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Physiology of granulocyte locomotion and its relation to defects of chemotaxis: a review' P C Wilkinson MD Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow GIl 6NT
Since leukocytes play an active role in sites of inflammation, and are thought to migrate to those sites by chemotactic locomotion, clinicians are naturally interested to know whether the leukocytes of their patients, especially those patients with a predisposition to infection, are able to locomote normally. For such studies the usual assay employed measures the locomotion of the patient's white cells through a filter towards a chemotactic substance. Other basically similar in vitro assays, e.g. of cell migration through agarose, can also be used. Another method is to abrade a small area of skin and to cover the abrasion with either a coverslip or a sterile chamber containing fluid, and to measure the influx of cells into the abrasion. While in one sense more physiological than the in vitro tests, since performed using the living tissue of the patient, this skin window technique is more difficult to interpret, since the whole complex of the body's reaction to injury has been activated; thus there are a large number of uncontrollable variables, and it is an oversimplification to suggest that a defect in cell accumulation in skin windows is proof of a defect of chemotaxis. Although the documentation of leukocyte locomotion defects is filling an ever-increasing number of pages in the journals, and such defects have been postulated in a large number of disease states, there is very little literature which really probes exactly what abnormalities of leukocyte physiology may underlie a defect of locomotion. For example, the distinction between defects of random locomotion and defects of chemotactic locomotion, of which much was made in many of the earliest reports (Miller et al. 1971), and which is certainly of considerable importance, was based on an imprecise understanding of the locomotor reactions of leukocytes, and frequently on inadequate methods (such as migration from buffy coats in capillary tubes). It is clear that two distinct reasons for defective locomotor responses can be distinguished, namely, abnormalities of the cells themselves, and abnormal generation of the factors (e.g. complement peptides inter alia) which attract them into sites of inflammation and infection. Defects in generation of these factors give rise to severe abnormalities of body defence but are not the primary concern of this communication. I wish, rather, to outline some important aspects of leukocyte physiology which act as determinants of their locomotor reactions, and to recapitulate briefly what is known about the mode of locomotion of these cells and the reactions which they can show in response to environmental chemical substances. Locomotion of different leukocyte types: some observations on lymphocytes
Neutrophil locomotion has been studied more frequently than that of any other cell type, both at a basic level and clinically, and much of the discussion which follows will be devoted to these cells. It is established that other myeloid cells, eosinophils and basophils, and various types of mononuclear phagocyte, including blood monocytes, alveolar and peritoneal macrophages, are also motile and can show chemotactic reactions. The lymphocyte has long been known as a motile cell, and it is somewhat surprising, in view of the fact that this cell type is now being investigated by more people more intensively than any other cell in the body, that the literature 1 Paper read to Section of Clinical Immunology & Allergy, 13 February 1978. Accepted 21 March 1978, updated 31 May 1979
0 1 41-0768/79/080606-06/$O 1.00/0
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on lymphocyte locomotion is scanty and that chemotactic reactions of lymphocytes have been only very recently described. One possible reason for this is that lymphocytes taken from blood or from lymph nodes, and tested directly, are not highly motile and respond rather feebly to chemoattractants. The lymphocytes seem to require to be stimulated in a way which is not understood before they become fully motile. This can be achieved easily by culture in vitro for 2 to 3 days in serum-containing media with or without mitogens (Wilkinson, Roberts et al. 1976, O'Neill & Parrott 1977). Likewise, lymphocytes from lymph nodes draining a site of antigenic challenge are more motile than those from unstimulated nodes (Russell et al. 1975). Another source of highly motile lymphocytes is the human cultured B-lymphoblast lines (Russell et al. 1975). Using these various populations, it can be established that lymphocytes respond to most of the chemotactic factors, e.g. activated serum, denatured proteins, formyl peptides, lymphokine chemotactic factors (Wilkinson, Russell et al. 1976 and unpublished observations, Ward et al. 1977), that neutrophils recognize. In addition, lymphocytes from primed lymph nodes are capable of locomotion to the priming antigen (Wilkinson et al. 1977) and polyclonal activators at low doses can induce locomotor responses in lymphocytes (Wilkinson, Roberts et al. 1976). One of the major problems in working with lymphocytes is their heterogeneity. Both T and B cells can locomote but there may be differences among subpopulations; for example, Parrott et al. (1978) have recently suggested that the human TVi subpopulation is more active in locomotion than the Ty subpopulation. Since Tp is a helper and T-y a suppressor population, this suggests that locomotor differences may parallel differences in other lymphocyte functions.
Visual observations of neutrophil leukocyte locomotion There are numerous descriptions of the locomotion of neutrophils in vitro, but many fewer of locomotion of the other blood cells. Nevertheless, it is probable that the locomotor process in all of these cells is similar. If a population of neutrophils is placed on a glass slide in the absence of protein, the cells flatten on the glass and do not move. If protein-coated glass is used or protein is added to the flattened cells, the cells begin to locomote. The first event observed is that the cells change from a rounded form to a polarized, elongated form, with a broad hyaline leading edge or lamellipodium which is rich in actin and myosin but which excludes the nucleus and cytoplasmic organelles (Figure 1). It must be emphasized that this polarization is an
g
C'*
-
.
