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T Lymphocytes and Neutrophil Granulocytes Differ in Regulatory Signaling and Migratory Dynamics with Regard to Spontaneous Locomotion and Chemotaxis
Cellular Immunology 199, 104 –114 (2000) doi:10.1006/cimm.1999.1605, available online at http://www.idealibrary.com on
T Lymphocytes and Neutrophil Granulocytes Differ in Regulatory Signaling and Migratory Dynamics with Regard to Spontaneous Locomotion and Chemotaxis Frank Entschladen, Matthias Gunzer, Chi Mi Scheuffele, Bernd Niggemann, and Kurt S. Za¨nker Institute for Immunology, Witten/Herdecke University, Stockumer Strasse 10, 58448 Witten, Germany Received October 18, 1999; accepted December 17, 1999
Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis. © 2000 Academic Press
INTRODUCTION Chemokines (chemoattractant cytokines) are peptides that mainly induce chemotactic movement of leukocytes but also have functions in trafficking and regulation of leukocyte effector function (1, 2). Chemokines are grouped into four subclasses. The CXC chemokines contain two cysteine residues in their aminoterminal domain, separated by a single aminoacid. The CC chemokines lack this separating aminoacid (1). The third group, known as C or XC chemokines, consists of one member, possessing only the second cysteine residue in the aminoterminal domain (3). The fourth class of chemokines, the CX 3C chemokines, are characterized by three separating amino acids between 0008-8749/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
the aminoterminal cysteines. Fractalkine is the only member of this class (4). All known chemokine receptors belong to the family of seven-helices receptors, which are coupled to G-protein-mediated signaling (5). Other chemoattractants like formylated peptides, e.g., formyl-methionyl-leucyl-phenylalanine (fMLP), the complement fragment C5a, or leukotriene B4, have no structural similarities to the chemokines described above but do bind to G-protein-coupled seven-helices receptors (6). The cellular receptor for the CXC chemokine stromal-cell-derived factor-1 (SDF-1) is CXCR4, expressed on monocytes, neutrophil granulocytes, and lymphocytes (7, 8). SDF-1 has chemotactic effects on lymphocytes and monocytes but not on neutrophil granulocytes (9). We used SDF-1 to stimulate T lymphocyte chemotaxis and fMLP to induce chemotaxis of neutrophil granulocytes. The induction of chemotaxis was accompanied by changes in dynamic parameters, i.e., the locomotory active fraction of a cell population, the velocity of migration, and frequency and duration of migration breaks. The functional role of the microtubule cytoskeleton in the migration of adherent cells, e.g., fibroblasts and tumor cells, has been studied extensively in the past: colcemid-induced microtubule disruption and Taxolinduced stabilization of the microtubule system both potently inhibit migration of adherent cells (10 –12). Recent results of nonadherent T lymphocytes suggest a distinct involvement of the microtubule system in the locomotion of these cells, underscoring different migration strategies in leukocytes and adherent cells (13). Microtubule stabilization by Taxol has only a minor effect on the locomotory activity of T lymphocytes, whereas disruption of microtubules induced by colcemid leads to an increase in migratory activity of T lymphocytes. The increase in locomotory activity is due to both the recruitment of previously nonlocomoting cells and an increase in the duration of locomotion, i.e., an increase in the number of minutes a cell is locomotory active within a certain period of observation (14).
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This means that upon colcemid treatment the cells reduce either frequency or duration of breaks performed during locomotory activity or both. Nikolai et al. (14) argue in accordance with other authors (15, 16) that the promigratory effect of microtubule disassembly in leukocytes is due to a functional interaction with the actin cytoskeleton, i.e., tubulin transduces a signal to the actin cytoskeleton. It has recently been shown that disassembly of the microtubule system regulates the actin cytoskeleton via a protein tyrosine kinase (PTK)-dependent signal transduction pathway (17). The actin cytoskeleton has been shown to be of major importance for T cell migration, since actin-destabilizing drugs inhibit uropod formation and cellular motility (18, 19). In contrast to T lymphocytes, reports on neutrophil granulocytes describe an inhibition of fMLP-induced migration by microtubule-stabilizing Taxol (20). Results on the effects of antitubulins are controversial. Different reports describe an inhibition of human neutrophil granulocyte chemotaxis at micromolar concentrations of colchicin, whereas lower concentrations are stimulatory (20). Spontaneous migration of neutrophil granulocytes is in contrast increased at micromolar concentrations of colchicine, nocodazole, or vinblastine (21). Differences between random movement and chemotaxis were also observed by Bandmann et al. (22). Neutrophil chemotaxis was inhibited by various antitubulins, whereas the basic locomotion was not affected by podophyllic acid ethylhydrazide (22). We tested the hypothesis of whether changes of locomotory behavior after induction of chemotaxis could be ascribed to changes of the regulatory signal transduction of migration and whether this signal transduction is similar or different in neutrophil granulocytes and T lymphocytes. We did this on the basis of the finding that spontaneous locomotion of T lymphocytes was regulated by PTK activity and was distinct from a second, protein kinase C (PKC)-dependent type of migration inducible by PKC-activating phorbol ester (23). We furthermore investigated whether changes in the microtubule cytoskeleton were involved in changes in locomotion from spontaneous, matrix-induced locomotion to chemotaxis. The rationale was the above-described result by Nikolai et al. (14) that colcemid treatment increased the part of locomotory active T lymphocytes within a population, the time each cell was locomotory active within the observation period, and the velocity of migration. Thus, colcemid induces changes in locomotory behavior similar to chemoattractants.
