Immunology Today, vol. 6, No. 9, 1985
273
Random locomotion, chemotaxis and chemokinesis. A guide to terms defining cell locomotion P. C. Wilkinson There is still confusion about what is meant by widely used terms for cellular locomotion such as chemokinesis and random locomotion and how these are distinguishedfrom chemotaxis. Also, most work on leukocyte locomotion up till now has been done with neutrophils, whose locomotion is easy to study in vitro, and is directed towards thefairly straightforward end of accumulation at sites of infection and tissue injury in vivo. In this article, Peter Wilkinson gives an outline of the classification of locomotor behaviour of cells1and of the end results expectedfrom eachform of behaviour. T h e locomotion of lymphocytes and mononuclear phagocytes can probably be classified in the same way as that ofneutrophils, but the migrations of these cells in vivo are complex and poorly understood. M o r e information is needed about problems such as the mechanism of cell locomotion in lymphocyte homing (to T and B areas or to the thymus, for example), or lymphocyte-accessory cell clustering, and it will be necessary to be precise about what m a y turn out to be quite complex locomotor responses in these and other immunological phenomena. There is an excellent summary of the theory of locomotor behaviour in Ref. 2 for those who wish to explore further. F r o m observations of moving cells, their locomotion can be classified as random or non-random. The question that then needs to be asked is what has caused the cells to respond by moving either randomly or in particular directions. A different set of words is used to define basic forms of locomotion on the one h a n d and responses to environmental stimuli on the other (Table 1). Misconceptions have arisen from mixing these two categories. For example, the word chemokinesis has been used synonymously with r a n d o m locomotion, whereas chemokinesis actually describes a change in the intensity of random locomotion induced by a chemical stimulus. Another difficulty stems from designating agents as 'chemotactic' or 'chemokinetic', even though the cells' responses depend on the conditions under which they meet that agent. Neutrophils in suspension, when stimulated by certain chemical agents, change from a spherical to an elongated, locomotor, shape 3-5 (Fig. 1). This shape change can only be translated into locomotion when the cells are allowed to make contact with a substratum on or through which the cells can move. The locomotor behaviour they then show depends on the local information they can obtain about the attractant. For example, what is its concentration, and is that concentration uniform or changing?
Random locomotion and chemokinesis Random locomotion is r a n d o m in direction and the axis of the moving cell is not oriented in relation to a stimulus. T h e term usually refers to locomotion along a p a t h that is randomly oriented. If a single leukocyte is tracked, it does not show the mathematician's random walk ( i . e . Brownian motion) but moves most of the time in a smoothly curving path, occasionally rounding up and
starting off again in a different direction. Obviously, if we sample a very short track, e.g. if a neutrophil (which when stimulated moves at about 15/am rain- i) is tracked for less than 1 to 2 minutes, it seems that the cell is following a straight path. However, if we track the cell for a longer time, it will be found that the displacement it makes becomes random in direction. This is called a 'persistent random walk' and is shown by all locomotor tissue cells that have been studied 6-8, W e can also consider r a n d o m locomotion of cell populations as a whole, rather than that of single cells. Here we find that the directions taken by the different cells wilt be random and that the displacement and eventual distribution of the whole population will be random. A kinesis is a change in the intensity of random locomotion. Cells can respond to external cues with alterations of speed or frequency of turning, but not direction, so that the displacement can still be defined as random. If the cue is chemical, the response is called chemokinesis. T h e kinesis response has two elements with different effects, viz. Orthokinesis in which the speed, or frequency, of locomotion is determined by the magnitude of the stimulus (Figs 2(a), 3(a)), and Klinokinesis in which the frequency or amount of turning is determined by the magnitude of the stimulus (Fig. 2(a)). TABLEI Terms used for basic locomotor behaviour
Terms used when this behaviour is expressed as a response to environmental stimuli
Random locomotion
Kinesis* (a) classifiedby change in behaviour Orthokinesi8 (change in speed) Klinokinesis (change in rate of turn)
(b) classifiedby nature of stimulus, e.g. chemokinesis Directional locomotion
Taxis : directional response to stimuli, e.g. Chemotaxis Phototaxis Magnetotaxis, etc. Contact guidance
Department of Bacteriology and Immunology, Western Infirmary, University of Glasgow, Glasgow G11 6NT, UK.
* T h e t w o classes o f k i n e s i s a r e n o t m u t u a l l y exclusive, T h u s , one c o u l d h a v e o r t h o - c h e m o k i n e s i s o r a k l i n o - p h o t o k i n e s i s , etc. 23.
an
© 1985, Elsevier Science Publishers B.V., Amsterdam O167- 4919/85/$02.00
274
Fig.