... ...
Figure 1. Interference-contrast photomicrograph of a locomoting blood neutrophil. The cell is moving from left to right. Note the thin hyaline anterior lamellipodium (arrowed: a) which excludes the cytoplasmic organelles found posteriorly to it in the main body of the cell. Note also the posterior retraction fibres (arrowed: b). x 2500. (Photograph by R B Allan)
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intrinsic property of locomoting cells and is not imposed by gradients or other stimuli from without. The polarized cells begin to move around but not in any particular direction, their locomotion being best described as a 'persistent random walk' (Allan & Wilkinson 1978). The locomotion is a crawling motion on a surface and involves sustained and controlled contractions and relaxations of microfilaments over a long period of time, giving rise to an amoeboid movement. Sustained locomotion poses especial problems, since one-off signals, such as are adequate to explain one-off events like exocytosis in mast cells, are inadequate to account for continued locomotion over a period of several hours. Chemical substances in the environment of the cell can influence this locomotion in various ways (Keller, Wilkinson et al. 1977). Firstly, chemical substances can determine the rate of cell locomotion or the frequency of turning of the cell, a reaction known as chemokinesis. For example, it is now clear that leukocytes show a dose-dependent increase in locomotion rate as the concentration of serum albumin in their environment is increased (Wilkinson et al. 1977, Wilkinson & Allan 1978a, Keller, Hess & Cottier 1977, Keller et al. 1978). It is likely that this is because albumin coating the surface on which the cell moves determines the degree of adhesion of the cells to the surface. Other proteins may play a similar role. Secondly, chemical substances can determine the direction of locomotion, a reaction known as chemotaxis. Chemotaxis alone would be likely to cause cells to migrate up a concentration gradient of a chemical to its source and to allow the cells to accumulate there. Chemokinesis alone would not do this, rather it would lead to dispersal of cells. A point which arises from the distinction between chemotaxis and chemokinesis is that the methods used for clinical studies often do not distinguish between these two reactions. Thus migration of cells through a filter towards a chemical placed in a lower chamber may be chemotactic, but it could also simply be that the chemical was causing an accelerated but nondirectional locomotion. In this case the cells would disperse according to the diffusion laws and, under chemokinetic conditions, many might travel through the filter. Special techniques are required to distinguish these reactions. The best ofthese filter assays is the 'checkerboard' assay of Zigmond & Hirsch (1973), in which a series of chambers is set up with various attractant concentrations above and below the filter. Even more satisfactory, but requiring more expertise, are visual assays, using time-lapse filming, in which the paths taken by individual cells can be determined directly. Using visual assays, it can be established that neutrophils respond to a chemotactic gradient by polarizing with the leading lamellipodium facing the gradient source before they begin to move (Zigmond 1974). They then move in a straight path to the gradient source. This means that the chemotactic gradient imposes an external constraint on the intrinsic polarization of the cell, so that the cell becomes orientated up-gradient. This is probably achieved by a 'spatial' gradient-detecting system by which the cell is able to determine that a greater number of receptors at the front of the cell have bound attractant than at the back. Neutrophils are able to detect 1% differences in attractant concentration across their diameter by these means (Zigmond 1977). One other finding which use of these assays has established is that many chemotactic factors, which cause cells to migrate in a gradient, can also act as chemokinetic agents, i.e. they influence both the rate and the direction in which cells move. This probably leads to more efficient accumulation of cells at gradient sources than chemotaxis alone. Possible physiological levels of locomotor defects From the above description, it is apparent that clinical defects of neutrophil locomotion might result from a disorder of the locomotor process itself; from defects in the ability to accelerate in response to chemokinetic signals or to adhere correctly to chemokinetic surfaces; or from defects in the direction-finding mechanism, i.e. in chemotaxis proper. The tests used to date have hardly been adequate to distinguish these, but the distinction is, in practice, possible by some variant of the checkerboard assay or by filming the cells of patients. Defects in chemotaxis itself could result from 'blindfolding' of the cells, due either to lack of receptors or to blockage of receptors by extrinsic material, e.g. from serum, thus reducing the efficiency of the gradient detection system. Defects of locomotion might be expected if there were
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abnormalities of signal transduction across the plasma membrane of the cell. I shall therefore briefly summarize what is known about recognition, transduction and the mechanochemical events of locomotion in leukocytes. These areas are under active investigation and the remarks below are likely to be outdated rapidly.
