Immunology 1990 69 127-133
Relation between locomotion, chemotaxis and clustering of immune cells P. C. WILKINSON Department of Bacteriology and Immunology, University of Glasgow (Western Infirmary), Glasgow
Acceptedfor publication 15 September 1989 SUMMARY Experiments were designed to discover whether locomotor or chemotactic events are needed for clustering of lymphocytes with accessory cells or, conversely, whether clustering precedes the activation of lymphocyte locomotion. The time-courses of clustering and locomotor activation were compared and the behaviour of moving cells during cluster formation was filmed. Human lymphocytes direct from blood were activated by culture for 24-48 hr with anti-CD3 antibody or in allogeneic mixed leucocyte reactions (AMLR). The proportion of clustered and locomotor lymphocytes was low at the beginning of culture. Clusters appeared during the first few hours, before the increase in numbers of locomotor lymphocytes. Filming gave no evidence that the cells attracted one another chemotactically to form clusters. Rather, cells made chance contact by random locomotion and then remained adherent, though lymphocytes very close (< 10 pm) to clusters did show increased pseudopod formation towards the cluster. However, the behaviour of motile lymphocytes responding to monocytes or macrophages given a phagocytic stimulus was different. Human monocytes which ingested opsonized zymosan released a material during but not following phagocytosis which caused an immediate increase in polar shape-change in lymphocytes. Macrophages from Corynebacterium parvum-induced mouse peritoneal exudates, given a phagocytic stimulus (opsonized Candida albicans), acted as sources of chemotactic gradients which attracted nearby lymphocytes to form clusters. This was due to brief release of a material immediately following phagocytosis, but after 15 min or so the macrophages no longer attracted nearby cells. These experiments suggest that, during induction of an immune response to a non-phagocytic stimulus, clusters form slowly by random contact followed by preferential adhesion. However, after phagocytosis, there may be a chemotactic response to the ingesting macrophage. This may help to focus lymphocytes onto macrophages which present microbial antigens.
INTRODUCTION
singly, been little studied. Petri, Braendstrup & Werdelin (1979) filmed cluster formation and suggested that lymphocytes actively sought out macrophages but they did not provide quantitative support for this idea. Clusters could form either by chemotactic attraction or by random contact, close cell-cell contact then inducing both locomotor activity and growth in the clustered cells. Activation of the locomotor capacity of lymphocytes is associated with activation of growth (Biberfeld 1971; McGregor & Logie, 1974). Although the proportion oflocomotor lymphocytes direct from human blood is low, both T cells (Wilkinson, 1986; Wilkinson & Higgins, 1987a) and B cells (Wilkinson & Higgins, 1987b; Wilkinson & Islam, 1989) show an increase in locomotor shapes, in invasion of collagen gels, and in responsiveness to chemoattractants (O'Neill & Parrott, 1977; ElNaggar, Van Epps & Williams, 1981) during the first 24 hr of culture with antigens or mitogens. These functions are acquired as the cells enter the GI phase of growth, increase in size, and synthesize increased amounts of RNA and protein. Both locomotor activation and clustering are therefore early events in immune activation. To investigate the causal relationship between them, it is helpful to investigate their temporal relationship. This is one of the purposes of this paper. There may be different mechanisms for clustering depending on
Heterotypic clustering of lymphocytes with accessory cells is essential for induction of immune responses, providing the contact necessary for T cells to recognize MHC-presented antigen and the close proximity required for cell-to-cell signalling of effector functions. Early studies documented the events of clustering by microscopic techniques including time-lapse cinematography. Lymphocytes were observed to attach to and circumnavigate the surfaces ofmacrophages in culture (Sharp & Burwell, 1960; Berman, 1966). In mixed leucocyte cultures, macrophage-associated lymphocytes increased in size and began to synthesize DNA (McFarland & Heilman, 1965; McFarland, Heilman & Moorhead, 1966). These activated lymphocytes were frequently in locomotor morphology. An antigen-dependent form of clustering was distinguished from, and shown to be stronger than, antigen-independent clustering (Lipsky & Rosenthal, 1973, 1975). The relation between locomotion and clustering has, surpriCorrespondence: Dr P. C. Wilkinson, Dept. of Bacteriology and Immunology, University of Glasgow (Western Infirmary), Glasgow GI I 6NT, U.K.
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whether an unactivated or a preactivated starting cell population is under study. These have been distinguished here. Lymphocytes from human blood which, unstimulated, have low locomotor activity, were activated by culture with anti-CD3 antibody or by AMLR. Anti-CD3 is a model for antigen-induced activation, though anti-CD3 clusters form by antibodymediated cross-linking between CD3 on the T cell and Fc receptors on the accessory cell rather than via the T-cell receptor. In a separate group of experiments, monocytes or macrophages were given a phagocytic stimulus. The locomotor behaviour of lymphocytes in response to products released during phagocytosis was examined using either precultured human blood mononuclear cells and measuring the effect of phagocytosis of opsonized zymosan by monocytes on lymphocyte locomotion, or using mononuclear cells from the mouse peritoneal cavity 4 days following immunological priming with Corynebacterium parvum. In this case, macrophages were allowed to ingest opsonized Candida albicans and the effect of phagocytosis on the subsequent locomotion of nearby lymphocytes towards the macrophages was filmed.
MATERIALS AND METHODS Reagents Hanks' buffered salt solution, RPMI-1640 and fetal calf serum (FCS) were from Flow, Rickmansworth, Herts. Human serum albumin (HSA) was from Behringwerke, Marburg, Germany. Anti-CD3 monoclonal antibody was from Orthoklein, Raritan, NJ (OKT3 IgG2a antibody). Zymosan and dibutyryl cyclic AMP were from Sigma, Poole, Dorset. Cyclosporin A was a gift from Dr A. W. Thomson, Department ofPathology, University of Aberdeen and was used as described by Wilkinson & Higgins (1987b). Anti-human Class II monoclonal antibody was from Sera Labs, Crawley, Sussex (Clone FMC 4). Class II + cells were scored by immunofluorescence using FITC-anti-mouse IgG as a second layer. Candida albicans blastospores were grown from clinical isolates by culture overnight on dextrose-peptone agar. 'Simultest' (FITC-anti-CD3+ phycoerythrin anti-CD19; Becton-Dickinson, Mountain View, CA) was used to distinguish T and B cells by fluorescence microscopy. Non-specific esterase (NSE) was used to identify monocytes/macrophages.