The peripheral blood mononuclear cell fraction was isolated from heparinized blood of healthy donors by density-gradient centrifugation using Ficoll–Hypaque (ICN, Meckenheim, Germany). CD4 ⫹ and CD8 ⫹ cells were positively selected (10 min, 4°C) using immunomagnetic beads coated with mouse anti-human CD4 or CD8 mAb (66.1 or ITI-5C2; Dynabeads, Dynal, Hamburg, Germany). Subsequently, cell-bound beads were detached (45 min; 20°C) using polyclonal anti-mouse Fab antibodies (Detachabead, Dynal). Purified CD4 ⫹ and CD8 ⫹ cells were ⬎98% CD3 ⫹ and 96 –99% CD4 ⫹ or CD8 ⫹, respectively, as detected by flow cytometry (FACSCalibur, Becton–Dickinson, San Jose, CA). More than 98% of the cells were viable, as assessed by propidium iodide staining and flow cytometry. As previously described, the isolation procedure did not alter the activation state of the cells (23). Results shown herein were obtained using CD4 ⫹ cells. Data were confirmed by experiments performed with CD8 ⫹ cells, which led to similar results. Neutrophil granulocytes were isolated from the pellet of a density-gradient centrifugation. The pellet containing neutrophil granulocytes and erythrocytes was mixed with platelet-depleted serum. This mixture was diluted 1:1.3 with a high-molecular-weight dextran solution (Makrodex, Fresenius, Bad Homburg, Germany) containing 0.01 M ethylendiaminetetraacetic acid. After 2 h erythrocytes had settled down and the neutrophil granulocyte containing supernatant was separated from the pellet. Contaminating erythrocytes were then removed by hypotonic lysis with 0.3% sodium chloride for 2 min on ice. The obtained purified neutrophil granulocytes contained less than 5% eosinophil granulocytes and lymphocytes, as determined by Pappenheim staining. Neutrophil granulocytes were used immediately after isolation. T lymphocytes were maintained overnight in RPMI supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum (Boehringer Mannheim, Mannheim, Germany), and penicillin (50 U/ml) and streptomycin (50 g/ml; Gibco, EggensteinLeopoldshafen, Germany). Neutrophil granulocytes developed their maximum spontaneous migratory activity directly after isolation, whereas T lymphocytes reached their maximum activity 12 to 24 h after isolation. Thus, the experiments with neutrophil granulocytes and T lymphocytes were performed at different time points in order to get optimal spontaneous migratory activity for both cell types.
MATERIALS AND METHODS
Collagen lattices were generated as previously described (25). In short, 2.5 ⫻ 10 5 cells were mixed with 100 l of buffered collagen solution (pH 7.4) containing 1.67 mg/ml collagen type I in minimal essential Eagle’s medium (Flow, McLean, VA). The collagen was bovine
Cell Isolation and Cultivation Human peripheral CD4 ⫹ and CD8 ⫹ cells were isolated from human blood as previously described (24).
Preparation of Three-Dimensional Collagen Lattices
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FIG. 1. Chemotaxis chamber. A central hole, 15 mm in diameter, was drilled into an acrylic glass microscope slide. This hole was covered on both sides with coverslips which were sealed with wax (paraffin:vaseline 1:1). On the upper side a gap was left to fill the chamber. From the anterior side, a 1-mm-wide channel was drilled into the acrylic glass slide. In this channel a glass capillary containing the chemoattractant source was inserted.