Immunology Today, vol. 6, No. 9, 1985
i
(a) Neutrophils which are unstimulated and are in suspension remain rounded. (b) On addition of formyl-Met-Leu-Phe, the cells (still in suspension) change shape and assume the typical locomotor form with broad anterior lamellipodium and narrow tail. There is no preferred direction of polarization. If these cellsare placed on a surface and allowed to move, they will still show no preferred polarization unless they can get directional information from their environment. In a gradient of f-Met-Leu-Phe, (c) the cells can obtain this information and then show an accurate polarization towards the gradient source (to the right of the figure) and move by chemotaxis towards it. (a) and (b) frorn Ref. 5. (Bars = 10 gm.) Note that orthokinesis a n d klinokinesis describe changes which are not restricted to chemical stimuli, b u t m a y also be induced by the physical properties of the e n v i r o n m e n t . T h u s , they are not always cttemokineses. Orthokinesis seems a simple reaction. Signals can make cells speed u p or slow down, stop or start. It is simple so long as the orthokinetic agent is present in the system in
isotropic concentration. As soon as the concentration becomes anisotropic, things get complicated. T o take a n example (Fig. 2(b)), cells are placed in a field with a b o u n d a r y down the middle. O n one side the cells are able to move very fast; on the other side, they can only move slowly. T h e experiment starts with the cells evenly distrib u t e d across both sides of the field. W h a t is likely to h a p p e n is that ceils on the fast side will cross the b o u n dary a n d t h e n g e t stuck on the slow side. Cells on the slow side will have difficulty in reaching the b o u n d a r y a n d crossing to the fast side. T h e e n d result will be cell a c c u m u l a t i o n on the slow, not the fast side. Note that this a c c u m u l a t i o n occurs even t h o u g h the paths traversed by the cells are r a n d o m in direction on both sides. A model of this sort has b e e n tested experimentally, using surfaceb o u n d i m m u n e complexes to trap r a n d o m l y m o v i n g neutrophils 9. T h e neutrophils do slow d o w n a n d accumulate slowly on crossing from a surface coated with a protein such as serum a l b u m i n to the i m m u n e complex-coated surface. A couple of i m p o r t a n t points arise from this. First, in chemotaxis assays, where a gradient is set up, a bias in s u b s e q u e n t cell distribution m a y well result from a chemotactic response to the gradient, b u t it can also result from a change in orthokinetic locomotion along the gradient. This makes it difficult to draw conclusions about the contribution of taxis or kinesis to cell a c c u m u l a t i o n u s i n g a n assay like the filter assay that gives no i n f o r m a t i o n about the speed or direction of cells. Secondly, cells that are m o v i n g randomly m a y show preferential adhesion to other cells on chance collision, so that clusters will form. T h u s the formation of clusters of i m m u n e cells (e. g. lymphocytes with accesso W cells) m a y result from r a n d o m locomotion with preferential adhesion, from chemotaxis, or from both. These are examples where a cell is slowed by meeting a sticky surface or a n o t h e r sticky cell: sticky perhaps because of specific receptors. A n opposite situation might occur if cells were m o v i n g through connective tissue by a non-adhesive m e c h a n i s m . Lymphocytes move through collagen gels b y p u s h i n g blebs into gaps in the fibrous matrix a n d u s i n g these to gain purchase s°. Lymphocytes are non-adhesive cells a n d this is a n o n adhesive method for moving, b u t iflymphocytes reached a l a c u n a in the gel where the n e a r b y fibrous matrix was sparse, they might no longer be able to find a t t a c h m e n t points a n d might accumulate because there was no m e c h a n i s m for them to move away. In s u m m a r y ,
275
Immunology Today, vol. 6, No. 9, 1985 Fig. 2. Locomotor responses.
Cell paths are represented as straight lines joined by dots, the dots being the position of the cell centre at fixed time intervals (e. g. 1 minute). (a) Random locomotion and kinesis
Klinokinesis
Orthokinesis
Fast-moving cell
Slow-moving cell
In orthokinesis, the cell traces a random path. The orthokinetic response causes the cell to travel from A to B faster or slower but does not change the path traversed by the cell.
Klinokinesis changes the amount of turning done by the cell.
(b) Orthokinesis at a boundary
(d) Contact guidance
Cells move in a preferred axis, but in either direction.
Cell moving fast
Cell moving slowly
The chance of crossing L ~ R is greater than R ~ L . Gradual accumulation on R. (c) Chemotaxis
To a distant (or linear) source, the locomotion appears unidirectional.
J't, To a small source, such as a microorganism, cells will move radially towards a centre.
orthokinesis in an anisotropic e n v i r o n m e n t can result in cell accumulation. This a c c u m u l a t i o n is likely to be slower t h a n that resulting f r o m chemotaxis. Klinokinesis T h e i m p o r t a n c e of reactions that influence the a m o u n t of t u r n i n g done by leukocytes has not b e e n evaluated. It w o u l d seem likely that if cells followed r a n d o m paths with a lot of turning, they w o u l d not get as far as if they followed straighter paths (Fig. 3(a)). T h e r e is some e x p e r i m e n t a l evidence for this. In supraoptimal, but u n i f o r m , concentrations of chemotactic factor such as f - M e t - L e u - P h e , neutrophils seem to t u r n m u c h m o r e t h a n in optimal concentrations 5'11. A t the latter concentrations, the cells polarize well, but at the higher concentration, each cell puts out several pseudopods and has no defined polarization. T h i s m a y be why neutrophil displacement is poor in s u p r a o p t i m a l concentrations of f-Met-Leu-Phe. N o t e that, i f a cell adapts to a stimulus by a decline in its response as the stimulus is continued, this m a y influence the effect of the stimulus on cell behaviour. Bacteria n o r m a l l y show t u m b l i n g behaviour. If exposed to a chemoattractant, they respond and show straight runs b u t eventually adapt and r e s u m e t u m b l i n g . A sequence of similar events leads to a flux of cells upgradient by klinokinesis with adaptation, which is m o r e or less the same as chemotaxis and is usually defined as such, the m a j o r difference b e i n g lack of morphological orientation towards the source. T h e effect of adaptation on leukocyte b e h a v i o u r is still b e i n g e v a l u a t e d t2. T h e fact that orthokinesis and klinokinesis have b e e n discussed separately does not m e a n that, in practice, a stimulus m a y not affect both. T h i s applies to the other reactions u n d e r discussion of course.
Immunology Today, vol. 6, No. 9, 1985
276
Fig. 3. Cell distributions expected from different locomotor responses. Effects of locomotor responses on cell distribution can be represented on vector scatter diagrams2in which the start-point of the cell population (or of each cell) is the origin and the displacement oft_he cells is represented by circles.
Cells moving fast or turning infrequently
Cells moving slowly or turning frequently
(a) Chemokinesis (orthokinesis) in isotropic environment. If cells move fast or slowly, they will disperse to a greater or lesser extent, but there will be no net displacement from the origin. For klinokinesis, m o r e t u r n i n g = less dispersion; less turning = more dispersion.
(c) Chemotaxis
(b) Orthokinesisin an anisotropic e n v i r o n m e n t can lead to a skew distribution. Here cells move faster on the R t (although their tracks are random). Each arrow represents displacement of a hypothetical cell,
(d) Contactguidance
Gradient source Guidance axis
Taxis
No Taxis
Cells move directionally (up they-axis), these will be net displacement of the population and accumulation at the gradient source.
Cells prefer they-axis to the x-axis, but move in two directions. There is no net displacement and no accumulation, though distribution is non-random.
