of hyaluronic acid on neutrophil adhesion.

The effects of hyaluronate on rabbit neutrophil adhesion were studied using a variety of techniques. Exogenous hyaluronate inhibited neutrophil aggregation under conditions of both turbulent flow and constant shear rate. Hyaluronate also inhibited neutrophil adhesion to glass. Inhibition was dose-dependent above 100 micrograms ml-1 and a minimum molecular weight for hyaluronate of 1 x 10(4) was required. These effects were not simply the result of increased bulk viscosity of the hyaluronate-containing medium, nor did they appear to be mediated by putative cell-surface receptor mechanisms. Instead, physical factors such as hindrance and/or changes in the interfacial free-energy exchange at the cell surface due to the unusual hydrodynamic properties of the hyaluronate molecule were considered to be more important. Since neutrophil migration in vivo occurs through hyaluronate-rich connective tissue matrices, the relevance of these findings for processes such as inflammation and wound healing is clear.

Animals↗