Human blood cells Blood was taken by venepuncture from healthy subjects. The mononuclear cell fraction was isolated on lymphocyte separation medium (Flow) and washed thrice with Hanks solution buffered to pH 7-4 with morpholinopropane sulphonate (MOPS) and containing HSA (10 mg/ml) (Hanks-HSA). Mouse peritoneal exudate cells These were provided by Dr J. H. Brock (this department) from peritoneal exudates prepared by injecting Corynebacterium parvum, strain 4982 (2 mg/mouse) 4 days previously into the peritoneal cavity of adult NIH strain mice. The peritoneal fluids were taken; cell counts showed 40-50% lymphocytes, 40-50% macrophages, up to 10% neutrophils and about 5% of dendritic cells. As documented in an earlier publication (Wilkinson, 1982) over 70% of the exudate lymphocytes were motile, probably because C. parvum acts as an immunological stimulus. Four-day exudates induced with a non-immunogenic stimulus, thio-
glycollate broth, contained lymphocytes that were mainly nonmotile. Also, the proportion of class II-positive macrophages, while variable, was always higher in C. parvum-induced exudates than in thioglycollate-induced exudates (J. H. Brock, personal communication). The peritoneal exudate cells were washed thrice and resuspended in RPMI + 20% heat-activated FCS. Methods for assaying clustering and locomotion Activation of human cells with anti-CD3. The mononuclear cell fraction was cultured at 5 x 105 cells/ml in Hanks-HSA without added serum in tissue culture plastic dishes (well size 1-6 x 17 cm; Flow). Anti-CD3 antibody was added at 25 ng/ml and culture was continued for 24-48 hr. Clustering was quantified using an inverted microscope (Nikon Diaphot TMD) with an eyepiece grid graticule and a x 20 phase contrast objective. The number of clusters per field in eight fields per well, defined as groups of three or more cells in close contact which could not be dissociated by gentle rocking, was scored. A baseline value for clustering was recorded within the first 2 hr of culture and thereafter at intervals up to 48 hr. Locomotor capacity was quantified by scoring the proportion of lymphocytes with morphological anteroposterior polarity. After appropriate incubation, cells were fixed with 2-5% glutaraldehyde, washed, 300 cells were counted and the number of spherical and polarized cells was scored. Cell viability was checked morphologically under phase contrast and was excellent in all experiments. AMLR. The mononuclear cell fractions of two blood samples from different normal persons were prepared separately. They were then mixed in equal proportions and cultured in Hanks-HSA without added stimulus as described above. Clustering and polarization were scored as described above. Effect on lymphocyte locomotion of phagocytosis of microorganisms by monocytes or macrophages. The mononuclear cell fraction from human blood was cultured either in Hanks-HSA or in RPM I+ FCS (20%) for 24-48 hr. The non-adherent cells were then removed, centrifuged, and the supernatant was retained. The non-adherent cells (mostly lymphocytes) were washed thrice in Hanks' solution and retained at 20° for use. The adherent cells (mostly monocytes) were also washed thee times and retained at 200. Zymosan particles (107/ml) were opsonized by incubation for 20 min at 370 with 20% autologous serum. They were then washed three times, resuspended in Hanks-HSA and incubated for 15 min at 37°. They were added (107 particles per well) to the adherent cells and incubated for 20-30 min at 37°. The supernatant containing non-phagocytosed zymosan, together with monocyte release products, was then removed and centrifuged at 2000 g. This supernatant (phagocytosis supernatant) was retained. After washing, the adherent cells were reincubated in Hanks-HSA for 30 min at 37°. This supernatant (postphagocytosis) also was centrifuged and retained. With each experiment the following controls were also set up: monocytes incubated for 30 min without zymosan; and opsonized zymosan incubated for 30 min at 370 without monocytes. The various supernatants were then tested for activity against the nonadherent lymphocyte population in a polarization assay. Lymphocytes were incubated at 370 for 30 min, fixed, and the proportion of polarized cells scored.
Locomotion, chemotaxis and clustering of immune cells Filming assays oflymphocyte locomotion. Filming was done using a metal filming chamber described elsewhere (Haston & Wilkinson, 1988). A coverslip was sealed to the lower surface of this chamber and cells either in Hanks-HSA or in Hanks-FCS (20%) were allowed to settle on the glass. The chamber, filled with appropriate medium, was sealed and placed on a microscope stage heated at 370 and the behaviour of the cells was filmed. The locomotion of the human mononuclear cell fraction was videotaped using a Panasonic 8050 time-lapse videorecorder. Long sequences of from 30 min to 4 hr were recorded during the whole culture period of 48 hr. The paths taken by moving lymphocytes were tracked and their interactions with other cells were recorded. Where adherence of a cell to a group of two or more cells was observed, the tape was run back and the direction and speed of locomotion of the cell or cells approaching to form a cluster was compared in the approach path (within 50 gm of the point of contact) with direction and speed in earlier parts of the tracks of the same cells. Filming of mouse peritoneal exudate cells. Mouse peritoneal exudate cells were filmed using time-lapse cinematography on 16 mm cine film (Kodak Plus-X-Reversal) with a Bolex H16 cinecamera and a lapse interval of 8 seconds between frames. Candida albicans blastospores preopsonized with fresh mouse serum (20%) were added at 2 5 x 105 spores/ml to the filming chamber which was immediately sealed and filming begun. The candida settled on the lower coverslip where some of them were phagocytosed by macrophages. This phagocytosis and the subsequent locomotor events were filmed. The direction and speed of locomotion of cells were analysed using methods described in earlier publications (Allan & Wilkinson, 1978; Haston & Wilkinson, 1988). Chemotaxis was quantified using the McCutcheon ratio (Dixon & McCutcheon, 1936), displacement divided by total distance travelled to the source. For a cell reaching a gradient source by travelling in a straight line, this ratio is + 1 *0. In the absence of any net displacement towards (or away from) the source, it is zero. RESULTS Relation between locomotion and clustering in human blood mononuclear cells The time-course of development of clusters and of lymphocytepolarized morphology during culture of human mononuclear cells in the presence of anti-CD3 is shown in Fig. Ia. The number of clusters increased during the first 10 hr and then reached a peak. By 10 hr there was little increase in the numbers of polarized cells and the major increase took place between 10 and 24 hr once clustering was fully developed. At 24 hr most clusters contained one or more class II-positive monocytes and a variable number of lymphocytes. About 10-15% of these were B cells and most of the remainder were T cells. A similar, but slower sequence of events was seen in the AMLR (Fig. lb) though the number of clusters was smaller than in the response to anti-CD3. These findings suggest that, in a first exposure to antigen or mitogen, clustering precedes locomotor activation. Agents that inhibit activation of lymphocyte locomotion reduced cluster formation. Clusters formed more slowly at 200 than at 370 (Fig. 2a) and not at all at 00. Culture with dibutyryl cyclic AMP (10-3M), which inhibits lymphocyte locomotion (unpublished observations in this laboratory) also reduced
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Figure 1. Time-course of formation of clusters (continuous lines) and proportion of lymphocytes in locomotor morphology (broken lines) in (a) anti-CD3, 25 ng/ml (mean + SEM, n = 5) and (b) the AMLR (mean of two experiments). For clarity, control curves for unmixed cells without an activator have been omitted. The number of clusters per field in these controls was 2-3 within 12-24 hr and the proportion of polarized cells at 24-48 hr was 10-15%.