Chemotactic recognition The problem of what leukocytes recognize is an intriguing one. Numerous chemotactic factors have been described and some of these have been defined. They include proteins, e.g. denatured proteins (Wilkinson & McKay 1974) or as- and ,B-casein, both of which are anionic amphipathic molecules, and large peptides such as the C5-derived complement peptides. More recently, some low-molecular-weight chemotactic factors have been isolated. These include fatty acids, especially hydroxylated or peroxidized unsaturated fatty acids (Sahu & Lynn 1977). One of these, HETE, is a hydroxylated arachidonic acid derivative believed to be released from aggregated platelets by the action of a lipoxygenase (Turner et al. 1975), so clearly of interest in inflammation. A number of small peptides have also been shown to be chemotactic. These include the eosinophil chemotactic factors Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu (Goetzl & Austen 1975) and the formylated peptides which attract neutrophils. This is a class of hydrophobic, anionic di- and tripeptides with the N-terminal blocked by formylation and a free C-terminus (Schiffmann et al. 1975, Showell et al. 1976). Many described in the literature are formyl-methionyl peptides, but N-terminal methionine does not seem essential and f-tyrosyl, fphenylalanyl and f-leucyl peptides are also active. Peptides such as f-Met-Leu-Phe or f-NorleuLeu-Phe are active at about 10. 9M, and show saturable binding to the surfaces of neutrophils with an association constant around 109 litres per mole (Aswanikumar et al. 1977, Williams et al. 1977). Thus there seem to be receptors for these peptides, though the receptors are able to bind quite a wide range of molecules. Earlier, from our studies of denatured or conjugated protein chemotactic factors, we had come to the conclusion that hydrophobicity - or rather, amphipathicity - of these proteins was an important determinant of their chemotactic activity (Wilkinson 1976) and studies with lipid-specific toxins suggested that membrane lipids were important in their binding to the cell surface (Wilkinson 1975). We have therefore done binding studies with denatured serum albumin which suggested that this, too, bound in a saturable fashion with a Ka of around 106 litres per mole and about 106 binding sites per cell (Wilkinson & Allan 1978b). Thus there are probably saturable binding sites for chemotactic factors of all types, though they may be catholic, rather than highly specific, in the molecules which they bind. While discussing chemotactic peptides, it is worth mentioning that the major peptide chemotactic activity from complement is C5 derived (C5a and similar peptides). Despite wide quotation as such, including citation in most textbooks, C3a is not a chemotactic factor (Fernandez et al. 1978). Stimulus response coupling The problem of how binding activates intracellular events is under active investigation. Recent studies have shown that addition of f-Met-Leu-Phe to neutrophils is followed immediately by an influx of Na+ and by fluxes of Ca2" (Naccache et al. 1977). Moreover, it has recently proved possible to insert microelectrodes into macrophages and to show that addition of chemotactic factor causes changes in transmembrane potential, namely, a brief depolarization followed by a longer hyperpolarization (Gallin & Gallin 1977). Leukocyte locomotion is mediated by the same proteins, actin and myosin, as muscle contraction. In muscle, contractile events are initiated by ion fluxes and the ability of myosin to bind to actin is controlled by the cytoplasmic level of Ca2 . This increases to allow contraction and the calcium is then pumped back into the sacroplasmic reticulum, allowing relaxation. It is not known how far analogies between tissue cells and muscle can be taken, but it is probable that ion fluxes will prove to be crucial to locomotion of leukocytes. There have also been some studies of the role of cyclic nucleotides in controlling leukocyte locomotion. These are probably best summarized as not yet pointing to any crucial role for these molecules.
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Actin-myosin filaments The major protein constituent of the leukocyte cytoplasm is actin. Myosin is also found there, but in smaller amounts. Microfilaments are formed of long strands of polymerized actin and, presumably, also of myosin. When polymerized these proteins give cytoplasm the consistency of a gel, and this has resuscitated the old idea that amoeboid locomotion involves sol-gel transformations in the cytoplasm which control cytoplasmic forward flow in the moving cell. Actin filaments certainly require to be anchored to membrane in order that the sliding of myosin on actin may provide sufficient purchase to result in a contraction, but the precise mechanism of this anchoring has not been established. Actin-containing microfilaments are not distributed uniformly in leukocytes, but are concentrated at sites of movement, i.e. in the leading lamellipodium, at sites of adhesion to surfaces, at sites of attachment of phagocytosable objects, etc. Thus, in an active cell, the filament assembly requires to be built up and dismantled rapidly in different places according to the cell's demands. Hartwig & Stossel (1975) and Stossel & Hartwig (1975) have suggested that a membrane-attached actin-binding protein can act as a nucleation point for actin filaments and thus, by redisposition of this actin-binding protein within the cytoplasm, networks of filaments can rapidly be built up or broken down at any given site in the cell. Furthermore, a child with a severe predisposition to infections and a locomotor defect was shown to have a defect in actin polymerization (Boxer et al. 1974). This remains an almost unique case in that the molecular disorder underlying the abnormality of locomotion was pinpointed. Much remains unknown about the detailed function of microfilaments. However, since they are crucial to such varied tasks as locomotion, phagocytosis, exocytosis, organelle fusion and mitosis, their control is likely to be exquisite. The role of microtubules in leukocyte locomotion is still unclear. These are widely regarded as serving as a cytoskeleton which maintains shape changes such as are required for leukocyte polarization. It has been suggested that microtubule depolymerization (e.g. by colchicine or vinblastine) does not paralyse locomotion, but does abolish chemotaxis by interfering with the cell's ability to polarize in gradients. This is probably too simple. Colchicine-treated leukocytes show bizarre shapes and a 'drunken-walk' type of locomotion, but they still orient accurately and respond to chemotactic gradients (Allan & Wilkinson 1978). It has recently been suggested that the underlying abnormality in Chediak-Higashi syndrome leukocytes is a defect of microtubules, and that this is the reason why these patients' neutrophils show defective locomotion (Boxer et al. 1976, Oliver & Zurier 1976). However, it is probably premature to assign a definite role to microtubules in locomotion.