dermal collagen (Cohesion, Palo Alto, CA) consisting of 95–98% type I collagen with the remainder being composed of type III collagen. The buffered collagen suspension was filled into self-constructed chemotaxis chambers and allowed to polymerize for 20 min at 37°C and a 5% CO 2 humidified atmosphere. For the direct observation of cellular chemotaxis, i.e., directional migration of individual cells, a novel chamber technique was developed (Fig. 1). A central hole, 15 mm wide, was drilled in an acrylic glass slide. Through one long side of the slide a 1-mm-wide channel was drilled to the edge of the central hole. The upper and lower sides of the central hole were sealed with cover slips, leaving a gap to fill the resulting chamber. The buffered collagen solution containing either T lymphocytes or neutrophil granulocytes was added to the chamber, covering the channel on the edge of the central hole. After polymerization, the chamber was sealed and migration was recorded by time-lapse videomicroscopy for 15 min. Subsequently, PBS as control or SDF-1 (Pharmingen, San Diego, CA) for the induction of T lymphocyte chemotaxis or fMLP (Sigma, Deisenhofen, Germany) for the induction of neutrophil granulocyte chemotaxis was added through the channel and the chemotactic behavior of the cells in the collagen matrix adjacent to the channel was recorded for the next 1 to 2 h. Functional involvement of PTKs and the PKC in the regulation of spontaneous locomotion and chemotaxis of neutrophil granulocytes and T lymphocytes was investigated by addition of the PTK inhibitor tyrphostin A23 and the PKC inhibitor calphostin C (both Calbiochem-Novabiochem, San Diego, CA) to the collagen lattices as described previously (23). Time-Lapse Videomicroscopy and Computer-Assisted Cell Tracking Cell locomotion within the three-dimensional collagen lattices was recorded by time-lapse videomicroscopy using four microscopy and recording devices simultaneously. For data analysis, 30 cells of each sample were randomly selected and two-dimensional
projections of the paths were digitized as x/y-coordinates in 1-min step intervals by computer-assisted cell tracking. From the obtained data migrated distance and direction of migration as well as time-related parameters such as migrated time, duration of migration breaks, and velocity were calculated for each single cell and the whole population. For the analysis of directionality, a modified form of a Markov-chain analysis (26) was established. The angle of each step of the cell paths was analyzed independently and the number of steps toward each quadrant of a coordinate system was counted. Therefore, the starting point of each step was set as the origin of the coordinate system. A random migration would have a 25% probability for the distribution of the steps into each quadrant. The number of steps toward or away from the site of addition of chemoattractants was determined by summing up all steps of the two quadrants adjacent to or opposite the source. The classical application of the Markov-chain analysis assumes that every step has the same length and therefore considers only the angles of the steps. Since the length of steps varies considerably, especially after the addition of fMLP, we added the length of steps directed into each quadrant, resulting in a net movement into each direction. Confocal Laser Scan Microscopy For immunofluorescent staining of the microtubule system, 50 l of a suspension of 3 ⫻ 10 5 neutrophil granulocytes or T lymphocytes in PBS or PBS containing 10 M fMLP or 1000 ng/ml SDF-1, respectively, was mixed with 100 l buffered collagen and the solution was transferred onto a coverslip. After 20 min of polymerization, locomotory activity was recorded for 10 min by time-lapse videomicroscopy and the respective migration paths were digitized as described above to determine the direction of migration prior to fixation. Subsequently, the cells were fixed with 3.7% formaldehyde (15 min, 20°C) and subsequently permeabilized with 0.5% Triton X-100 (10 min, 20°C). Samples were then incubated with 10 g/ml (1 h, 20°C) of mouse anti-␣-tubulin antibody B-5-1-2 (Sigma). After being washed with PBS, samples were incubated (1 h, 20°C) with 10 g/ml of lissamine rhodamine B sulfonylchloride-conjugated Fab fragments, specific for mouse Fc␥ (The Jackson Laboratories, Westgrove, PA). After a further washing step the coverslips were inverted and mounted on slides. Confocal laser scanning microscopy using a Leica TCS 4D microscope was performed as previously described (14, 27). Flow Cytometry Assembled tubulin in the cells was measured as described previously (14). In brief, 1 ⫻ 10 6 neutrophil granulocytes or T lymphocytes were treated for 1 h at
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37°C with 10 nM fMLP or 1000 ng/ml SDF-1, respectively. As positive and negative controls, cells were treated with 15 M Taxol (paclitaxel; Sigma) or 1 g/ml colcemid (Demecolcine; Sigma), respectively. After incubation, the cells were fixed (4% paraformaldehyde, 15 min, 20°C), permeabilized (0.5% Triton X-100, 10 min, 20°C), and immunostained for tubulin using 10 g/ml of mouse anti-␣-tubulin antibody B-5-1-2 as described for confocal laser scan microscopy. The mean fluorescence intensity was a direct measure of the assembled tubulin, since tubulin that was not bound to microtubules was washed out during Triton X-100 permeabilization. Immunoblotting The total amount of cellular ␣-tubulin was analyzed by immunoblotting as described previously (23). Neutrophil granulocytes and T lymphocytes (0.1 ⫻ 10 6 to 2 ⫻ 10 6) were lysed in Laemmli sample buffer (10 min, 95°C). Proteins were separated using polyacrylamide electophoresis according to the method of Laemmli (28) and were transferred to an Immobilion-P membrane (Millipore, Bedford, MA). After blocking of the membranes with 5% dry milk powder (1 h, 20°C), membranes were incubated with 1 g/ml of mouse anti-␣tubulin antibody B-5-1-2 (1 h, 20°C) and subsequently with a peroxidase-linked anti-mouse antibody (1 g/ ml, 1 h, 20°C). After incubation of the membrane with chemiluminescence substrate (Boehringer Mannheim; 2 min, 20°C) the chemiluminescence signal was detected by exposure to a Kodak X-OMAT AR film sheet (Sigma). RESULTS The Migratory Response of T Lymphocytes to SDF-1 and of Neutrophil Granulocytes to fMLP Was Chemotactic After placing 1000 ng/ml SDF-1 into the capillary of the chemotaxis chamber, T lymphocytes showed a chemotactic response (Fig. 2A). Modified Markov-chain analysis revealed that within the first 15 min 70.5% of the steps, with the length of the steps taken into account, were directed toward the chemoattractant source, whereas only 29.5% were orientated in the opposite direction. For the following 15 min only 53.3% of the migrated steps were oriented toward the source (Fig. 2C), probably because the gradient had disappeared due to diffusion. The addition of 10 nM fMLP to collagen lattices with neutrophil granulocytes resulted in a pronounced chemotactic response also. Within the first 15 min after addition of fMLP 77.8% of all steps were directed toward the source and only 22.2% were oriented to the opposite direction (Fig. 2B). Again the fMLP gradient probably disappeared due to diffusion, resulting in a nearly randomly distributed migration of
FIG. 2. Vector diagrams of chemotactic response. The migration paths of T lymphocytes in response to SDF-1 (A and C) and of neutrophil granulocytes in response to fMLP (B and D) were divided into 15-min intervals. Starting points of each interval of the paths were moved into the center and the number of steps toward the chemoattractant source and away from it was analyzed, taking the varying length of steps into consideration. The arrows indicate the direction from which 1000 ng/ml SDF-1 (A) and 10 nM fMLP (C) were added. Thirty cells were analyzed for each vector diagram. Data shown are representative of three independent experiments performed with neutrophil granulocytes and T lymphocytes from different donors.