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Figure 2. (a) Time-course of cluster formation in response to anti-CD3 (25 ng/ml). Cells cultured at 370 ( x ), 200 (0) or in the presence of dibutyryl cyclic AMP (10-3M) at 370 (O). Results of a single experiment. (b) Time-course of cluster formation in response to anti-CD3 in the absence ( x ) and presence (0) of cyclosporin, I pg/ml. Mean of three experiments + SEM.
clustering (Fig. 2a). Cyclosporin inhibits anti-CD3-activated lymphocyte locomotion (Wilkinson & Higgins, 1987b; Fig. 3) and moderately reduced the number of clusters formed (Fig. 2b). These results support the view that active locomotion contributes to cluster formation. Visual observations
Time-lapse videorecording of cell locomotion during the formation of clusters both in response to anti-CD3 and in an AMLR showed that at the beginning of culture, the number of motile lymphocytes was low; about 10% of the total in Hanks-HSA and 20% in FCS. Many of the translocating cells during the first few hours were monocytes, though many of these became sessile as culture continued. Clusters formed slowly and randomly. Locomotor cells coming into contact with one another sometimes remained attached but the cells showed no change in turning behaviour or speed as they neared one another. However, lymphocytes close to a cluster often showed increased protrusive and ruffling activity at the anterior pole and formed
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Figure 4. Sequential images from a time-lapse film showing behaviour of a lymphocyte (shaded) approaching a cluster. Numerals indicate time in seconds. The straight line represents a fixed point in space relative to which cell movement can be judged. Cells incubated in anti-CD3 (25 ng/ ml). The shaded lymphocyte had been migrating randomly. On approaching the cluster, it put out a broad anterior pseudopod (seen at 40 and 55 seconds) which contacted the cluster (65 seconds). The lymphocyte then began to translocate rapidly round the edge of the cluster.
Figure 3. Appearance of a (25 ng/ml)
there
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many
cyclosporin
mononuclear cell culture after 24 hr in aCD3
the absence (a)
in
lymphocytes
or presence
in
but that most of the cells
the presence of clusters in both, in
are
though
spherical
in the absence of
in its presence. Note
total cluster numbers
were
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cyclosporin (b).
in the direction of the
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(b) of cyclosporin. Note that
locomnotor morphology
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( 80% NSE +) were given opsonized zymosan as a phagocytic stimulus and the various supernatants were tested for their capacity to induce polarization ofnon-adherent lymphocytes. Results are shown in Table 1. Phagocytosis supernatants caused increased polarization of lymphocytes whereas supernatants of zymosan alone, or of monocytes alone, incubated for 30 min did not. However, supernatants from monocytes alone after culture for 24 hr did increase lymphocyte polarization, possibly due to release of a material at a low level by resting monocytes which is accelerated by phagocytosis. Table 2 shows that the pulse of release during phagocytosis is short-lived. After completion of phagocytosis and removal of free zymosan, the post-phagocytic supernatants had no activity. The attractant in supernatants of monocytes was not characterized biochemically, though preliminary experiments have shown that it is destroyed by boiling and is retained within a dialysis sac. Attempts were made to demonstrate the effects of this attractant on lymphocyte locomotion by filming, as this is the best method for showing chemotaxis of cells to a gradient source. The most satisfactory lymphocyte population for filming assays proved to be that from the peritoneal cavity of the mouse following challenge with C. parvum, as these were both more adherent and more motile than lymphocytes from human blood.
and
added in
were
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a
can be short-
Chemotaxis of mouse peritoneal lymphocytes towards phagocytosing macrophages Following phagocytosis of candida by macrophages in C. parvum-induced exudates, some macrophages released a pulse of attractant which caused nearby lymphocytes to migrate directionally towards the macrophage and to form a cluster around it (Fig. 5). During brief film sequences, the number of lymphocytes joining such clusters increased rapidly (Table 3). A traced record of an event in which eight lymphocytes within a radius of 100 im of a macrophage migrated in radially to form a cluster is shown in Fig. 6. After about 15 min no further response was seen. Lymphocytes responded chemotactically only to macrophages and not to free, unphagocytosed Candida. Staining the clusters after fixation showed that they contained 50-70% lymphocytes, 20-30% macrophages and 5-10% neutrophils. Cells of dendritic morphology were also present in small numbers. It was easier to distinguish lymphocytes from neutrophils in filter assays than in films of living cells. The released
Locomotion, chemotaxis and clustering of immune cells
131
Table 1. Effect of supernatants from phagocytosing monocytes on morphological polarization of lymphocytes P value
Supernatant added to lymphocytes (370 30 min)
Percentage polarized lymphocytes* (mean + SEM)
n
99 + 13
6
18 3+2 6 22-9+3-4
5 6
0-02 3 cells) at the beginning and at the end of the film.
hour. It is hard to think of a chemotactic mechanism that would operate so gradually, and the lengthy stretches of videotape gave no evidence for chemotaxis operating at a distance, though there was evidence of directional protrusive behaviour by cells close to a cluster. This could mean that cells such as monocytes release a very weak signal whose directionality is quickly lost by diffusion. Nevertheless, the presence of such a material in uniform concentration could still signal an increase in lymphocyte speed (chemokinesis). In these experiments locomotor activation of lymphocytes was seen only after clusters had formed. Earlier observers showed that lymphocytes which had joined clusters grew in size and became motile (McFarland et al., 1966; McFarland & Schechter, 1970;' Lipsky & Rosenthal, 1975) which suggests that the signal for locomotor activation is transmitted by close cell-to-cell contact. However, the time sequence does not prove that clustering is essential for locomotor activation. This remains to be tested. Our working hypothesis at present, based on this and earlier work, is that GO lymphocytes are poorly motile and that locomotor capacity is acquired as the cells enter GI and begin to grow. Entry of T cells into GI requires contact with appropriate accessory cells, thus contact is a prerequisite for locomotor activation. A completely different pattern of behaviour was observed when locomotor lymphocytes were exposed either to the supernatants from phagocytosing monocytes or to gradients diffusing from macrophages during phagocytosis. Here lymphocyte locomotion and chemotaxis were activated rapidly, and lymphocytes migrated in to cluster round macrophages in groups of 10 or so within 20 min. This appeared to be due to transient release of an attractant. The attractant molecules involved may not be different from those in, e.g., anti-CD3induced clustering. Phagocytosis may simply trigger massive release of attractants which are released at low levels under resting conditions. We do not know what these molecules are. A potential candidate would be IL-8 (neutrophil activating protein) which is an attractant for T lymphocytes (Larsen et al., 1989) as well as for neutrophils (Yoshimura et al., 1987). In the mouse peritoneal exudates studied here, neutrophils as well as lymphocytes responded to the macrophage-released factor.