Immunology Today, vol. 6",No. 9, 1985
Directional locomotion, chemotaxis and contact guidance In directional locomotion, there is a preference for, or avoidance of, a particular direction or directions (though in this article, I shall ignore avoidance of particular directions). Two responses to the environment are known to cause directional locomotion in leukocytes; chemotaxis and contact guidance. Chemotaxis is a reaction by which the direction of locomotion of cells is determined by a field (e.g. a gradient) of a chemical stimulus and which, in leukocytes, is characterized by morphological orientation and directional locomotion of cells towards the source of the stimulus (Figs l(c), 2(c)). Note that there are other nonchemical taxes in non-immunological cells, e.g. phototaxis. Chemotaxis is by far the best studied of the behavioural reactions under discussion. It is an excellent mechanism for cell accumulation (Figs 2(c), 3(c)), and provides a likely explanation for the rapid influx of neutrophils into acute inflammatory lesions. O n the other hand, mechanisms for clustering immune cells, Where the n u m b e r of cells in a cluster is small, m a y be quite subtle, with short-range or transient signals. T h e defining features of chemotaxis, morphological orientation and directional locomotion, are easily demonstrable visually in neutrophils in gradients of factors such as C5a or f - M e t - L e u - P h e . These features are also demonstrable in mononuclear phagocytes 13 and probably in lymphocytes ~4, though in both cases there m a y be responding and non-responding subpopulations of blood or lymph node cells~5-~7.M a n y putative chemotactic factors have been documented using filter assays, but without clear evidence for the two essential features of chemotaxis, orientation and directional locomotion. In some cases, the checkerboard filter assay 18 has been used to demonstrate a chemotactic response. Some of the problems with this are discussed below. Contact guidance is a reaction by which the direction of locomotion of cells is determined by the shape, arrangement or curvature of the substratum. It is probably a response to physical, rather than chemical, properties of the environment. Developmental biologists have long debated the idea that migrations of cells can be directed by the patterning of the surfaces across which they move 19. A favourite substratum for such studies has been a fibrous gel of collagen which has been aligned by tension so that the collagen fibres run more or less parallel. Like other cell-types, neutrophils and lymphocytes migrating through collagen or fibrin gels prefer to migrate in the axis of alignment of the fibres2°. This is directional locomotion but not unidirectional (Figs 2(d), 3(d)). T h e cells are as free to move from A to B as from B to A in the preferred axis. Thus cells will not accumulate. Tissue patterning and contact guidance are probably important determinants of cell locomotion in vivo and influence the capacity of cells to carry out other locomotor responses. For example, neutrophil chemotactic responses are more efficient in the axis of a guidance field than at right angles to that axis ~.
Practical problems The distinctions between different locomotor res-
277 ponses seem to be clear, so why is there still confusion? O n e answer lies in the difficulties in distinguishing the different reactions experimentally. Addition of fM e t - L e u - P h e at optimal concentration to neutrophils in suspension increases the proportion of cells in locomotor shape from 5% to about 95% 2. Since these cells are floating, they cannot move. If placed on a suitable substratum, they will move rapidly and at random in an isotropic concentration of f - M e t - L e u - P h e (orthokinesis). If placed in a gradient, they will move directionally towards the gradient source (chemotaxis). Finally, as mentioned above, raising the f - M e t - L e u - P h e concentration may increase the frequency of turning and thus have a klinokinetic effect. So the response that the cells make to a single, defined factor differs with the experimental conditions. To distinguish a taxis, kinesis, or guidance response, we need to be able to measure cell speed, turning frequency, orientation and direction, i.e. ideally, we need to film the cells and analyse their tracks. If problems about the sorting-out of immune cells are to be solved, this will have to be done. However, at present, most workers are concerned with chemotaxis and want to answer the question 'Is this a chemotactic factor and is this a chemotactic response?' The simplest visual assay for answering this question directly is the orientation assay22, which does not require filming, but is not sensitive when the chemotactic response is not strong. More commonly an answer is attempted using an assay in which what is studied is not the behaviour of moving cells but the distribution they have achieved at the end of a chosen time. Inferences about cell behaviour from the final cell distribution are bound to be indirect. If cells move through a filter, or under agarose , in a gradient of a stimulus, this does not prove that they have shown a chemotactic response. For instance, attractants have a considerable effect in increasing the proportion of moving cells, an orthokinetic effect. A way round the difficulty of distinguishing chemotaxis from chemokinesis has been to use a series of chambers, some with different isotropic concentrations, some With different gradients of attractant across the fdter, i.e. the checkerboard filter assay18. This has been useful, though in many instances it has been misused since the calculation 18'24which permits comparison of the expected response of cells moving randomly in a gradient with the response obtained experimentally has been ignored. It is also probably invalid when using heterogeneous cell populations. Another problem arises in the clinical laboratory where 'chemotaxis' is measured in patients. Most of these patients probably do not have defects of chemotaxis as properly defined, but cells that for unknown reasons move poorly. It does seem important in the present state of the art to supplement indirect assays with visual assays which give direct information about cell behaviour. Only thus is it possible to evaluate the effects that the numerous reported 'chemotactic' factors actually have on cell locomotion. This will be especially important for unravelling the behaviour of cells of mixed lineages and stages of development such as lymphocytes. The end results of the different responses discussed here are so different that this is not simply an academic exercise. [~
278
Immunology Today, vol. 6, No. 9, 1985
Acknowledgement I thank Wendy Haston, Hansuli Keller and John Lackie for suggesting improvements in this manuscript.