the neutrophil granulocytes in the following 15 min (Fig. 2D). Only 53.4% of the steps were directed toward the source, whereas 46.6% were aimed in the opposite direction. The Locomotory Behavior of T Lymphocytes and of Neutrophil Granulocytes Characteristically Changes after Induction of Chemotaxis After incorporation within the three-dimensional collagen matrix both T lymphocytes and neutrophil granulocytes developed spontaneous locomotory activity (Table 1). After the induction of chemotaxis of T lymphocytes with 1000 ng/ml SDF-1, the locomotory activity significantly and dose-dependently increased from 19.5 to 64.6% of the cell population. In neutrophil granulocytes, induction of chemotaxis with 10 nM fMLP significantly increased the locomotory activity from 13.7% spontaneously migrating cells to 67.0% of the population. The induction of chemotaxis in neutrophil granulocytes was accompanied by a significant increase of the velocity (actual mean speed during migratory activity excluding breaks) from 3.9 to 12.7 m/ min, whereas the velocity of T lymphocytes remained nearly unchanged at around 9 m/min after induction of chemotaxis (Table 1). Both cell types changed their migratory behavior concerning the frequency and duration of breaks (Table 1). Migrating cells were not permanently locomotory active. Spontaneously migrating neutrophil granulocytes performed on average 4.5 breaks per h, each break lasting 9.9 min. After induc-
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TABLE 1 Differences in Dynamic Parameters of Spontaneous Locomotion and Migration in Response to Chemoattractants of T Lymphocytes and Neutrophil Granulocytes Velocity (m/min)
Locomoting cells (%)
T lymphocytes (n ⫽ 5) Neutrophil granulocytes (n ⫽ 6)
Spontaneous
Induced
Spontaneous
Induced
19.5 (⫾14.2) 13.7 (⫾10.6)
64.6 (⫾24.3)* 67.0 (⫾18.2)‡
8.2 (⫾0.6) 3.9 (⫾2.3)
9.5 (⫾1.4) 12.7 (⫾3.1)‡
Frequency of breaks (h ⫺1)
T lymphocytes (n ⫽ 5) Neutrophil granulocytes (n ⫽ 7)
Duration of breaks (min)
Spontaneous
Induced
Spontaneous
Induced
3.0 (⫾1.0) 4.5 (⫾1.0)
2.8 (⫾0.3) 2.4 (⫾1.1)†
13.0 (⫾3.0) 9.9 (⫾3.1)
8.8 (⫾1.6)* 6.8 (⫾3.7)
Note. n, number of independent experiments. Thirty cells were analyzed per experiment. Statistical significance of changes was calculated using Student’s t test. * P ⬍ 0.05. † P ⬍ 0.005. ‡ P ⬍ 0.0005.