0~~~1
Figure 6. Chemotaxis of mouse lymphocytes towards a macrophage following phagocytosis by the latter. The macrophage (shaded) is shown at the centre of the figure with an attached lymphocyte. The cell outlines of these and of eight nearby lymphocytes have been drawn in their positions at the time of phagocytosis. Cell tracks were plotted by dotting the cell centres at 80-second intervals and joining the dots. The numerals are times in minutes after phagocytosis. There was a lag before most of the cells began to respond. The mean speed + SEM over the final 8 min of the track was 36+031 gm/min. The mean chemotactic ratio, displacement/total distance travelled was +089+0±04 indicating a strong directional response towards the gradient source. Bar= 10 pm.
Another factor with similar activity in our laboratory is LCF, the T-lymphocyte chemotactic factor described by Potter & Van Epps (1987). Whatever the attractant turns out to be, this rapid attraction of lymphocytes may be a useful device for clustering lymphocytes round a macrophage which has ingested a microorganism and is presenting microbial antigens on its surface. In this paper, an exhaustive exploration of the nature of the accessory cell required to activate lymphocyte locomotion was not made. In anti-CD3-activation this cell must be FcR+, presumably a monocyte (Wilkinson & Higgins, 1987a). Most of the clusters contained monocytes at their centre. Monocytes and macrophages are obviously also the attractant accessory cells in the phagocytosis experiments. However, in the AMLR, dendritic cells may be more important (Steinman & Witmer, 1978; Austyn, 1987) and cells of dendritic morphology certainly moved into and were present in clusters in these experiments. REFERENCES ALLAN R.B. & WILKINSON P.C. (1978) A visual analysis of chemotactic and chemokinetic locomotion of human neutrophil leucocytes. Exp. Cell Res. 111, 191. AUSTYN J.M. (1987) Lymphoid dendritic cells. Immunology 62, 161. BERMAN L. (1966) Lymphocytes and macrophages in vitro. Their activities in relation to functions of small lymphocytes. Lab. Invest. 15, 1084. BIBERFELD P. (1971) Uropod formation in phytohaemagglutinin (PHA) stimulated lymphocytes. Exp. Cell Res. 66, 433. DIXON H.M. & MCCUTCHEON M. (1936) Chemotropism of leukocytes
Locomotion, chemotaxis and clustering of immune cells in relation to their rate of locomotion. Proc. Soc. Exp. Biol. Med. 34, 173. EL-NAGGAR A.L., VAN Epps D.E. & WILLIAMS R.C. (1981) Effect of culturing on the human lymphocyte locomotor response to casein, C5a and f-met-leu-phe. Cell Immunol. 60, 43. HASTON W.S. & WILKINSON P.C. (1988) Visual methods for measuring leukocyte locomotion. Meth. Enzymol. 162, 17. LARSEN C.G., ANDERSON A.O., APPELLA E., OPPENHEIM J.J. & MATSUSHIMA K. (1989) The neutrophil activating protein (NAP-1) is also chemotactic for T lymphocytes. Science, 243, 1464. LIPSKY P.E. & ROSENTHAL A.S. (1973) Macrophage lymphocyte interaction. I. Characteristics of the antigen-independent binding of guinea-pig thymocytes and lymphocytes to syngeneic macrophages. J. exp. Med. 138, 900. LIPSKY P.E. & ROSENTHAL A.S. (1975) Macrophage-lymphocyte interaction. II. Antigen-mediated physical interactions between immune guinea-pig lymph node lymphocytes and syngeneic macrophages. J. exp. Med. 141, 138. McFARLAND W. & HEILMAN D.H. (1965) Lymphocyte foot appendage: its role in lymphocyte function and in immunological reactions. Nature (Lond.), 205, 887. McFARLAND W., HEILMAN D.H. & MOORHEAD J.F. (1966) Functional anatomy of the lymphocyte in immunological reactions in vitro. J. exp. Med. 124, 851. McFARLAND W. & SCHECHTER G.P. (1970) The lymphocyte in immunological reactions in vitro. Ultrastructural studies. Blood, 35, 683. McGREGOR D.D. & LOGIE P.S. (1974) The mediator of cellular immunity. VII. Localization of sensitized lymphocytes in inflammatory exudates. J. exp. Med. 139, 1415. O'NEILL G.J. & PARROTT D.M.V. (1977) Locomotion of human lymphoid cells. I. Effect of culture and Con A on T and non-T lymphocytes. Cell Immunol. 33, 257.
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PETRI J., BRAENDSTRUP 0. & WERDELIN 0. (1979) Macrophagelymphocyte clusters in the immune response to soluble protein antigens in vitro. VIII. Cinephotomicrographic studies. Scand. J. Immunol. 10, 493. POTTER J.W. & VAN Epps D.E. (1986) Human T-lymphocyte chemotactic activity: nature and production in response to antigen. Cell Immunol. 97, 59. SHARP J.A. & BURWELL R.G. (1960) Interaction 'peripolesis' of macrophages and lymphocytes after skin homografting or challenge with soluble antigens. Nature (Lond.), 188, 474. STEINMAN R.M. & WITMER M.D. (1978) Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proc. natl. Acad. Sci. U.S.A. 75, 5132. WILKINSON P.C. (1982) Visual observation of chemotaxis and chemotropism in mouse macrophages. Immunobiology, 161, 376. WILKINSON P.C. (1986) The locomotor capacity of human lymphocytes and its enhancement by cell growth. Immunology, 57, 281. WILKINSON P.C. & HIGGINS A. (1987a) OKT3-activated locomotion of human blood lymphocytes: a phenomenon requiring contact of T cells with Fc-receptor-bearing cells. Immunology, 60, 445. WILKINSON P.C. & HIGGINs A. (1987b) Cyclosporin A inhibits mitogenactivated but not phorbol ester-activated locomotion of human lymphocytes. Immunology, 61, 311. WILKINSON P.C. & ISLAM L.N. (1989) Recombinant interleukin 4 and gamma interferon activate locomotor capacity in human B lymphocytes. Immunology, 67, 237. YosHIMURA T., MATsusHImA K., TANAKA S., ROBINSON E.A., APPELLA E., OPPENHEIM J.J. & LEONARD E.J. (1987) Purification of human monocyte-derived neutrophil chemotactic factor that shares sequence homology with other host defense cytokines. Proc. natl. Acad. Sci. U.S.A. 84, 9233.