References 1 2 3 4
Keller, H. U., Wilkinson, P. C., Abercrombie, M. et al. (1977) Clin. Exp. Immunol. 27,377-380 Dunn, G. A. (1981) In Biologyof the ChemotacticResponse(Lackie, J. M. and Wilkinson, P. C. eds), pp. 1-26 C.U.P., Cambridge Keller, H. U., Naef, A. and Zimmermann, A. (1984) Exp. Cell.Research 153, 173-185 Smith, C. W., Hollers, J. C., Patrick, R. A. and Hassett, C. (1979) J. Clin. Invest. 63,221-229 Shields, J. M. and Haston, W. S. (1985).]: CellSci. 74, 75-93 Gall, M. H. and Boone, C. W. (1970)Biophys.J. 10, 980-993 Nossall, R. and Zigmond, S. H. (1976)Biophys.J. 16, 1171-1182 Allan, R. B. and Wilkinson, P. C. (1978)Exp. CellRes. 111,191-203 Wilkinson, P. C., Lackie, J. H., Forrester, J. V. and Dunn, G. A. (1984)J, CellBiol. 99, 1761-1768
El
}
Monoelonal Antibody Technology: The Production and Characterisation of Rodent and Human Hybridomas (Laboratory Techniques in Biochemistry and Molecular Biology; Vol. 13) by Ailsa Campbell(series editors R. 1t. Burdon and R. H. van Knippenberg), Elsevier Biomedical Press, 1984. $26.50/Dfl. 69. O0 (xiv + 265) I S B N 0 444 80575 3 I read Ailsa Campbell's book on M o n o clonal Antibody Technology with a feeling of gratitude. This is t h e book to r e c o m m e n d to those who wish to know how to make monoclonal antibodies. Whilst there are now m a n y books and reviews in the literature on monoclonal antibody technology, most deal either with specialized or advanced appfica: tions of the reagents or are introductory reviews that cover the subject rather too briefly. This volume aims to cover most aspects o f hybridoma technology from the practical standpoint. It is directed primarily at the 'amateur immunologist': at those scientists who wish to enter hybridoma technology to solve problems in their own particular sphere, and, as such, it hits its mark. T h e book goes into considerable, yet highly readable, detail on the subjects of immunization, the selection and maintenance o f the cell lines, techniques for detection ofmonoclonal antibodies, cell fusion, the production and purification of the monoclonal antibodies, and the analysis and use of reagents. It covers mouse and rat hybridomas; it deals very briefly with the subject of T-cell hybridomas and also gives a discussion of h u m a n monoclonal antibody production as it exists cur-
I0 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Haston, W. S., Shields, J. M. and Wilkinson, P. C, (1982).f CelIBiol. 92,747-752 Keller, H. U. and Meier, G. BloodCells, in press Zigmond, S. H. and Sullivan, S. J. (1979)J. CellBiol. 82,517-527 Wilkinson, P. C. (1982) Immunobiology, 161,376-384 Wilkinson, P. C. (1985)J. Immunol. Methods, 76, 105-120 Cianciolo, G.J. and Snyderman, R. (1981)J. Clin. Invest. 67, 60-68 Falk, W. and Leonard, E. J. (1980) Infect. Immunity29, 953-959 Parrott, D. M. V. and Wilkinson, P. C. (1981) Progr. Allergy, 28, 193-284 Zigmond, S. H. and Hirsch, J. G. (1973)J. Exp. Med 137,387-410 Dunn, G. A. (1982) in CellBehaviour(Benairs, R., Curtis, A. S. G. and Dunn, G. A., eds) pp. 247-280, Cambridge Univ. Press, Cambridge Wilkinson, P. C., Shields,J. M. andHaston, W. S. (1982)Exp. CellRes. 140, 55-62 Wilkinson, P. C. andLackie, J.M.(1983)Exp. CellRes. 145,255-264 Zigmond, S. H. (1977).]. CellBiol. 75,606-616 Fraenkel, G. S. and Gunn, D. L. (1961) The Orientation of Animals, Dover, New York Lauffenburger, D. A., Rothman, C. and Zigmond, S. H. (1983) J. Immunol. 131,940-947
rently. T h e book aims to be as comprehensive as possible and has m a n a g e d to cover not only the basic procedures used in D r Campbell's own laboratory but points out some of the variations in technique and methodology that can be encountered. She gives a brief but clear explanation of the reasons for each step and the theoretical background which would help any scientist without extensive working knowledge in immunology to perform these techniques. Some idea of the usefulness of the book can be gained by noting that the subject headings include such matters as 'contamination' and 'how to cope with a fusion which is too successful'. T h e chapter on screening methods for the detection of hybridomas is particularly instructive and although I could quibble over some points (for example I would emphasize the use of immunohistological screening for cellular antigen specificities rather more than does Dr. Campbell) it would be unfair to say that such points are anything more than a difference in opinion. I particularly liked her chapter on animal handling. This is something that is rarely dealt with and I found this whole chapter both clear and concise. I also liked her cost analysis and felt that her continued emphasis on the disadvantages as well as the advantages ofmonoclonal antibodies to be extremely important. As she points out, it is
Idiotypy in Biology and Medicine edited by 1-1. Kohler, jr. Urbain and P. A. Cazenave, Academic Press Inc., 1983. £62.50 (464pages) I S B N 1 241 17780 8 This volume on idiotypy and its biological significance is well-organized and timely. Its appearance in 1984 coincides with the 10th anniversary of the publication of Neils J e r n e ' s now classic paper
important for anyone entering this field to first ask themselves 'what advantages may be realistically gained by the use of this technique a n d . . . (would) conventional a n t i s e r a . . , suffice'. I f I have any criticisms, it is in the area of the application of monoclonal antibodies, Admittedly this falls outside the remit of this book, but I would have liked just a little more emphasis on the advantages to be gained from using these reagents over those obtained with polyclonal antisera and such discussions would also have amplified some of the information on the characteristics of monoclonal antibodies. Such a criticism, however, is akin to complaining of the lack of wildfowl in a Bombay duck. T h e only other criticism is of the publishers: I feel that the price of the paper-back at $26.50 is high and the price of the hardback at $73 is prohibitive. However, as a valuable addition to the book-shelf I would r e c o m m e n d this book, not only to people wishing to learn about hybridoma technology but also to those already working in the field, as I feel there are few of us who could not learn something from it. ALISON GOODALL Department of Immunology, Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG, UK.