Conclusion From the brief account given above, it will be recognized that in theory defects of leukocyte locomotion could result from abnormalities at any of the stages in conversion of a sensory signal to a motor response and from defects in the molecular apparatus for locomotion. Indeed, it is fairly easy to induce such defects in vitro by incubating normal leukocytes with drugs which interfere with leukocytes at various levels. For example, anaesthetic agents, inductional, inhalational or local, cause a reversible paralysis of leukocyte locomotion (Moudgil et al. 1977); these agents are known to reduce membrane permeability, and thus the efficiency of ion transfer across the membrane, as well as causing disappearance of submembranous filaments as seen by electron microscopy. Inhibitors of glycolytic pathways also inhibit locomotion (Carruthers 1967), perhaps because they deprive the cell of energy sources used in locomotion. Myosin is an adenosine triphosphatase and requires ATP for its function. Leukocytes infected with influenza or herpes simplex viruses show depressed locomotion (Kleinermann et al. 1974, Larson & Blades 1976, Rabson et al. 1977), an effect which can apparently be reversed with the drug, levamisole, which raises intracellular levels of cyclic guanosine monophosphate. Recent results from our laboratory and from Kay et al. (1978) suggest that immunoglobulin or immune complexes bound to the Fc receptors on the cell surface may interfere with chemotactic responses. Polymeric IgA and IgA myeloma proteins bind to neutrophils and act as especially good inhibitors of leukocyte chemotaxis (Van Epps & Williams 1976).
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By these approaches it is possible to dissect the various levels at which the locomotor process can be interfered with. It is to be hoped that thorough investigation of the defective granulocytes found in various disease states will provide an equally fruitful approach. Fairly sophisticated techniques will be required, such as have not been applied extensively yet, if the sites of the lesions in the cells of these patients are to be established with precision.
References Allan R B & Wilkinson P C (1978) Experimental Cell Research 111, 191 Aswanikumar S, Corcoran B, Schiffmann E, Day A R, Freer R J, Showell H J, Becker E L & Pert C B (1977) Biochemical and Biophysical Research Communications 74, 810 Boxer L A, Hedley-Whyte E T & Stossel T P (1974) New England Journal of Medicine 291, 1093 Boxer L A, Watanage A M, Rister M, Besch H R, Allen J & Baehner R L (1976) New England Journal of Medicine 295, 1041 Carruthers B M (1967) Canadian Journal ofPhysiology and Pharmacology 45, 269 Fernandez H N, Henson P M, Otani A & Hugli T E (1978) Journal of Immunology 120, 102 Gallin E K & Gallin J I (1977) Journal of Cell Biology 75, 277 Goetzl E J & Austen K F (1975) Proceedings of the National Academy of Sciences 70, 2916 Hartwig J H & Stossel T P (1975) Journal of Biological Chemistry 250, 5699 Kay N E, Bumol T F & Douglas S D (1978) Journal of Laboratory and Clinical Medicine 91, 850 Keller H U, Hess M W & Cottier H (1977) Experientia 33, 1386 Keller H U, Wilkinson P C, Abercrombie M, Becker E L, Hirsch J G, Miller M E, Ramsey W S & Zigmond S H (1977) Clinical and Experimental Immunology 27, 377 Keller H U, Wissler J H, Hess M W & Cottier H (1978) European Journal of Immunology 8, 1 Kleinermann E S, Snyderman R & Daniels G A (1974) Journal of Immunology 113, 1562 Larson H E & Blades R (1976) Lancet i, 283 Miller M E, Oski F A & Harris M B (1971) Lancet i, 665 Moudgil G C, Allan R B, Russell R J & Wilkinson P C (1977) British Journal of Anaesthesia 49, 97 Naccache P H, Showell H J, Becker E L & Sha'afi R I (1977) Journal of Cell Biology 73, 428 Oliver J M & Zurier R B (1976) Journal of Clinical Investigation 57, 1239 O'Neill G J & Parrott D M V (1977) Cellular Immunology 33, 257 Parrott D M V, Good R A, O'Neill G J & Gupta S (1978) Proceedings of the National Academy of Sciences 75, 2392 Rabson A R, Whiting D A, Anderson R, Glover A & Koornhof H J (1977) Journal of Infectious Diseases 135, 113 Russell R J, Wilkinson P C, Sless F & Parrott D M V (1975) Nature 256, 646 Sahu S & Lynn W S (1977) Inflammation 2, 47 Schiffmann E, Corcoran B A & Wahl S M (1975) Proceedings of the National Academy of Sciences 72, 1059 Showeil H J, Freer R J, Zigmond S H, Schiffmann E, Aswanikumar S, Corcoran B & Becker E L (1976) Journal of Experimental Medicine 143, 1154 Stossel T P & Hartwig J H (1975) Journal of Cell Biology 68, 602 Turner S R, Tainer J A & Lynn W S (1975) Nature 257, 680 Van Epps D E & Williams R (1976) Journal of Experimental Medicine 144, 1227 Ward P A, Unanue E R, Goralnick S J & Schreiner G F (1977) Journal of Immunology 119, 416 Wilkinson P C (1975) Nature 255, 485 Wilkinson P C (1976) Clinical and Experimental Immunology 25, 280 Wilkinson P C & Allan R B (1978a) In: Leukocyte Chemotaxis. Ed, J I Gallin and P G Quie. Raven Press, NY; pp 1-24 Wilkinson P C & Allan R B (1978b) Molecular and Cellular Biochemistry 20, 25 Wilkinson P C & McKay I C (1974) Antibiotics and Chemotherapy 19, 421 Wilkinson P C, Parrot D M V, Russell R J & Sless F (1977) Journal of Experimental Medicine 145, 1158 Wilkinson P C, Roberts J A, Russell R J & McLoughlin M (1976) Clinical and Experimental Immunology 25, 280 Wilkinson P C, Russell R J, Pumphrey R S H, Sless F & Parrott D M V (1976) Agents and Actions 6, 243 Williams L T, Snyderman R, Pike M C & Lefkowitz R J (1977) Proceedings of the National Academy of Sciences 74, 1204 Zigmond S H (1974) Nature 249, 450 Zigmond S H (1977) Journal of Cell Biology 75, 606 Zigmond S H & Hirsch J G (1973) Journal of Experimental Medicine 137, 387