Relation between locomotion, chemotaxis and clustering of immune cells P. C. WILKINSON Department of Bacteriology and Immunology, University of Glasgow (Western Infirmary), Glasgow
Acceptedfor publication 15 September 1989 SUMMARY Experiments were designed to discover whether locomotor or chemotactic events are needed for clustering of lymphocytes with accessory cells or, conversely, whether clustering precedes the activation of lymphocyte locomotion. The time-courses of clustering and locomotor activation were compared and the behaviour of moving cells during cluster formation was filmed. Human lymphocytes direct from blood were activated by culture for 24-48 hr with anti-CD3 antibody or in allogeneic mixed leucocyte reactions (AMLR). The proportion of clustered and locomotor lymphocytes was low at the beginning of culture. Clusters appeared during the first few hours, before the increase in numbers of locomotor lymphocytes. Filming gave no evidence that the cells attracted one another chemotactically to form clusters. Rather, cells made chance contact by random locomotion and then remained adherent, though lymphocytes very close (< 10 pm) to clusters did show increased pseudopod formation towards the cluster. However, the behaviour of motile lymphocytes responding to monocytes or macrophages given a phagocytic stimulus was different. Human monocytes which ingested opsonized zymosan released a material during but not following phagocytosis which caused an immediate increase in polar shape-change in lymphocytes. Macrophages from Corynebacterium parvum-induced mouse peritoneal exudates, given a phagocytic stimulus (opsonized Candida albicans), acted as sources of chemotactic gradients which attracted nearby lymphocytes to form clusters. This was due to brief release of a material immediately following phagocytosis, but after 15 min or so the macrophages no longer attracted nearby cells. These experiments suggest that, during induction of an immune response to a non-phagocytic stimulus, clusters form slowly by random contact followed by preferential adhesion. However, after phagocytosis, there may be a chemotactic response to the ingesting macrophage. This may help to focus lymphocytes onto macrophages which present microbial antigens.
INTRODUCTION
singly, been little studied. Petri, Braendstrup & Werdelin (1979) filmed cluster formation and suggested that lymphocytes actively sought out macrophages but they did not provide quantitative support for this idea. Clusters could form either by chemotactic attraction or by random contact, close cell-cell contact then inducing both locomotor activity and growth in the clustered cells. Activation of the locomotor capacity of lymphocytes is associated with activation of growth (Biberfeld 1971; McGregor & Logie, 1974). Although the proportion oflocomotor lymphocytes direct from human blood is low, both T cells (Wilkinson, 1986; Wilkinson & Higgins, 1987a) and B cells (Wilkinson & Higgins, 1987b; Wilkinson & Islam, 1989) show an increase in locomotor shapes, in invasion of collagen gels, and in responsiveness to chemoattractants (O'Neill & Parrott, 1977; ElNaggar, Van Epps & Williams, 1981) during the first 24 hr of culture with antigens or mitogens. These functions are acquired as the cells enter the GI phase of growth, increase in size, and synthesize increased amounts of RNA and protein. Both locomotor activation and clustering are therefore early events in immune activation. To investigate the causal relationship between them, it is helpful to investigate their temporal relationship. This is one of the purposes of this paper. There may be different mechanisms for clustering depending on
Heterotypic clustering of lymphocytes with accessory cells is essential for induction of immune responses, providing the contact necessary for T cells to recognize MHC-presented antigen and the close proximity required for cell-to-cell signalling of effector functions. Early studies documented the events of clustering by microscopic techniques including time-lapse cinematography. Lymphocytes were observed to attach to and circumnavigate the surfaces ofmacrophages in culture (Sharp & Burwell, 1960; Berman, 1966). In mixed leucocyte cultures, macrophage-associated lymphocytes increased in size and began to synthesize DNA (McFarland & Heilman, 1965; McFarland, Heilman & Moorhead, 1966). These activated lymphocytes were frequently in locomotor morphology. An antigen-dependent form of clustering was distinguished from, and shown to be stronger than, antigen-independent clustering (Lipsky & Rosenthal, 1973, 1975). The relation between locomotion and clustering has, surpriCorrespondence: Dr P. C. Wilkinson, Dept. of Bacteriology and Immunology, University of Glasgow (Western Infirmary), Glasgow GI I 6NT, U.K.
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whether an unactivated or a preactivated starting cell population is under study. These have been distinguished here. Lymphocytes from human blood which, unstimulated, have low locomotor activity, were activated by culture with anti-CD3 antibody or by AMLR. Anti-CD3 is a model for antigen-induced activation, though anti-CD3 clusters form by antibodymediated cross-linking between CD3 on the T cell and Fc receptors on the accessory cell rather than via the T-cell receptor. In a separate group of experiments, monocytes or macrophages were given a phagocytic stimulus. The locomotor behaviour of lymphocytes in response to products released during phagocytosis was examined using either precultured human blood mononuclear cells and measuring the effect of phagocytosis of opsonized zymosan by monocytes on lymphocyte locomotion, or using mononuclear cells from the mouse peritoneal cavity 4 days following immunological priming with Corynebacterium parvum. In this case, macrophages were allowed to ingest opsonized Candida albicans and the effect of phagocytosis on the subsequent locomotion of nearby lymphocytes towards the macrophages was filmed.
MATERIALS AND METHODS Reagents Hanks' buffered salt solution, RPMI-1640 and fetal calf serum (FCS) were from Flow, Rickmansworth, Herts. Human serum albumin (HSA) was from Behringwerke, Marburg, Germany. Anti-CD3 monoclonal antibody was from Orthoklein, Raritan, NJ (OKT3 IgG2a antibody). Zymosan and dibutyryl cyclic AMP were from Sigma, Poole, Dorset. Cyclosporin A was a gift from Dr A. W. Thomson, Department ofPathology, University of Aberdeen and was used as described by Wilkinson & Higgins (1987b). Anti-human Class II monoclonal antibody was from Sera Labs, Crawley, Sussex (Clone FMC 4). Class II + cells were scored by immunofluorescence using FITC-anti-mouse IgG as a second layer. Candida albicans blastospores were grown from clinical isolates by culture overnight on dextrose-peptone agar. 'Simultest' (FITC-anti-CD3+ phycoerythrin anti-CD19; Becton-Dickinson, Mountain View, CA) was used to distinguish T and B cells by fluorescence microscopy. Non-specific esterase (NSE) was used to identify monocytes/macrophages.