in the Annales d'Immunologie Institut Pasteur. It is also the year in which the Nobel prize committee recognized J e r n e ' s creative and powerful concept Of immunological networks. Edited by three recognized leaders in network investigation the volume contains 23 chapters written by experts, many of w h o m have experimentally confirmed some of the major predictions of the J e r n e hypothesis. T h e volume is organized into three sections that deal
273
Random locomotion, chemotaxis and chemokinesis. A guide to terms defining cell locomotion P. C. Wilkinson There is still confusion about what is meant by widely used terms for cellular locomotion such as chemokinesis and random locomotion and how these are distinguishedfrom chemotaxis. Also, most work on leukocyte locomotion up till now has been done with neutrophils, whose locomotion is easy to study in vitro, and is directed towards thefairly straightforward end of accumulation at sites of infection and tissue injury in vivo. In this article, Peter Wilkinson gives an outline of the classification of locomotor behaviour of cells1and of the end results expectedfrom eachform of behaviour. T h e locomotion of lymphocytes and mononuclear phagocytes can probably be classified in the same way as that ofneutrophils, but the migrations of these cells in vivo are complex and poorly understood. M o r e information is needed about problems such as the mechanism of cell locomotion in lymphocyte homing (to T and B areas or to the thymus, for example), or lymphocyte-accessory cell clustering, and it will be necessary to be precise about what m a y turn out to be quite complex locomotor responses in these and other immunological phenomena. There is an excellent summary of the theory of locomotor behaviour in Ref. 2 for those who wish to explore further. F r o m observations of moving cells, their locomotion can be classified as random or non-random. The question that then needs to be asked is what has caused the cells to respond by moving either randomly or in particular directions. A different set of words is used to define basic forms of locomotion on the one h a n d and responses to environmental stimuli on the other (Table 1). Misconceptions have arisen from mixing these two categories. For example, the word chemokinesis has been used synonymously with r a n d o m locomotion, whereas chemokinesis actually describes a change in the intensity of random locomotion induced by a chemical stimulus. Another difficulty stems from designating agents as 'chemotactic' or 'chemokinetic', even though the cells' responses depend on the conditions under which they meet that agent. Neutrophils in suspension, when stimulated by certain chemical agents, change from a spherical to an elongated, locomotor, shape 3-5 (Fig. 1). This shape change can only be translated into locomotion when the cells are allowed to make contact with a substratum on or through which the cells can move. The locomotor behaviour they then show depends on the local information they can obtain about the attractant. For example, what is its concentration, and is that concentration uniform or changing?
Random locomotion and chemokinesis Random locomotion is r a n d o m in direction and the axis of the moving cell is not oriented in relation to a stimulus. T h e term usually refers to locomotion along a p a t h that is randomly oriented. If a single leukocyte is tracked, it does not show the mathematician's random walk ( i . e . Brownian motion) but moves most of the time in a smoothly curving path, occasionally rounding up and
starting off again in a different direction. Obviously, if we sample a very short track, e.g. if a neutrophil (which when stimulated moves at about 15/am rain- i) is tracked for less than 1 to 2 minutes, it seems that the cell is following a straight path. However, if we track the cell for a longer time, it will be found that the displacement it makes becomes random in direction. This is called a 'persistent random walk' and is shown by all locomotor tissue cells that have been studied 6-8, W e can also consider r a n d o m locomotion of cell populations as a whole, rather than that of single cells. Here we find that the directions taken by the different cells wilt be random and that the displacement and eventual distribution of the whole population will be random. A kinesis is a change in the intensity of random locomotion. Cells can respond to external cues with alterations of speed or frequency of turning, but not direction, so that the displacement can still be defined as random. If the cue is chemical, the response is called chemokinesis. T h e kinesis response has two elements with different effects, viz. Orthokinesis in which the speed, or frequency, of locomotion is determined by the magnitude of the stimulus (Figs 2(a), 3(a)), and Klinokinesis in which the frequency or amount of turning is determined by the magnitude of the stimulus (Fig. 2(a)). TABLEI Terms used for basic locomotor behaviour
Terms used when this behaviour is expressed as a response to environmental stimuli
Random locomotion
Kinesis* (a) classifiedby change in behaviour Orthokinesi8 (change in speed) Klinokinesis (change in rate of turn)
(b) classifiedby nature of stimulus, e.g. chemokinesis Directional locomotion
Taxis : directional response to stimuli, e.g. Chemotaxis Phototaxis Magnetotaxis, etc. Contact guidance
Department of Bacteriology and Immunology, Western Infirmary, University of Glasgow, Glasgow G11 6NT, UK.
* T h e t w o classes o f k i n e s i s a r e n o t m u t u a l l y exclusive, T h u s , one c o u l d h a v e o r t h o - c h e m o k i n e s i s o r a k l i n o - p h o t o k i n e s i s , etc. 23.
an
© 1985, Elsevier Science Publishers B.V., Amsterdam O167- 4919/85/$02.00
274
Fig.