tion of chemotaxis, the frequency of breaks was significantly reduced to 2.4 per h, whereas the decrease of the duration of breaks to 6.8 min was not a statistically significant change. T lymphocytes revealed a distinct pattern of changes. During spontaneous locomotory activity, the cells performed 3.0 breaks per h, each lasting an average of 13.0 min. After the induction of chemotaxis using SDF-1, the frequency of breaks was unchanged (2.8 per h), but the duration of breaks was significantly reduced to 8.8 min (Table 1). Spontaneous Migration and Chemotaxis of T Lymphocytes and Neutrophil Granulocytes Are Differentially Regulated by PTKs and the PKC We have reported previously that spontaneous migration of T lymphocytes depends on PTK activity, but not on PKC activity, as was investigated by inhibition of enzyme activity using the specific inhibitors tyrphostin A23 and calphostin C, respectively (23). In contrast to spontaneous locomotion, SDF-1-induced locomotion of T lymphocytes was dependent on both PTK and PKC activity (Figs. 3A and 3C). The inhibition of PTK activity led to a substantial reduction in the number of cells that migrated. However, the migration dynamics concerning velocity as well as frequency and duration of breaks were similar to spontaneous locomotion. The SDF-1-induced migratory activity of T lymphocytes was reduced to spontaneous migratory activity after inhibition of the PKC and was decreased to 10% after inhibition of PTK activity. Similar results were obtained using the CXC chemokine interleukin-8 and the class C chemokine lymphotactin (data not shown). The herein used concentrations of the PKC inhibitor calphostin C (20 nM) and of the PTK inhibitor tyrphostin A23 (100 M) were shown to be the most effective
concentrations in that they did not influence cell viability (23). These concentrations were also used to investigate the regulation of neutrophil granulocytes (Figs. 3B and 3D). Tyrphostin A23 reduced fMLP-induced locomotion of neutrophil granulocytes from nearly 100 to 80%, but did not influence spontaneous migration of these cells (Fig. 3B). On the other hand, calphostin C nearly completely abolished fMLP-induced locomotion, but had only a little effect on spontaneous locomotion (Fig. 3D). The effects of both kinase inhibitors on the migration of neutrophil granulocytes were not due to cell death, as was assesssed by propidium iodide staining and flow cytometry 24 h after the end of the migration experiments. Interestingly, although the chemotactic response of neutrophil granulocytes to fMLP and of T lymphocytes to SDF-1 disappeared after 15 min (Fig. 2), the migratory activity of the cells remained constant on a high level of activity of nearly 100% (neutrophil granulocytes) and over 60% (T lymphocytes) for the following h (Fig. 3). Therefore, the ability of chemokines to induce migratory activity can occur independently from the property to guide the directionality of cell movement. The Organization of the Microtubule System Does Not Change after the Induction of Chemotaxis in T Lymphocytes and Neutrophil Granulocytes In spherical, nonlocomoting cells the microtubule organizing center (MTOC) was located at the periphery of the cell with the mictotubules surrounding the nucleus (Fig. 4A). In migrating T lymphocytes, the MTOC was located in the trailing uropod with microtubules radiating into the cell body (Fig. 4B). Occasionally, the MTOC was found at the leading part of the locomoting cell (Fig. 4C). After treatment with SDF-1, no changes
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FIG. 3. Involvement of PTKs and the PKC in spontaneous locomotion (white symbols) and in migration in response to chemoattractans (black symbols). SDF-1 (1000 ng/ml) (T lymphocytes; A and C) and fMLP (10 nM) (neutrophil granulocytes; B and D) were used as chemoattractants. In A and B 100 M of the PTK-inhibitor tyrphostin A23 (TA) was added, in C and D 20 nM of the PKC-inhibitor calphostin C was added (CC). Graphs represent mean values of three independent experiments (90 cells were analyzed) with neutrophil granulocytes and T lymphocytes from different donors.
in the microtubule organization were observed (Fig. 4D equal with 4B). In contrast, the microtubule system of neutrophil granulocytes was not as well organized as was that of T lymphocytes (Figs. 4E– 4G). Tubulin revealed a clustered, polarized distribution during nonlocomoting (Fig. 4E) and in spontaneously migrating neutrophil granulocytes (Fig. 4F). After induction of locomotion using 10 nM fMLP no changes in the tubulin distribution were observed (Fig. 4G). In accordance with the findings of Chiplonkar et al. (29) the microtubule system of neutrophil granulocytes was predominantly located in the leading front of the migrating cells. In accordance with our observations made by confocal microscopy, we did not detect any significant changes in the microtubule cytoskeleton assembly analyzed by flow cytometry (Fig. 5). The mean fluorescence intensity of anti-tubulin antibody bound was not changed after SDF-1 treatment of T lymphocytes (Fig. 5A) or fMLP-treatment of neutrophil granulocytes (Fig. 5B) compared to PBS-treated control cells. Colcemid and Taxol served as negative and positive controls
in order to avoid the possibility of technical errors. Whereas ␣-tubulin assembly significantly changed after treatment of T lymphocytes with colcemid or Taxol, only a tendency for changes in assembly was observed in neutrophil granulocytes. The statistically nonsignificant effect of colcemid or Taxol on the tubulin assembly of neutrophil granulocytes (Fig. 5B) compared to T lymphocytes (Fig. 5A) results from the lower overall tubulin content of neutrophils (Fig. 5C) as was assessed by immunoblotting. DISCUSSION The migratory behavior of both T lymphocytes and neutrophil granulocytes changes upon treatment with chemoattractant substances in terms of migratory activity of the population, velocity of migration, and frequency and duration of breaks. These distinct types of locomotion (spontaneous, matrix-induced locomotion vs chemokine-induced migration) are based on distinct molecular signaling states in both cell types. Chemotaxis depends on the activity of the PKC, whereas
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FIG. 4. Distribution of tubulin in T lymphocytes (A to D) and neutrophil granulocytes (E to G). The cells were incubated within collagen matrices for 30 min with or without a chemoattractant. Subsequently the cells were fixed, permeabilized, and immunostained with the anti-tubulin antibody B-5-1-2. Three-channel confocal microscopy was performed for reflection contrast (upper), fluorescence image (middle), and transmission light (lower). A: Nonlocomoting T lymphocyte; B and C: spontaneously locomoting T lymphocytes; D: migratory activity of
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FIG. 5. Flow cytometric analysis of assembled tubulin in T lymphocytes and neutrophil granulocytes. Changes in cellular tubulin content were assessed 1 h after incubation with 1000 ng/ml SDF-1 (T lymphocytes; A) and 10 nM fMLP (neutrophil granulocytes; B). The mean fluorescence intensity of the control (black bars) was adjusted close to 250 in the setup procedure prior to acquisition of data. Taxol (15 M) and colcemid (1 g/ml) were used as positive and negative controls, respectively. Bars are mean values and standard deviation of three independent experiments with cells from different donors. Statistically significant changes are marked by asterisks (P ⬍ 0.05) as was calculated by Student’s t test. C: Total amount of cellular ␣-tubulin of neutrophil granulocytes and T lymphocytes was compared by immunoblotting. As indicated, four different cell concentrations (0.1 to 2 ⫻ 10 6) of each cell type were applied per lane.