Journal of the Royal Society of Medicine Volume 72 August 1979
Physiology of granulocyte locomotion and its relation to defects of chemotaxis: a review' P C Wilkinson MD Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow GIl 6NT
Since leukocytes play an active role in sites of inflammation, and are thought to migrate to those sites by chemotactic locomotion, clinicians are naturally interested to know whether the leukocytes of their patients, especially those patients with a predisposition to infection, are able to locomote normally. For such studies the usual assay employed measures the locomotion of the patient's white cells through a filter towards a chemotactic substance. Other basically similar in vitro assays, e.g. of cell migration through agarose, can also be used. Another method is to abrade a small area of skin and to cover the abrasion with either a coverslip or a sterile chamber containing fluid, and to measure the influx of cells into the abrasion. While in one sense more physiological than the in vitro tests, since performed using the living tissue of the patient, this skin window technique is more difficult to interpret, since the whole complex of the body's reaction to injury has been activated; thus there are a large number of uncontrollable variables, and it is an oversimplification to suggest that a defect in cell accumulation in skin windows is proof of a defect of chemotaxis. Although the documentation of leukocyte locomotion defects is filling an ever-increasing number of pages in the journals, and such defects have been postulated in a large number of disease states, there is very little literature which really probes exactly what abnormalities of leukocyte physiology may underlie a defect of locomotion. For example, the distinction between defects of random locomotion and defects of chemotactic locomotion, of which much was made in many of the earliest reports (Miller et al. 1971), and which is certainly of considerable importance, was based on an imprecise understanding of the locomotor reactions of leukocytes, and frequently on inadequate methods (such as migration from buffy coats in capillary tubes). It is clear that two distinct reasons for defective locomotor responses can be distinguished, namely, abnormalities of the cells themselves, and abnormal generation of the factors (e.g. complement peptides inter alia) which attract them into sites of inflammation and infection. Defects in generation of these factors give rise to severe abnormalities of body defence but are not the primary concern of this communication. I wish, rather, to outline some important aspects of leukocyte physiology which act as determinants of their locomotor reactions, and to recapitulate briefly what is known about the mode of locomotion of these cells and the reactions which they can show in response to environmental chemical substances. Locomotion of different leukocyte types: some observations on lymphocytes
Neutrophil locomotion has been studied more frequently than that of any other cell type, both at a basic level and clinically, and much of the discussion which follows will be devoted to these cells. It is established that other myeloid cells, eosinophils and basophils, and various types of mononuclear phagocyte, including blood monocytes, alveolar and peritoneal macrophages, are also motile and can show chemotactic reactions. The lymphocyte has long been known as a motile cell, and it is somewhat surprising, in view of the fact that this cell type is now being investigated by more people more intensively than any other cell in the body, that the literature 1 Paper read to Section of Clinical Immunology & Allergy, 13 February 1978. Accepted 21 March 1978, updated 31 May 1979
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on lymphocyte locomotion is scanty and that chemotactic reactions of lymphocytes have been only very recently described. One possible reason for this is that lymphocytes taken from blood or from lymph nodes, and tested directly, are not highly motile and respond rather feebly to chemoattractants. The lymphocytes seem to require to be stimulated in a way which is not understood before they become fully motile. This can be achieved easily by culture in vitro for 2 to 3 days in serum-containing media with or without mitogens (Wilkinson, Roberts et al. 1976, O'Neill & Parrott 1977). Likewise, lymphocytes from lymph nodes draining a site of antigenic challenge are more motile than those from unstimulated nodes (Russell et al. 1975). Another source of highly motile lymphocytes is the human cultured B-lymphoblast lines (Russell et al. 1975). Using these various populations, it can be established that lymphocytes respond to most of the chemotactic factors, e.g. activated serum, denatured proteins, formyl peptides, lymphokine chemotactic factors (Wilkinson, Russell et al. 1976 and unpublished observations, Ward et al. 1977), that neutrophils recognize. In addition, lymphocytes from primed lymph nodes are capable of locomotion to the priming antigen (Wilkinson et al. 1977) and polyclonal activators at low doses can induce locomotor responses in lymphocytes (Wilkinson, Roberts et al. 1976). One of the major problems in working with lymphocytes is their heterogeneity. Both T and B cells can locomote but there may be differences among subpopulations; for example, Parrott et al. (1978) have recently suggested that the human TVi subpopulation is more active in locomotion than the Ty subpopulation. Since Tp is a helper and T-y a suppressor population, this suggests that locomotor differences may parallel differences in other lymphocyte functions.