Immunology Today, vol. 6, No. 9, 1985
i
(a) Neutrophils which are unstimulated and are in suspension remain rounded. (b) On addition of formyl-Met-Leu-Phe, the cells (still in suspension) change shape and assume the typical locomotor form with broad anterior lamellipodium and narrow tail. There is no preferred direction of polarization. If these cellsare placed on a surface and allowed to move, they will still show no preferred polarization unless they can get directional information from their environment. In a gradient of f-Met-Leu-Phe, (c) the cells can obtain this information and then show an accurate polarization towards the gradient source (to the right of the figure) and move by chemotaxis towards it. (a) and (b) frorn Ref. 5. (Bars = 10 gm.) Note that orthokinesis a n d klinokinesis describe changes which are not restricted to chemical stimuli, b u t m a y also be induced by the physical properties of the e n v i r o n m e n t . T h u s , they are not always cttemokineses. Orthokinesis seems a simple reaction. Signals can make cells speed u p or slow down, stop or start. It is simple so long as the orthokinetic agent is present in the system in
isotropic concentration. As soon as the concentration becomes anisotropic, things get complicated. T o take a n example (Fig. 2(b)), cells are placed in a field with a b o u n d a r y down the middle. O n one side the cells are able to move very fast; on the other side, they can only move slowly. T h e experiment starts with the cells evenly distrib u t e d across both sides of the field. W h a t is likely to h a p p e n is that ceils on the fast side will cross the b o u n dary a n d t h e n g e t stuck on the slow side. Cells on the slow side will have difficulty in reaching the b o u n d a r y a n d crossing to the fast side. T h e e n d result will be cell a c c u m u l a t i o n on the slow, not the fast side. Note that this a c c u m u l a t i o n occurs even t h o u g h the paths traversed by the cells are r a n d o m in direction on both sides. A model of this sort has b e e n tested experimentally, using surfaceb o u n d i m m u n e complexes to trap r a n d o m l y m o v i n g neutrophils 9. T h e neutrophils do slow d o w n a n d accumulate slowly on crossing from a surface coated with a protein such as serum a l b u m i n to the i m m u n e complex-coated surface. A couple of i m p o r t a n t points arise from this. First, in chemotaxis assays, where a gradient is set up, a bias in s u b s e q u e n t cell distribution m a y well result from a chemotactic response to the gradient, b u t it can also result from a change in orthokinetic locomotion along the gradient. This makes it difficult to draw conclusions about the contribution of taxis or kinesis to cell a c c u m u l a t i o n u s i n g a n assay like the filter assay that gives no i n f o r m a t i o n about the speed or direction of cells. Secondly, cells that are m o v i n g randomly m a y show preferential adhesion to other cells on chance collision, so that clusters will form. T h u s the formation of clusters of i m m u n e cells (e. g. lymphocytes with accesso W cells) m a y result from r a n d o m locomotion with preferential adhesion, from chemotaxis, or from both. These are examples where a cell is slowed by meeting a sticky surface or a n o t h e r sticky cell: sticky perhaps because of specific receptors. A n opposite situation might occur if cells were m o v i n g through connective tissue by a non-adhesive m e c h a n i s m . Lymphocytes move through collagen gels b y p u s h i n g blebs into gaps in the fibrous matrix a n d u s i n g these to gain purchase s°. Lymphocytes are non-adhesive cells a n d this is a n o n adhesive method for moving, b u t iflymphocytes reached a l a c u n a in the gel where the n e a r b y fibrous matrix was sparse, they might no longer be able to find a t t a c h m e n t points a n d might accumulate because there was no m e c h a n i s m for them to move away. In s u m m a r y ,
275
Immunology Today, vol. 6, No. 9, 1985 Fig. 2. Locomotor responses.
Cell paths are represented as straight lines joined by dots, the dots being the position of the cell centre at fixed time intervals (e. g. 1 minute). (a) Random locomotion and kinesis
Klinokinesis
Orthokinesis
Fast-moving cell
Slow-moving cell
In orthokinesis, the cell traces a random path. The orthokinetic response causes the cell to travel from A to B faster or slower but does not change the path traversed by the cell.
Klinokinesis changes the amount of turning done by the cell.
(b) Orthokinesis at a boundary
(d) Contact guidance
Cells move in a preferred axis, but in either direction.
Cell moving fast
Cell moving slowly
The chance of crossing L ~ R is greater than R ~ L . Gradual accumulation on R. (c) Chemotaxis
To a distant (or linear) source, the locomotion appears unidirectional.
J't, To a small source, such as a microorganism, cells will move radially towards a centre.
orthokinesis in an anisotropic e n v i r o n m e n t can result in cell accumulation. This a c c u m u l a t i o n is likely to be slower t h a n that resulting f r o m chemotaxis. Klinokinesis T h e i m p o r t a n c e of reactions that influence the a m o u n t of t u r n i n g done by leukocytes has not b e e n evaluated. It w o u l d seem likely that if cells followed r a n d o m paths with a lot of turning, they w o u l d not get as far as if they followed straighter paths (Fig. 3(a)). T h e r e is some e x p e r i m e n t a l evidence for this. In supraoptimal, but u n i f o r m , concentrations of chemotactic factor such as f - M e t - L e u - P h e , neutrophils seem to t u r n m u c h m o r e t h a n in optimal concentrations 5'11. A t the latter concentrations, the cells polarize well, but at the higher concentration, each cell puts out several pseudopods and has no defined polarization. T h i s m a y be why neutrophil displacement is poor in s u p r a o p t i m a l concentrations of f-Met-Leu-Phe. N o t e that, i f a cell adapts to a stimulus by a decline in its response as the stimulus is continued, this m a y influence the effect of the stimulus on cell behaviour. Bacteria n o r m a l l y show t u m b l i n g behaviour. If exposed to a chemoattractant, they respond and show straight runs b u t eventually adapt and r e s u m e t u m b l i n g . A sequence of similar events leads to a flux of cells upgradient by klinokinesis with adaptation, which is m o r e or less the same as chemotaxis and is usually defined as such, the m a j o r difference b e i n g lack of morphological orientation towards the source. T h e effect of adaptation on leukocyte b e h a v i o u r is still b e i n g e v a l u a t e d t2. T h e fact that orthokinesis and klinokinesis have b e e n discussed separately does not m e a n that, in practice, a stimulus m a y not affect both. T h i s applies to the other reactions u n d e r discussion of course.
Immunology Today, vol. 6, No. 9, 1985
276
Fig. 3. Cell distributions expected from different locomotor responses. Effects of locomotor responses on cell distribution can be represented on vector scatter diagrams2in which the start-point of the cell population (or of each cell) is the origin and the displacement oft_he cells is represented by circles.
Cells moving fast or turning infrequently
Cells moving slowly or turning frequently
(a) Chemokinesis (orthokinesis) in isotropic environment. If cells move fast or slowly, they will disperse to a greater or lesser extent, but there will be no net displacement from the origin. For klinokinesis, m o r e t u r n i n g = less dispersion; less turning = more dispersion.
(c) Chemotaxis
(b) Orthokinesisin an anisotropic e n v i r o n m e n t can lead to a skew distribution. Here cells move faster on the R t (although their tracks are random). Each arrow represents displacement of a hypothetical cell,
(d) Contactguidance
Gradient source Guidance axis
Taxis
No Taxis
Cells move directionally (up they-axis), these will be net displacement of the population and accumulation at the gradient source.
Cells prefer they-axis to the x-axis, but move in two directions. There is no net displacement and no accumulation, though distribution is non-random.