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spontaneous locomotion is PKC-independent. The G-protein-mediated engagement of phospholipases and downstream activation of the PKC is supposed to be a pathway commonly used by chemokines (30). Concerning PTK activity, neutrophil granulocytes and T lymphocytes are regulated in different ways. Both types of locomotory activity (spontaneous locomotion and chemotaxis) of T lymphocytes are PTK dependent. We have previously reported on the crucial role of PTK activity in T lymphocyte locomotion (23). Spontaneous locomotion of neutrophil granulocytes is independent of this group of kinases. Evidence for an involvement of PTK activity in fMLP-mediated signals was provided by Ptasznik et al. (31), who showed that the Shc adaptor protein was phosphorylated on tyrosine by the Lyn tyrosine kinase in response to chemoattractant receptor activation. However, PTK-independent pathways must also contribute to the regulation of fMLP-induced migration of neutrophil granulocytes, since inhibition of PTK activity did only partially inhibit this type of locomotion in our migration assay. The tubulin cytoskeleton of migrating T lymphocytes consists of an MTOC in the uropod with microtubules radiating into the cell body (32). In accordance with findings of Volkov et al. (33) we found the PKC in colocalization with the MTOC (23). After treatment with the chemokine SDF-1 no changes of the microtubule cytoskeleton formation were observed by confocal laser scan microscopy and flow cytometry, and the velocity of migration was only marginally increased. On the other hand, colcemid treatment leads to a detectable reduction of microtubules in migrating T lymphocytes accompanied by a highly significant increase in velocity (14). The authors discuss that an increase in velocity using colcemid might be due to a signal provided by tubulin disassembly rather than an increased motility of the cells reached by a breakdown of the microtubule cytoskeleton in spontaneous, matrix-induced migration. In monocytes, tubulin disassembly leads to PTK-mediated signaling on the actin cytoskeleton (17), which could have hinted at an involvement of tubulin-mediated PTK-activity in the regulation of both types of T lymphocyte locomotion or induced locomotion of neutrophil granulocytes. But since the assembly of the microtubule cytoskeleton did not change after induction of chemotaxis in both cell types, we exclude an involvement of this signaling in the regulation of locomotory activity induced by chemoattractants. We therefore follow the argumentation of Volkov et al. (33) that the PKC might be involved in modulations of the actin cytoskeleton, as was shown in T
T lymphocytes was induced using 1000 ng/ml SDF-1; E: nonlocomoting neutrophil granulocyte; F: spontaneously migrating neutrophil granulocyte; G: migratory activity of neutrophil granulocytes was induced using 10 nM fMLP. Arrows indicate the direction of migration prior to fixation as was assessed by time-lapse videomicroscopy and digitization of the individual paths. Size of images is 33 ⫻ 33 m 2.
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lymphocytes as well as neutrophil granulocytes (34, 35), but we do not have any evidence that the PKC regulates chemotaxis via effects on the microtubule cytoskeleton. Volkov et al. (33) deduced from their results that the PKC might be involved in the regulation of microtubule-dependent motility. This discrepancy of Volkov and colleagues’ results (33) might be explained by different experimental setups that were chosen to observe cell polarisation. Volkov et al. (33) engaged integrin signaling by crosslinking the leukocyte function-associated antigen-1, whereas we showed that migration of T lymphocytes within a three-dimensional collagen matrix occurs independently of  1 integrins (27). Thus, engaging integrin signaling by receptor crosslinking induces an additional signal transduction pathway that is not needed for the migration of T lymphocytes within three-dimensional collagen matrices. As discussed above, the presented flow cytometric analyses of microtubule formations do not suggest a direct involvement of tubulin polymerization/depolymerization in the regulation of migration. However, tubulin undergoes numerous posttranslational modifications (36), which might lead to changes in the binding capacity of tubulin-associated proteins. The -chain-associated protein (ZAP-70) binds to tubulin in Jurkat T cells (37) and was shown to be required for leukocyte function-associated antigen-1-mediated migration of a T cell hybridoma (38). ZAP-70 was found to be associated to the focal adhesion kinase and paxillin in migrating T cells (39), which are important regulatory elements of actin-based migration. Thus, the microtubule cytoskeleton is associated to the migratory machinery, but we have no evidence for functional involvement so far. In this context it might be noteworthy that ZAP-70 binds to the CD3 -chain of the T cell receptor complex and CD3 crosslinking leads to a pronounced induction