Visual observations of neutrophil leukocyte locomotion There are numerous descriptions of the locomotion of neutrophils in vitro, but many fewer of locomotion of the other blood cells. Nevertheless, it is probable that the locomotor process in all of these cells is similar. If a population of neutrophils is placed on a glass slide in the absence of protein, the cells flatten on the glass and do not move. If protein-coated glass is used or protein is added to the flattened cells, the cells begin to locomote. The first event observed is that the cells change from a rounded form to a polarized, elongated form, with a broad hyaline leading edge or lamellipodium which is rich in actin and myosin but which excludes the nucleus and cytoplasmic organelles (Figure 1). It must be emphasized that this polarization is an
g
C'*
-
.
... ...
Figure 1. Interference-contrast photomicrograph of a locomoting blood neutrophil. The cell is moving from left to right. Note the thin hyaline anterior lamellipodium (arrowed: a) which excludes the cytoplasmic organelles found posteriorly to it in the main body of the cell. Note also the posterior retraction fibres (arrowed: b). x 2500. (Photograph by R B Allan)
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intrinsic property of locomoting cells and is not imposed by gradients or other stimuli from without. The polarized cells begin to move around but not in any particular direction, their locomotion being best described as a 'persistent random walk' (Allan & Wilkinson 1978). The locomotion is a crawling motion on a surface and involves sustained and controlled contractions and relaxations of microfilaments over a long period of time, giving rise to an amoeboid movement. Sustained locomotion poses especial problems, since one-off signals, such as are adequate to explain one-off events like exocytosis in mast cells, are inadequate to account for continued locomotion over a period of several hours. Chemical substances in the environment of the cell can influence this locomotion in various ways (Keller, Wilkinson et al. 1977). Firstly, chemical substances can determine the rate of cell locomotion or the frequency of turning of the cell, a reaction known as chemokinesis. For example, it is now clear that leukocytes show a dose-dependent increase in locomotion rate as the concentration of serum albumin in their environment is increased (Wilkinson et al. 1977, Wilkinson & Allan 1978a, Keller, Hess & Cottier 1977, Keller et al. 1978). It is likely that this is because albumin coating the surface on which the cell moves determines the degree of adhesion of the cells to the surface. Other proteins may play a similar role. Secondly, chemical substances can determine the direction of locomotion, a reaction known as chemotaxis. Chemotaxis alone would be likely to cause cells to migrate up a concentration gradient of a chemical to its source and to allow the cells to accumulate there. Chemokinesis alone would not do this, rather it would lead to dispersal of cells. A point which arises from the distinction between chemotaxis and chemokinesis is that the methods used for clinical studies often do not distinguish between these two reactions. Thus migration of cells through a filter towards a chemical placed in a lower chamber may be chemotactic, but it could also simply be that the chemical was causing an accelerated but nondirectional locomotion. In this case the cells would disperse according to the diffusion laws and, under chemokinetic conditions, many might travel through the filter. Special techniques are required to distinguish these reactions. The best ofthese filter assays is the 'checkerboard' assay of Zigmond & Hirsch (1973), in which a series of chambers is set up with various attractant concentrations above and below the filter. Even more satisfactory, but requiring more expertise, are visual assays, using time-lapse filming, in which the paths taken by individual cells can be determined directly. Using visual assays, it can be established that neutrophils respond to a chemotactic gradient by polarizing with the leading lamellipodium facing the gradient source before they begin to move (Zigmond 1974). They then move in a straight path to the gradient source. This means that the chemotactic gradient imposes an external constraint on the intrinsic polarization of the cell, so that the cell becomes orientated up-gradient. This is probably achieved by a 'spatial' gradient-detecting system by which the cell is able to determine that a greater number of receptors at the front of the cell have bound attractant than at the back. Neutrophils are able to detect 1% differences in attractant concentration across their diameter by these means (Zigmond 1977). One other finding which use of these assays has established is that many chemotactic factors, which cause cells to migrate in a gradient, can also act as chemokinetic agents, i.e. they influence both the rate and the direction in which cells move. This probably leads to