Human blood cells Blood was taken by venepuncture from healthy subjects. The mononuclear cell fraction was isolated on lymphocyte separation medium (Flow) and washed thrice with Hanks solution buffered to pH 7-4 with morpholinopropane sulphonate (MOPS) and containing HSA (10 mg/ml) (Hanks-HSA). Mouse peritoneal exudate cells These were provided by Dr J. H. Brock (this department) from peritoneal exudates prepared by injecting Corynebacterium parvum, strain 4982 (2 mg/mouse) 4 days previously into the peritoneal cavity of adult NIH strain mice. The peritoneal fluids were taken; cell counts showed 40-50% lymphocytes, 40-50% macrophages, up to 10% neutrophils and about 5% of dendritic cells. As documented in an earlier publication (Wilkinson, 1982) over 70% of the exudate lymphocytes were motile, probably because C. parvum acts as an immunological stimulus. Four-day exudates induced with a non-immunogenic stimulus, thio-
glycollate broth, contained lymphocytes that were mainly nonmotile. Also, the proportion of class II-positive macrophages, while variable, was always higher in C. parvum-induced exudates than in thioglycollate-induced exudates (J. H. Brock, personal communication). The peritoneal exudate cells were washed thrice and resuspended in RPMI + 20% heat-activated FCS. Methods for assaying clustering and locomotion Activation of human cells with anti-CD3. The mononuclear cell fraction was cultured at 5 x 105 cells/ml in Hanks-HSA without added serum in tissue culture plastic dishes (well size 1-6 x 17 cm; Flow). Anti-CD3 antibody was added at 25 ng/ml and culture was continued for 24-48 hr. Clustering was quantified using an inverted microscope (Nikon Diaphot TMD) with an eyepiece grid graticule and a x 20 phase contrast objective. The number of clusters per field in eight fields per well, defined as groups of three or more cells in close contact which could not be dissociated by gentle rocking, was scored. A baseline value for clustering was recorded within the first 2 hr of culture and thereafter at intervals up to 48 hr. Locomotor capacity was quantified by scoring the proportion of lymphocytes with morphological anteroposterior polarity. After appropriate incubation, cells were fixed with 2-5% glutaraldehyde, washed, 300 cells were counted and the number of spherical and polarized cells was scored. Cell viability was checked morphologically under phase contrast and was excellent in all experiments. AMLR. The mononuclear cell fractions of two blood samples from different normal persons were prepared separately. They were then mixed in equal proportions and cultured in Hanks-HSA without added stimulus as described above. Clustering and polarization were scored as described above. Effect on lymphocyte locomotion of phagocytosis of microorganisms by monocytes or macrophages. The mononuclear cell fraction from human blood was cultured either in Hanks-HSA or in RPM I+ FCS (20%) for 24-48 hr. The non-adherent cells were then removed, centrifuged, and the supernatant was retained. The non-adherent cells (mostly lymphocytes) were washed thrice in Hanks' solution and retained at 20° for use. The adherent cells (mostly monocytes) were also washed thee times and retained at 200. Zymosan particles (107/ml) were opsonized by incubation for 20 min at 370 with 20% autologous serum. They were then washed three times, resuspended in Hanks-HSA and incubated for 15 min at 37°. They were added (107 particles per well) to the adherent cells and incubated for 20-30 min at 37°. The supernatant containing non-phagocytosed zymosan, together with monocyte release products, was then removed and centrifuged at 2000 g. This supernatant (phagocytosis supernatant) was retained. After washing, the adherent cells were reincubated in Hanks-HSA for 30 min at 37°. This supernatant (postphagocytosis) also was centrifuged and retained. With each experiment the following controls were also set up: monocytes incubated for 30 min without zymosan; and opsonized zymosan incubated for 30 min at 370 without monocytes. The various supernatants were then tested for activity against the nonadherent lymphocyte population in a polarization assay. Lymphocytes were incubated at 370 for 30 min, fixed, and the proportion of polarized cells scored.
Locomotion, chemotaxis and clustering of immune cells Filming assays oflymphocyte locomotion. Filming was done using a metal filming chamber described elsewhere (Haston & Wilkinson, 1988). A coverslip was sealed to the lower surface of this chamber and cells either in Hanks-HSA or in Hanks-FCS (20%) were allowed to settle on the glass. The chamber, filled with appropriate medium, was sealed and placed on a microscope stage heated at 370 and the behaviour of the cells was filmed. The locomotion of the human mononuclear cell fraction was videotaped using a Panasonic 8050 time-lapse videorecorder. Long sequences of from 30 min to 4 hr were recorded during the whole culture period of 48 hr. The paths taken by moving lymphocytes were tracked and their interactions with other cells were recorded. Where adherence of a cell to a group of two or more cells was observed, the tape was run back and the direction and speed of locomotion of the cell or cells approaching to form a cluster was compared in the approach path (within 50 gm of the point of contact) with direction and speed in earlier parts of the tracks of the same cells. Filming of mouse peritoneal exudate cells. Mouse peritoneal exudate cells were filmed using time-lapse cinematography on 16 mm cine film (Kodak Plus-X-Reversal) with a Bolex H16 cinecamera and a lapse interval of 8 seconds between frames. Candida albicans blastospores preopsonized with fresh mouse serum (20%) were added at 2 5 x 105 spores/ml to the filming chamber which was immediately sealed and filming begun. The candida settled on the lower coverslip where some of them were phagocytosed by macrophages. This phagocytosis and the subsequent locomotor events were filmed. The direction and speed of locomotion of cells were analysed using methods described in earlier publications (Allan & Wilkinson, 1978; Haston & Wilkinson, 1988). Chemotaxis was quantified using the McCutcheon ratio (Dixon & McCutcheon, 1936), displacement divided by total distance travelled to the source. For a cell reaching a gradient source by travelling in a straight line, this ratio is + 1 *0. In the absence of any net displacement towards (or away from) the source, it is zero. RESULTS Relation between locomotion and clustering in human blood mononuclear cells The time-course of development of clusters and of lymphocytepolarized morphology during culture of human mononuclear cells in the presence of anti-CD3 is shown in Fig. Ia. The number of clusters increased during the first 10 hr and then reached a peak. By 10 hr there was little increase in the numbers of polarized cells and the major increase took place between 10 and 24 hr once clustering was fully developed. At 24 hr most clusters contained one or more class II-positive monocytes and a variable number of lymphocytes. About 10-15% of these were B cells and most of the remainder were T cells. A similar, but slower sequence of events was seen in the AMLR (Fig. lb) though the number of clusters was smaller than in the response to anti-CD3. These findings suggest that, in a first exposure to antigen or mitogen, clustering precedes locomotor activation. Agents that inhibit activation of lymphocyte locomotion reduced cluster formation. Clusters formed more slowly at 200 than at 370 (Fig. 2a) and not at all at 00. Culture with dibutyryl cyclic AMP (10-3M), which inhibits lymphocyte locomotion (unpublished observations in this laboratory) also reduced
(a)
129 (b)
to
2
o 40 E 30 ~2O
o' 20
CO
10I2
12
24
36
48
Time (hr)
Figure 1. Time-course of formation of clusters (continuous lines) and proportion of lymphocytes in locomotor morphology (broken lines) in (a) anti-CD3, 25 ng/ml (mean + SEM, n = 5) and (b) the AMLR (mean of two experiments). For clarity, control curves for unmixed cells without an activator have been omitted. The number of clusters per field in these controls was 2-3 within 12-24 hr and the proportion of polarized cells at 24-48 hr was 10-15%.