of T cell migration (40). Therefore, T cell receptor-mediated signaling might have influence on both the actin cytoskeleton in terms of migration and the microtubule cytoskeleton in terms of proliferation. For the fMLP-induced locomotion of neutrophil granulocytes we hypothesize the following signal transduction pathway: fMLP-mediated activation of heterotrimeric G proteins leads to activation of the phospholipases C  1 and  2 and thereby to generation of diacylglycerol and inositol-1,4,5-triphosphate. Inositol1,4,5-triphosphate induces a calcium release from intracellular stores, whereas diacylglycerol activates the PKC. Preliminary results of our group show that the PKC is located in the anterior part of migrating neutrophil granulocytes and that the intracellular calcium concentration is increased in this part of the cell compared to the posterior part. Both activation of the PKC and increased calcium lead to release of actin filaments from the myristoylated alanine-rich C kinase substrate
and from gelsolin resulting in increased actin polymerization, which would be the motivating force for migration of neutrophil granulocytes. Occasionally, we observed T lymphocytes with a MTOC at the leading edge of the migrating cells instead of the usual distribution in the uropod. We presume that these cells are undergoing a directional change, but we do not know whether this altered localization causes a change of directionality or is a consequence of it. It might well be that reorientation of the MTOC is involved in changes in the direction of migration in T lymphocytes. Compared to T lymphocytes the microtubule cytoskeleton of migrating neutrophil granulocytes is less well organized. Tubulin was found in a grainy distribution at the leading part of both spontaneously and fMLP-induced locomoting neutrophil granulocytes. T lymphocytes probably need the microtubule cytoskeleton for stabilizing the directionality of migration and, since these cells are still able to proliferate after activation via the T cell receptor, they need the microtubule cytoskeleton to build the mitotic spindle during mitosis. Neutrophil granulocytes are postmitotic cells that are not able to undergo mitosis. They do not need mitotic machinery for their function as host defense. The advantage of this minor microtubule architecture in combination with the segmented nucleus might be a high cellular flexibility, which enables the neutrophil granulocytes to migrate faster than other leukocytes do, e.g., T lymphocytes, and to infiltrate even dense tissues due to their high morphological dynamic. Both cell types increase locomotory activity after treatment with chemoattractants in a characteristic, distinct manner. While T lymphocytes significantly reduce the duration of their migration breaks, neutrophil granulocytes significantly reduce the frequency of migration breaks, thus belonging to different locomotory phenotypes. These different locomotory phenotypes might be due to the differences shown in the regulatory signal transduction of T lymphocytes and neutrophil granulocytes and additionally to differences in cell– matrix interactions in the process of migration. A strong argument for differences in cell–matrix interactions is given by differences in integrin dependence of migration. The spontaneous migration of T lymphocytes within three-dimensional collagen lattices has been shown to be independent of  1 integrins (27). Furthermore, SDF-1-induced migration of T cell hybridomas through intercellular adhesion molecule1-coated filters was independent of the leukocyte function-associated antigen-1 at 100 ng/ml of this chemokine (41). On the other hand, fMLP leads to  1-integrin-mediated adherence of neutrophil granulocytes to fibrin (42, 43) and blocking of  1 integrins inhibited migration of neutrophil granulocytes into extravascular tissue, as was investigated by Werr et al. (44) using intravital time-lapse videomicroscopy.
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In summary, spontaneous migration of T lymphocytes and neutrophil granulocytes is different in regulatory signal transduction. This difference is reflected in distinct locomotory phenotypes of the cells. Furthermore, both cell types characteristically change locomotory behavior and intracellular regulatory signaling after the induction of chemotaxis. As we have proposed before (45), neutrophil granulocytes and T lymphocytes have at least two distinct molecular signaling states: a matrix-induced, spontaneous type of locomotion and a type of migration in response to chemoattractants. Alterations between spontaneous locomotion and chemotaxis do not involve changes in the microtubule cytoskeleton. These findings, described here for T lymphocytes and neutrophil granulocytes, reflect the different roles of these cells in the inflammatory response.
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ACKNOWLEDGMENTS We thank Britta Reubke-Gothe and Ilka Stechert for expert technical assistance. This work was supported by the Fritz-Bender-Foundation, Munich, Germany.