more efficient accumulation of cells at gradient sources than chemotaxis alone. Possible physiological levels of locomotor defects From the above description, it is apparent that clinical defects of neutrophil locomotion might result from a disorder of the locomotor process itself; from defects in the ability to accelerate in response to chemokinetic signals or to adhere correctly to chemokinetic surfaces; or from defects in the direction-finding mechanism, i.e. in chemotaxis proper. The tests used to date have hardly been adequate to distinguish these, but the distinction is, in practice, possible by some variant of the checkerboard assay or by filming the cells of patients. Defects in chemotaxis itself could result from 'blindfolding' of the cells, due either to lack of receptors or to blockage of receptors by extrinsic material, e.g. from serum, thus reducing the efficiency of the gradient detection system. Defects of locomotion might be expected if there were
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abnormalities of signal transduction across the plasma membrane of the cell. I shall therefore briefly summarize what is known about recognition, transduction and the mechanochemical events of locomotion in leukocytes. These areas are under active investigation and the remarks below are likely to be outdated rapidly.
Chemotactic recognition The problem of what leukocytes recognize is an intriguing one. Numerous chemotactic factors have been described and some of these have been defined. They include proteins, e.g. denatured proteins (Wilkinson & McKay 1974) or as- and ,B-casein, both of which are anionic amphipathic molecules, and large peptides such as the C5-derived complement peptides. More recently, some low-molecular-weight chemotactic factors have been isolated. These include fatty acids, especially hydroxylated or peroxidized unsaturated fatty acids (Sahu & Lynn 1977). One of these, HETE, is a hydroxylated arachidonic acid derivative believed to be released from aggregated platelets by the action of a lipoxygenase (Turner et al. 1975), so clearly of interest in inflammation. A number of small peptides have also been shown to be chemotactic. These include the eosinophil chemotactic factors Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu (Goetzl & Austen 1975) and the formylated peptides which attract neutrophils. This is a class of hydrophobic, anionic di- and tripeptides with the N-terminal blocked by formylation and a free C-terminus (Schiffmann et al. 1975, Showell et al. 1976). Many described in the literature are formyl-methionyl peptides, but N-terminal methionine does not seem essential and f-tyrosyl, fphenylalanyl and f-leucyl peptides are also active. Peptides such as f-Met-Leu-Phe or f-NorleuLeu-Phe are active at about 10. 9M, and show saturable binding to the surfaces of neutrophils with an association constant around 109 litres per mole (Aswanikumar et al. 1977, Williams et al. 1977). Thus there seem to be receptors for these peptides, though the receptors are able to bind quite a wide range of molecules. Earlier, from our studies of denatured or conjugated protein chemotactic factors, we had come to the conclusion that hydrophobicity - or rather, amphipathicity - of these proteins was an important determinant of their chemotactic activity (Wilkinson 1976) and studies with lipid-specific toxins suggested that membrane lipids were important in their binding to the cell surface (Wilkinson 1975). We have therefore done binding studies with denatured serum albumin which suggested that this, too, bound in a saturable fashion with a Ka of around 106 litres per mole and about 106 binding sites per cell (Wilkinson & Allan 1978b). Thus there are probably saturable binding sites for chemotactic factors of all types, though they may be catholic, rather than highly specific, in the molecules which they bind. While discussing chemotactic peptides, it is worth mentioning that the major peptide chemotactic activity from complement is C5 derived (C5a and similar peptides). Despite wide quotation as such, including citation in most textbooks, C3a is not a chemotactic factor (Fernandez et al. 1978). Stimulus response coupling The problem of how binding activates intracellular events is under active investigation. Recent studies have shown that addition of f-Met-Leu-Phe to neutrophils is followed immediately by an influx of Na+ and by fluxes of Ca2" (Naccache et al. 1977). Moreover, it has recently proved possible to insert microelectrodes into macrophages and to show that addition of chemotactic factor causes changes in transmembrane potential, namely, a brief depolarization followed by a longer hyperpolarization (Gallin & Gallin 1977). Leukocyte locomotion is mediated by the same proteins, actin and myosin, as muscle contraction. In muscle, contractile events are initiated by ion fluxes and the ability of myosin to bind to actin is controlled by the cytoplasmic level of Ca2 . This increases to allow contraction and the calcium is then pumped back into the sacroplasmic reticulum, allowing relaxation. It is not known how far analogies between tissue cells and muscle can be taken, but it is probable that ion fluxes will prove to be crucial to locomotion of leukocytes. There have also been some studies of the role of cyclic nucleotides in controlling leukocyte locomotion. These are probably best summarized as not yet pointing to any crucial role for these molecules.