(a)
(b)
Z
0
U,
C-
6
12
18
24
Time (hr)
Figure 2. (a) Time-course of cluster formation in response to anti-CD3 (25 ng/ml). Cells cultured at 370 ( x ), 200 (0) or in the presence of dibutyryl cyclic AMP (10-3M) at 370 (O). Results of a single experiment. (b) Time-course of cluster formation in response to anti-CD3 in the absence ( x ) and presence (0) of cyclosporin, I pg/ml. Mean of three experiments + SEM.
clustering (Fig. 2a). Cyclosporin inhibits anti-CD3-activated lymphocyte locomotion (Wilkinson & Higgins, 1987b; Fig. 3) and moderately reduced the number of clusters formed (Fig. 2b). These results support the view that active locomotion contributes to cluster formation. Visual observations
Time-lapse videorecording of cell locomotion during the formation of clusters both in response to anti-CD3 and in an AMLR showed that at the beginning of culture, the number of motile lymphocytes was low; about 10% of the total in Hanks-HSA and 20% in FCS. Many of the translocating cells during the first few hours were monocytes, though many of these became sessile as culture continued. Clusters formed slowly and randomly. Locomotor cells coming into contact with one another sometimes remained attached but the cells showed no change in turning behaviour or speed as they neared one another. However, lymphocytes close to a cluster often showed increased protrusive and ruffling activity at the anterior pole and formed
130
P. C. Wilkinson --07.
0
40
55
65
80
104
Figure 4. Sequential images from a time-lapse film showing behaviour of a lymphocyte (shaded) approaching a cluster. Numerals indicate time in seconds. The straight line represents a fixed point in space relative to which cell movement can be judged. Cells incubated in anti-CD3 (25 ng/ ml). The shaded lymphocyte had been migrating randomly. On approaching the cluster, it put out a broad anterior pseudopod (seen at 40 and 55 seconds) which contacted the cluster (65 seconds). The lymphocyte then began to translocate rapidly round the edge of the cluster.
Figure 3. Appearance of a (25 ng/ml)
there
are
many
cyclosporin
mononuclear cell culture after 24 hr in aCD3
the absence (a)
in
lymphocytes
or presence
in
but that most of the cells
the presence of clusters in both, in
are
though
spherical
in the absence of
in its presence. Note
total cluster numbers
were
lower
cyclosporin (b).
in the direction of the
phase-dense pseudopods at
(b) of cyclosporin. Note that
locomnotor morphology
very short range
flattened
( 80% NSE +) were given opsonized zymosan as a phagocytic stimulus and the various supernatants were tested for their capacity to induce polarization ofnon-adherent lymphocytes. Results are shown in Table 1. Phagocytosis supernatants caused increased polarization of lymphocytes whereas supernatants of zymosan alone, or of monocytes alone, incubated for 30 min did not. However, supernatants from monocytes alone after culture for 24 hr did increase lymphocyte polarization, possibly due to release of a material at a low level by resting monocytes which is accelerated by phagocytosis. Table 2 shows that the pulse of release during phagocytosis is short-lived. After completion of phagocytosis and removal of free zymosan, the post-phagocytic supernatants had no activity. The attractant in supernatants of monocytes was not characterized biochemically, though preliminary experiments have shown that it is destroyed by boiling and is retained within a dialysis sac. Attempts were made to demonstrate the effects of this attractant on lymphocyte locomotion by filming, as this is the best method for showing chemotaxis of cells to a gradient source. The most satisfactory lymphocyte population for filming assays proved to be that from the peritoneal cavity of the mouse following challenge with C. parvum, as these were both more adherent and more motile than lymphocytes from human blood.
and
added in
were
of FCS
a
can be short-
Chemotaxis of mouse peritoneal lymphocytes towards phagocytosing macrophages Following phagocytosis of candida by macrophages in C. parvum-induced exudates, some macrophages released a pulse of attractant which caused nearby lymphocytes to migrate directionally towards the macrophage and to form a cluster around it (Fig. 5). During brief film sequences, the number of lymphocytes joining such clusters increased rapidly (Table 3). A traced record of an event in which eight lymphocytes within a radius of 100 im of a macrophage migrated in radially to form a cluster is shown in Fig. 6. After about 15 min no further response was seen. Lymphocytes responded chemotactically only to macrophages and not to free, unphagocytosed Candida. Staining the clusters after fixation showed that they contained 50-70% lymphocytes, 20-30% macrophages and 5-10% neutrophils. Cells of dendritic morphology were also present in small numbers. It was easier to distinguish lymphocytes from neutrophils in filter assays than in films of living cells. The released
Locomotion, chemotaxis and clustering of immune cells
131
Table 1. Effect of supernatants from phagocytosing monocytes on morphological polarization of lymphocytes P value
Supernatant added to lymphocytes (370 30 min)
Percentage polarized lymphocytes* (mean + SEM)
n
99 + 13
6
18 3+2 6 22-9+3-4
5 6
0-02 3 cells) at the beginning and at the end of the film.
hour. It is hard to think of a chemotactic mechanism that would operate so gradually, and the lengthy stretches of videotape gave no evidence for chemotaxis operating at a distance, though there was evidence of directional protrusive behaviour by cells close to a cluster. This could mean that cells such as monocytes release a very weak signal whose directionality is quickly lost by diffusion. Nevertheless, the presence of such a material in uniform concentration could still signal an increase in lymphocyte speed (chemokinesis). In these experiments locomotor activation of lymphocytes was seen only after clusters had formed. Earlier observers showed that lymphocytes which had joined clusters grew in size and became motile (McFarland et al., 1966; McFarland & Schechter, 1970;' Lipsky & Rosenthal, 1975) which suggests that the signal for locomotor activation is transmitted by close cell-to-cell contact. However, the time sequence does not prove that clustering is essential for locomotor activation. This remains to be tested. Our working hypothesis at present, based on this and earlier work, is that GO lymphocytes are poorly motile and that locomotor capacity is acquired as the cells enter GI and begin to grow. Entry of T cells into GI requires contact with appropriate accessory cells, thus contact is a prerequisite for locomotor activation. A completely different pattern of behaviour was observed when locomotor lymphocytes were exposed either to the supernatants from phagocytosing monocytes or to gradients diffusing from macrophages during phagocytosis. Here lymphocyte locomotion and chemotaxis were activated rapidly, and lymphocytes migrated in to cluster round macrophages in groups of 10 or so within 20 min. This appeared to be due to transient release of an attractant. The attractant molecules involved may not be different from those in, e.g., anti-CD3induced clustering. Phagocytosis may simply trigger massive release of attractants which are released at low levels under resting conditions. We do not know what these molecules are. A potential candidate would be IL-8 (neutrophil activating protein) which is an attractant for T lymphocytes (Larsen et al., 1989) as well as for neutrophils (Yoshimura et al., 1987). In the mouse peritoneal exudates studied here, neutrophils as well as lymphocytes responded to the macrophage-released factor.