Immunology Today, vol. 6",No. 9, 1985
Directional locomotion, chemotaxis and contact guidance In directional locomotion, there is a preference for, or avoidance of, a particular direction or directions (though in this article, I shall ignore avoidance of particular directions). Two responses to the environment are known to cause directional locomotion in leukocytes; chemotaxis and contact guidance. Chemotaxis is a reaction by which the direction of locomotion of cells is determined by a field (e.g. a gradient) of a chemical stimulus and which, in leukocytes, is characterized by morphological orientation and directional locomotion of cells towards the source of the stimulus (Figs l(c), 2(c)). Note that there are other nonchemical taxes in non-immunological cells, e.g. phototaxis. Chemotaxis is by far the best studied of the behavioural reactions under discussion. It is an excellent mechanism for cell accumulation (Figs 2(c), 3(c)), and provides a likely explanation for the rapid influx of neutrophils into acute inflammatory lesions. O n the other hand, mechanisms for clustering immune cells, Where the n u m b e r of cells in a cluster is small, m a y be quite subtle, with short-range or transient signals. T h e defining features of chemotaxis, morphological orientation and directional locomotion, are easily demonstrable visually in neutrophils in gradients of factors such as C5a or f - M e t - L e u - P h e . These features are also demonstrable in mononuclear phagocytes 13 and probably in lymphocytes ~4, though in both cases there m a y be responding and non-responding subpopulations of blood or lymph node cells~5-~7.M a n y putative chemotactic factors have been documented using filter assays, but without clear evidence for the two essential features of chemotaxis, orientation and directional locomotion. In some cases, the checkerboard filter assay 18 has been used to demonstrate a chemotactic response. Some of the problems with this are discussed below. Contact guidance is a reaction by which the direction of locomotion of cells is determined by the shape, arrangement or curvature of the substratum. It is probably a response to physical, rather than chemical, properties of the environment. Developmental biologists have long debated the idea that migrations of cells can be directed by the patterning of the surfaces across which they move 19. A favourite substratum for such studies has been a fibrous gel of collagen which has been aligned by tension so that the collagen fibres run more or less parallel. Like other cell-types, neutrophils and lymphocytes migrating through collagen or fibrin gels prefer to migrate in the axis of alignment of the fibres2°. This is directional locomotion but not unidirectional (Figs 2(d), 3(d)). T h e cells are as free to move from A to B as from B to A in the preferred axis. Thus cells will not accumulate. Tissue patterning and contact guidance are probably important determinants of cell locomotion in vivo and influence the capacity of cells to carry out other locomotor responses. For example, neutrophil chemotactic responses are more efficient in the axis of a guidance field than at right angles to that axis ~.
Practical problems The distinctions between different locomotor res-
277 ponses seem to be clear, so why is there still confusion? O n e answer lies in the difficulties in distinguishing the different reactions experimentally. Addition of fM e t - L e u - P h e at optimal concentration to neutrophils in suspension increases the proportion of cells in locomotor shape from 5% to about 95% 2. Since these cells are floating, they cannot move. If placed on a suitable substratum, they will move rapidly and at random in an isotropic concentration of f - M e t - L e u - P h e (orthokinesis). If placed in a gradient, they will move directionally towards the gradient source (chemotaxis). Finally, as mentioned above, raising the f - M e t - L e u - P h e concentration may increase the frequency of turning and thus have a klinokinetic effect. So the response that the cells make to a single, defined factor differs with the experimental conditions. To distinguish a taxis, kinesis, or guidance response, we need to be able to measure cell speed, turning frequency, orientation and direction, i.e. ideally, we need to film the cells and analyse their tracks. If problems about the sorting-out of immune cells are to be solved, this will have to be done. However, at present, most workers are concerned with chemotaxis and want to answer the question 'Is this a chemotactic factor and is this a chemotactic response?' The simplest visual assay for answering this question directly is the orientation assay22, which does not require filming, but is not sensitive when the chemotactic response is not strong. More commonly an answer is attempted using an assay in which what is studied is not the behaviour of moving cells but the distribution they have achieved at the end of a chosen time. Inferences about cell behaviour from the final cell distribution are bound to be indirect. If cells move through a filter, or under agarose , in a gradient of a stimulus, this does not prove that they have shown a chemotactic response. For instance, attractants have a considerable effect in increasing the proportion of moving cells, an orthokinetic effect. A way round the difficulty of distinguishing chemotaxis from chemokinesis has been to use a series of chambers, some with different isotropic concentrations, some With different gradients of attractant across the fdter, i.e. the checkerboard filter assay18. This has been useful, though in many instances it has been misused since the calculation 18'24which permits comparison of the expected response of cells moving randomly in a gradient with the response obtained experimentally has been ignored. It is also probably invalid when using heterogeneous cell populations. Another problem arises in the clinical laboratory where 'chemotaxis' is measured in patients. Most of these patients probably do not have defects of chemotaxis as properly defined, but cells that for unknown reasons move poorly. It does seem important in the present state of the art to supplement indirect assays with visual assays which give direct information about cell behaviour. Only thus is it possible to evaluate the effects that the numerous reported 'chemotactic' factors actually have on cell locomotion. This will be especially important for unravelling the behaviour of cells of mixed lineages and stages of development such as lymphocytes. The end results of the different responses discussed here are so different that this is not simply an academic exercise. [~
278
Immunology Today, vol. 6, No. 9, 1985
Acknowledgement I thank Wendy Haston, Hansuli Keller and John Lackie for suggesting improvements in this manuscript.