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Actin-myosin filaments The major protein constituent of the leukocyte cytoplasm is actin. Myosin is also found there, but in smaller amounts. Microfilaments are formed of long strands of polymerized actin and, presumably, also of myosin. When polymerized these proteins give cytoplasm the consistency of a gel, and this has resuscitated the old idea that amoeboid locomotion involves sol-gel transformations in the cytoplasm which control cytoplasmic forward flow in the moving cell. Actin filaments certainly require to be anchored to membrane in order that the sliding of myosin on actin may provide sufficient purchase to result in a contraction, but the precise mechanism of this anchoring has not been established. Actin-containing microfilaments are not distributed uniformly in leukocytes, but are concentrated at sites of movement, i.e. in the leading lamellipodium, at sites of adhesion to surfaces, at sites of attachment of phagocytosable objects, etc. Thus, in an active cell, the filament assembly requires to be built up and dismantled rapidly in different places according to the cell's demands. Hartwig & Stossel (1975) and Stossel & Hartwig (1975) have suggested that a membrane-attached actin-binding protein can act as a nucleation point for actin filaments and thus, by redisposition of this actin-binding protein within the cytoplasm, networks of filaments can rapidly be built up or broken down at any given site in the cell. Furthermore, a child with a severe predisposition to infections and a locomotor defect was shown to have a defect in actin polymerization (Boxer et al. 1974). This remains an almost unique case in that the molecular disorder underlying the abnormality of locomotion was pinpointed. Much remains unknown about the detailed function of microfilaments. However, since they are crucial to such varied tasks as locomotion, phagocytosis, exocytosis, organelle fusion and mitosis, their control is likely to be exquisite. The role of microtubules in leukocyte locomotion is still unclear. These are widely regarded as serving as a cytoskeleton which maintains shape changes such as are required for leukocyte polarization. It has been suggested that microtubule depolymerization (e.g. by colchicine or vinblastine) does not paralyse locomotion, but does abolish chemotaxis by interfering with the cell's ability to polarize in gradients. This is probably too simple. Colchicine-treated leukocytes show bizarre shapes and a 'drunken-walk' type of locomotion, but they still orient accurately and respond to chemotactic gradients (Allan & Wilkinson 1978). It has recently been suggested that the underlying abnormality in Chediak-Higashi syndrome leukocytes is a defect of microtubules, and that this is the reason why these patients' neutrophils show defective locomotion (Boxer et al. 1976, Oliver & Zurier 1976). However, it is probably premature to assign a definite role to microtubules in locomotion.
Conclusion From the brief account given above, it will be recognized that in theory defects of leukocyte locomotion could result from abnormalities at any of the stages in conversion of a sensory signal to a motor response and from defects in the molecular apparatus for locomotion. Indeed, it is fairly easy to induce such defects in vitro by incubating normal leukocytes with drugs which interfere with leukocytes at various levels. For example, anaesthetic agents, inductional, inhalational or local, cause a reversible paralysis of leukocyte locomotion (Moudgil et al. 1977); these agents are known to reduce membrane permeability, and thus the efficiency of ion transfer across the membrane, as well as causing disappearance of submembranous filaments as seen by electron microscopy. Inhibitors of glycolytic pathways also inhibit locomotion (Carruthers 1967), perhaps because they deprive the cell of energy sources used in locomotion. Myosin is an adenosine triphosphatase and requires ATP for its function. Leukocytes infected with influenza or herpes simplex viruses show depressed locomotion (Kleinermann et al. 1974, Larson & Blades 1976, Rabson et al. 1977), an effect which can apparently be reversed with the drug, levamisole, which raises intracellular levels of cyclic guanosine monophosphate. Recent results from our laboratory and from Kay et al. (1978) suggest that immunoglobulin or immune complexes bound to the Fc receptors on the cell surface may interfere with chemotactic responses. Polymeric IgA and IgA myeloma proteins bind to neutrophils and act as especially good inhibitors of leukocyte chemotaxis (Van Epps & Williams 1976).
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By these approaches it is possible to dissect the various levels at which the locomotor process can be interfered with. It is to be hoped that thorough investigation of the defective granulocytes found in various disease states will provide an equally fruitful approach. Fairly sophisticated techniques will be required, such as have not been applied extensively yet, if the sites of the lesions in the cells of these patients are to be established with precision.
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