0~~~1
Figure 6. Chemotaxis of mouse lymphocytes towards a macrophage following phagocytosis by the latter. The macrophage (shaded) is shown at the centre of the figure with an attached lymphocyte. The cell outlines of these and of eight nearby lymphocytes have been drawn in their positions at the time of phagocytosis. Cell tracks were plotted by dotting the cell centres at 80-second intervals and joining the dots. The numerals are times in minutes after phagocytosis. There was a lag before most of the cells began to respond. The mean speed + SEM over the final 8 min of the track was 36+031 gm/min. The mean chemotactic ratio, displacement/total distance travelled was +089+0±04 indicating a strong directional response towards the gradient source. Bar= 10 pm.
Another factor with similar activity in our laboratory is LCF, the T-lymphocyte chemotactic factor described by Potter & Van Epps (1987). Whatever the attractant turns out to be, this rapid attraction of lymphocytes may be a useful device for clustering lymphocytes round a macrophage which has ingested a microorganism and is presenting microbial antigens on its surface. In this paper, an exhaustive exploration of the nature of the accessory cell required to activate lymphocyte locomotion was not made. In anti-CD3-activation this cell must be FcR+, presumably a monocyte (Wilkinson & Higgins, 1987a). Most of the clusters contained monocytes at their centre. Monocytes and macrophages are obviously also the attractant accessory cells in the phagocytosis experiments. However, in the AMLR, dendritic cells may be more important (Steinman & Witmer, 1978; Austyn, 1987) and cells of dendritic morphology certainly moved into and were present in clusters in these experiments. REFERENCES ALLAN R.B. & WILKINSON P.C. (1978) A visual analysis of chemotactic and chemokinetic locomotion of human neutrophil leucocytes. Exp. Cell Res. 111, 191. AUSTYN J.M. (1987) Lymphoid dendritic cells. Immunology 62, 161. BERMAN L. (1966) Lymphocytes and macrophages in vitro. Their activities in relation to functions of small lymphocytes. Lab. Invest. 15, 1084. BIBERFELD P. (1971) Uropod formation in phytohaemagglutinin (PHA) stimulated lymphocytes. Exp. Cell Res. 66, 433. DIXON H.M. & MCCUTCHEON M. (1936) Chemotropism of leukocytes
Locomotion, chemotaxis and clustering of immune cells in relation to their rate of locomotion. Proc. Soc. Exp. Biol. Med. 34, 173. EL-NAGGAR A.L., VAN Epps D.E. & WILLIAMS R.C. (1981) Effect of culturing on the human lymphocyte locomotor response to casein, C5a and f-met-leu-phe. Cell Immunol. 60, 43. HASTON W.S. & WILKINSON P.C. (1988) Visual methods for measuring leukocyte locomotion. Meth. Enzymol. 162, 17. LARSEN C.G., ANDERSON A.O., APPELLA E., OPPENHEIM J.J. & MATSUSHIMA K. (1989) The neutrophil activating protein (NAP-1) is also chemotactic for T lymphocytes. Science, 243, 1464. LIPSKY P.E. & ROSENTHAL A.S. (1973) Macrophage lymphocyte interaction. I. Characteristics of the antigen-independent binding of guinea-pig thymocytes and lymphocytes to syngeneic macrophages. J. exp. Med. 138, 900. LIPSKY P.E. & ROSENTHAL A.S. (1975) Macrophage-lymphocyte interaction. II. Antigen-mediated physical interactions between immune guinea-pig lymph node lymphocytes and syngeneic macrophages. J. exp. Med. 141, 138. McFARLAND W. & HEILMAN D.H. (1965) Lymphocyte foot appendage: its role in lymphocyte function and in immunological reactions. Nature (Lond.), 205, 887. McFARLAND W., HEILMAN D.H. & MOORHEAD J.F. (1966) Functional anatomy of the lymphocyte in immunological reactions in vitro. J. exp. Med. 124, 851. McFARLAND W. & SCHECHTER G.P. (1970) The lymphocyte in immunological reactions in vitro. Ultrastructural studies. Blood, 35, 683. McGREGOR D.D. & LOGIE P.S. (1974) The mediator of cellular immunity. VII. Localization of sensitized lymphocytes in inflammatory exudates. J. exp. Med. 139, 1415. O'NEILL G.J. & PARROTT D.M.V. (1977) Locomotion of human lymphoid cells. I. Effect of culture and Con A on T and non-T lymphocytes. Cell Immunol. 33, 257.
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PETRI J., BRAENDSTRUP 0. & WERDELIN 0. (1979) Macrophagelymphocyte clusters in the immune response to soluble protein antigens in vitro. VIII. Cinephotomicrographic studies. Scand. J. Immunol. 10, 493. POTTER J.W. & VAN Epps D.E. (1986) Human T-lymphocyte chemotactic activity: nature and production in response to antigen. Cell Immunol. 97, 59. SHARP J.A. & BURWELL R.G. (1960) Interaction 'peripolesis' of macrophages and lymphocytes after skin homografting or challenge with soluble antigens. Nature (Lond.), 188, 474. STEINMAN R.M. & WITMER M.D. (1978) Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proc. natl. Acad. Sci. U.S.A. 75, 5132. WILKINSON P.C. (1982) Visual observation of chemotaxis and chemotropism in mouse macrophages. Immunobiology, 161, 376. WILKINSON P.C. (1986) The locomotor capacity of human lymphocytes and its enhancement by cell growth. Immunology, 57, 281. WILKINSON P.C. & HIGGINS A. (1987a) OKT3-activated locomotion of human blood lymphocytes: a phenomenon requiring contact of T cells with Fc-receptor-bearing cells. Immunology, 60, 445. WILKINSON P.C. & HIGGINs A. (1987b) Cyclosporin A inhibits mitogenactivated but not phorbol ester-activated locomotion of human lymphocytes. Immunology, 61, 311. WILKINSON P.C. & ISLAM L.N. (1989) Recombinant interleukin 4 and gamma interferon activate locomotor capacity in human B lymphocytes. Immunology, 67, 237. YosHIMURA T., MATsusHImA K., TANAKA S., ROBINSON E.A., APPELLA E., OPPENHEIM J.J. & LEONARD E.J. (1987) Purification of human monocyte-derived neutrophil chemotactic factor that shares sequence homology with other host defense cytokines. Proc. natl. Acad. Sci. U.S.A. 84, 9233.
