Assays of leukocyte locomotion and chemotaxis
Introduction
Locomotion is generally agreed to be important for the accumulation of leukocytes at sites of inflammation and infection. It must also be important, though very little is known about this, for the complex migrations of immune cells through lymphoid tissues and from one tissue to another, e.g. egress of mature cells from bone marrow, ingress and egress in the thymus, sorting out of B and T cells in lymphoid tissues, or the homing of blood monocytes to become macrophages with diverse functions in diverse organs. Major functions of the immune system depend on the right cells being at the right place at the right time. The interactions necessary for a T-dependent immune response require contact of accessory cells with antigen-specific T and B cells and these cells may have to migrate considerable distances to meet. On contact with antigen, dendritic cells migrate from the periphery (e.g. skin) to the lymph nodes. The signals for these important migrations are shrouded in mystery at present. Is chemotaxis needed or not? Apart from inflammation, we know remarkably little about locomotor behaviour of cells of the immune system. This is a field waiting to be exploited. To do this successfully, reliable techniques are needed and probably new techniques will need to be devised.
After 100 years during which nothing was known about the first event of inflammation, namely the adhesion of leukocytes to vascular endothelium, the past 10 years have seen enormous advances in the understanding of adhesion and the molecules that control it. Leukocytes in contact with endothelium must detect chemotactic factors generated in the extravascular tissues, but these are likely to be washed away by the fast-flowing blood stream. However, if chemotactic factors bind to the surfaces of vascular endothelial cells, they can be detected as surface molecules rather than in solution (Huber et al., 1991, Tanaka et al., 1993). Much work is needed to substantiate this idea. Some believe that all, or nearly all, chemotaxis is explicable as a response to surface-bound molecules, or even that the only chemotactic event of major importance is transmigration through endothelium. While not denying the importance of the latter, anyone who has watched time-lapse films of phagocytes chasing micro-organisms (Fig. 1) will know that leukocytes have a lot of direction-finding to do after leaving vessels and, provided that they can gain purchase for forward movement, they can do it well on a very wide variety of surfaces or matrices, biological and artificial. Chemotactic responses to bacteria, studied by Metchnikoff (1893)are neglected at present, though there is still much to be discovered. The motor events seen in the cell on stimulation with an attractant can all occur in suspension (Shields and Haston, 1985), so they do not depend on adhesion. Subsequent locomotion may require adhesion or, in a three-dimensional (3-D) matrix, it may not (Haston et al., 1982). Assays are required both to allow the events of adhesion and locomotion to be studied in isolation and also to try to reproduce the real-life situation by putting them back together.
Section snippets
Terminology
In order to be clear about what our methods are measuring, we need clear definitions of the phenomena under study. Readers should be aware that there are internationally agreed definitions of the locomotor reactions of cells and a brief discussion of these is given below. What follows are comments rather than the precise definitions, which will be found elsewhere (Keller et al., 1977). Arriving at these definitions was possible only from the information given by visual studies of locomotor
Historical
Scientists, like journalists, are excited about today's (or tomorrow's) news, but uninterested in history. Since few readers will be familiar with the early work and since some of the methods that are still useful now have been in use for most of this century, it will be appropriate to introduce the review with some history (which may be defined for present purposes as an account of events that took place before the foundation of the Journal of Immunological Methods).
Leber (1888)an
The filter assay
At the time of publication of the first issue of the Journal of Immunological Methods, the micropore filter assay was the method of choice for most workers studying leukocyte locomotion and chemotaxis. It still is: this despite its imperfections and despite the fact that a number of alternatives are available. If this were all to be said and if this were a satisfactory state of affairs, there would be little point in writing a review on developments in the field during the past 25 years. But
Assays using collagen or fibrin gels
These assays are gaining in popularity. Seeding leukocytes onto the upper surface of a gel of collagen (Brown, 1982, Haston et al., 1982, Schor et al., 1983) or fibrin (Wilkinson and Lackie, 1983, Ciano et al., 1986, Lanir et al., 1988) and studying their ability to invade (Fig. 3a) in the presence or absence of attractants incorporated into the gel uses the same principle as the filter assay. It also has several advantages over that assay, notably that it provides a physiologically relevant
The shape-change assay. Morphological polarization of locomotor cells
This is the simplest assay of all and one of the most reliable (Haston and Shields, 1985). It is especially useful for analysis of leukocytes taken from blood and is based on the fact that non-motile blood leukocytes are spherical. If exposed to a chemoattractant, they go through a sequence of shape-changes beginning within a few seconds and complete in a few minutes (Zigmond and Sullivan, 1979, Shields and Haston, 1985, McKay et al., 1991) which result in the characteristic head–tail
The orientation assay
Zigmond (1977)introduced a chamber which allowed a direct demonstration of chemotaxis. This was a plastic or glass slide rather like a white-cell counting chamber with two troughs on either side of a bridge 1 mm in diameter. Cells were allowed to adhere to a coverslip which was inverted over the troughs and bridge. One of the troughs was filled with an attractant, the other with a control solution, thus allowing formation of a gradient across the bridge. Within 15–30 min, neutrophils became
Observation and analysis of cell movement
This brings us back to the early workers who first introduced time-lapse cinematography. Now that microscopes have been improved, computers have been introduced and time-lapse videorecording has replaced cinematography, filming and analysis of cell movement is much easier than it was for the pioneers. Many people think that filming cells is anecdotal and non-quantitative; something to fill in ten minutes in a long meeting. There is much more to it than this: the late Michael Abercrombie and the
Recreating the physiological environment
The assays discussed above were for the most part devised to simplify a complex situation and to provide methods to study locomotion separately from all the other events of an inflammatory or immune response. There is now a need to bring things together again and, for example, to study locomotion on and through vascular endothelium or through extracellular matrices. Migration in collagen or fibrin gels is a model for the latter function. Many investigators are now interested in developing
Conclusions
Cell locomotion is a complex form of behaviour varying in time and space. It is modified by the absolute concentration and by concentration gradients of chemical substances as well as by the patterning and architecture of tissues, the adhesive nature of the surfaces on which the cells move and whether the cells are moving on a 2-D surface (as on a vessel wall) or a 3-D matrix (as in extracellular tissues). No single assay system is capable of giving a true measure of all these influences. The
Acknowledgements
The author's work is supported by the Wellcome Trust.
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