References 1 2 3 4
Keller, H. U., Wilkinson, P. C., Abercrombie, M. et al. (1977) Clin. Exp. Immunol. 27,377-380 Dunn, G. A. (1981) In Biologyof the ChemotacticResponse(Lackie, J. M. and Wilkinson, P. C. eds), pp. 1-26 C.U.P., Cambridge Keller, H. U., Naef, A. and Zimmermann, A. (1984) Exp. Cell.Research 153, 173-185 Smith, C. W., Hollers, J. C., Patrick, R. A. and Hassett, C. (1979) J. Clin. Invest. 63,221-229 Shields, J. M. and Haston, W. S. (1985).]: CellSci. 74, 75-93 Gall, M. H. and Boone, C. W. (1970)Biophys.J. 10, 980-993 Nossall, R. and Zigmond, S. H. (1976)Biophys.J. 16, 1171-1182 Allan, R. B. and Wilkinson, P. C. (1978)Exp. CellRes. 111,191-203 Wilkinson, P. C., Lackie, J. H., Forrester, J. V. and Dunn, G. A. (1984)J, CellBiol. 99, 1761-1768
El
}
Monoelonal Antibody Technology: The Production and Characterisation of Rodent and Human Hybridomas (Laboratory Techniques in Biochemistry and Molecular Biology; Vol. 13) by Ailsa Campbell(series editors R. 1t. Burdon and R. H. van Knippenberg), Elsevier Biomedical Press, 1984. $26.50/Dfl. 69. O0 (xiv + 265) I S B N 0 444 80575 3 I read Ailsa Campbell's book on M o n o clonal Antibody Technology with a feeling of gratitude. This is t h e book to r e c o m m e n d to those who wish to know how to make monoclonal antibodies. Whilst there are now m a n y books and reviews in the literature on monoclonal antibody technology, most deal either with specialized or advanced appfica: tions of the reagents or are introductory reviews that cover the subject rather too briefly. This volume aims to cover most aspects o f hybridoma technology from the practical standpoint. It is directed primarily at the 'amateur immunologist': at those scientists who wish to enter hybridoma technology to solve problems in their own particular sphere, and, as such, it hits its mark. T h e book goes into considerable, yet highly readable, detail on the subjects of immunization, the selection and maintenance o f the cell lines, techniques for detection ofmonoclonal antibodies, cell fusion, the production and purification of the monoclonal antibodies, and the analysis and use of reagents. It covers mouse and rat hybridomas; it deals very briefly with the subject of T-cell hybridomas and also gives a discussion of h u m a n monoclonal antibody production as it exists cur-
I0 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Haston, W. S., Shields, J. M. and Wilkinson, P. C, (1982).f CelIBiol. 92,747-752 Keller, H. U. and Meier, G. BloodCells, in press Zigmond, S. H. and Sullivan, S. J. (1979)J. CellBiol. 82,517-527 Wilkinson, P. C. (1982) Immunobiology, 161,376-384 Wilkinson, P. C. (1985)J. Immunol. Methods, 76, 105-120 Cianciolo, G.J. and Snyderman, R. (1981)J. Clin. Invest. 67, 60-68 Falk, W. and Leonard, E. J. (1980) Infect. Immunity29, 953-959 Parrott, D. M. V. and Wilkinson, P. C. (1981) Progr. Allergy, 28, 193-284 Zigmond, S. H. and Hirsch, J. G. (1973)J. Exp. Med 137,387-410 Dunn, G. A. (1982) in CellBehaviour(Benairs, R., Curtis, A. S. G. and Dunn, G. A., eds) pp. 247-280, Cambridge Univ. Press, Cambridge Wilkinson, P. C., Shields,J. M. andHaston, W. S. (1982)Exp. CellRes. 140, 55-62 Wilkinson, P. C. andLackie, J.M.(1983)Exp. CellRes. 145,255-264 Zigmond, S. H. (1977).]. CellBiol. 75,606-616 Fraenkel, G. S. and Gunn, D. L. (1961) The Orientation of Animals, Dover, New York Lauffenburger, D. A., Rothman, C. and Zigmond, S. H. (1983) J. Immunol. 131,940-947
rently. T h e book aims to be as comprehensive as possible and has m a n a g e d to cover not only the basic procedures used in D r Campbell's own laboratory but points out some of the variations in technique and methodology that can be encountered. She gives a brief but clear explanation of the reasons for each step and the theoretical background which would help any scientist without extensive working knowledge in immunology to perform these techniques. Some idea of the usefulness of the book can be gained by noting that the subject headings include such matters as 'contamination' and 'how to cope with a fusion which is too successful'. T h e chapter on screening methods for the detection of hybridomas is particularly instructive and although I could quibble over some points (for example I would emphasize the use of immunohistological screening for cellular antigen specificities rather more than does Dr. Campbell) it would be unfair to say that such points are anything more than a difference in opinion. I particularly liked her chapter on animal handling. This is something that is rarely dealt with and I found this whole chapter both clear and concise. I also liked her cost analysis and felt that her continued emphasis on the disadvantages as well as the advantages ofmonoclonal antibodies to be extremely important. As she points out, it is
Idiotypy in Biology and Medicine edited by 1-1. Kohler, jr. Urbain and P. A. Cazenave, Academic Press Inc., 1983. £62.50 (464pages) I S B N 1 241 17780 8 This volume on idiotypy and its biological significance is well-organized and timely. Its appearance in 1984 coincides with the 10th anniversary of the publication of Neils J e r n e ' s now classic paper
important for anyone entering this field to first ask themselves 'what advantages may be realistically gained by the use of this technique a n d . . . (would) conventional a n t i s e r a . . , suffice'. I f I have any criticisms, it is in the area of the application of monoclonal antibodies, Admittedly this falls outside the remit of this book, but I would have liked just a little more emphasis on the advantages to be gained from using these reagents over those obtained with polyclonal antisera and such discussions would also have amplified some of the information on the characteristics of monoclonal antibodies. Such a criticism, however, is akin to complaining of the lack of wildfowl in a Bombay duck. T h e only other criticism is of the publishers: I feel that the price of the paper-back at $26.50 is high and the price of the hardback at $73 is prohibitive. However, as a valuable addition to the book-shelf I would r e c o m m e n d this book, not only to people wishing to learn about hybridoma technology but also to those already working in the field, as I feel there are few of us who could not learn something from it. ALISON GOODALL Department of Immunology, Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG, UK.
in the Annales d'Immunologie Institut Pasteur. It is also the year in which the Nobel prize committee recognized J e r n e ' s creative and powerful concept Of immunological networks. Edited by three recognized leaders in network investigation the volume contains 23 chapters written by experts, many of w h o m have experimentally confirmed some of the major predictions of the J e r n e hypothesis. T h e volume is organized into three sections that deal