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T Lymphocytes and Neutrophil Granulocytes Differ in Regulatory Signaling and Migratory Dynamics with Regard to Spontaneous Locomotion and Chemotaxis Frank Entschladen, Matthias Gunzer, Chi Mi Scheuffele, Bernd Niggemann, and Kurt S. Za¨nker Institute for Immunology, Witten/Herdecke University, Stockumer Strasse 10, 58448 Witten, Germany Received October 18, 1999; accepted December 17, 1999
Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis. © 2000 Academic Press
INTRODUCTION Chemokines (chemoattractant cytokines) are peptides that mainly induce chemotactic movement of leukocytes but also have functions in trafficking and regulation of leukocyte effector function (1, 2). Chemokines are grouped into four subclasses. The CXC chemokines contain two cysteine residues in their aminoterminal domain, separated by a single aminoacid. The CC chemokines lack this separating aminoacid (1). The third group, known as C or XC chemokines, consists of one member, possessing only the second cysteine residue in the aminoterminal domain (3). The fourth class of chemokines, the CX 3C chemokines, are characterized by three separating amino acids between 0008-8749/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
the aminoterminal cysteines. Fractalkine is the only member of this class (4). All known chemokine receptors belong to the family of seven-helices receptors, which are coupled to G-protein-mediated signaling (5). Other chemoattractants like formylated peptides, e.g., formyl-methionyl-leucyl-phenylalanine (fMLP), the complement fragment C5a, or leukotriene B4, have no structural similarities to the chemokines described above but do bind to G-protein-coupled seven-helices receptors (6). The cellular receptor for the CXC chemokine stromal-cell-derived factor-1 (SDF-1) is CXCR4, expressed on monocytes, neutrophil granulocytes, and lymphocytes (7, 8). SDF-1 has chemotactic effects on lymphocytes and monocytes but not on neutrophil granulocytes (9). We used SDF-1 to stimulate T lymphocyte chemotaxis and fMLP to induce chemotaxis of neutrophil granulocytes. The induction of chemotaxis was accompanied by changes in dynamic parameters, i.e., the locomotory active fraction of a cell population, the velocity of migration, and frequency and duration of migration breaks. The functional role of the microtubule cytoskeleton in the migration of adherent cells, e.g., fibroblasts and tumor cells, has been studied extensively in the past: colcemid-induced microtubule disruption and Taxolinduced stabilization of the microtubule system both potently inhibit migration of adherent cells (10 –12). Recent results of nonadherent T lymphocytes suggest a distinct involvement of the microtubule system in the locomotion of these cells, underscoring different migration strategies in leukocytes and adherent cells (13). Microtubule stabilization by Taxol has only a minor effect on the locomotory activity of T lymphocytes, whereas disruption of microtubules induced by colcemid leads to an increase in migratory activity of T lymphocytes. The increase in locomotory activity is due to both the recruitment of previously nonlocomoting cells and an increase in the duration of locomotion, i.e., an increase in the number of minutes a cell is locomotory active within a certain period of observation (14).
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This means that upon colcemid treatment the cells reduce either frequency or duration of breaks performed during locomotory activity or both. Nikolai et al. (14) argue in accordance with other authors (15, 16) that the promigratory effect of microtubule disassembly in leukocytes is due to a functional interaction with the actin cytoskeleton, i.e., tubulin transduces a signal to the actin cytoskeleton. It has recently been shown that disassembly of the microtubule system regulates the actin cytoskeleton via a protein tyrosine kinase (PTK)-dependent signal transduction pathway (17). The actin cytoskeleton has been shown to be of major importance for T cell migration, since actin-destabilizing drugs inhibit uropod formation and cellular motility (18, 19). In contrast to T lymphocytes, reports on neutrophil granulocytes describe an inhibition of fMLP-induced migration by microtubule-stabilizing Taxol (20). Results on the effects of antitubulins are controversial. Different reports describe an inhibition of human neutrophil granulocyte chemotaxis at micromolar concentrations of colchicin, whereas lower concentrations are stimulatory (20). Spontaneous migration of neutrophil granulocytes is in contrast increased at micromolar concentrations of colchicine, nocodazole, or vinblastine (21). Differences between random movement and chemotaxis were also observed by Bandmann et al. (22). Neutrophil chemotaxis was inhibited by various antitubulins, whereas the basic locomotion was not affected by podophyllic acid ethylhydrazide (22). We tested the hypothesis of whether changes of locomotory behavior after induction of chemotaxis could be ascribed to changes of the regulatory signal transduction of migration and whether this signal transduction is similar or different in neutrophil granulocytes and T lymphocytes. We did this on the basis of the finding that spontaneous locomotion of T lymphocytes was regulated by PTK activity and was distinct from a second, protein kinase C (PKC)-dependent type of migration inducible by PKC-activating phorbol ester (23). We furthermore investigated whether changes in the microtubule cytoskeleton were involved in changes in locomotion from spontaneous, matrix-induced locomotion to chemotaxis. The rationale was the above-described result by Nikolai et al. (14) that colcemid treatment increased the part of locomotory active T lymphocytes within a population, the time each cell was locomotory active within the observation period, and the velocity of migration. Thus, colcemid induces changes in locomotory behavior similar to chemoattractants.
The peripheral blood mononuclear cell fraction was isolated from heparinized blood of healthy donors by density-gradient centrifugation using Ficoll–Hypaque (ICN, Meckenheim, Germany). CD4 ⫹ and CD8 ⫹ cells were positively selected (10 min, 4°C) using immunomagnetic beads coated with mouse anti-human CD4 or CD8 mAb (66.1 or ITI-5C2; Dynabeads, Dynal, Hamburg, Germany). Subsequently, cell-bound beads were detached (45 min; 20°C) using polyclonal anti-mouse Fab antibodies (Detachabead, Dynal). Purified CD4 ⫹ and CD8 ⫹ cells were ⬎98% CD3 ⫹ and 96 –99% CD4 ⫹ or CD8 ⫹, respectively, as detected by flow cytometry (FACSCalibur, Becton–Dickinson, San Jose, CA). More than 98% of the cells were viable, as assessed by propidium iodide staining and flow cytometry. As previously described, the isolation procedure did not alter the activation state of the cells (23). Results shown herein were obtained using CD4 ⫹ cells. Data were confirmed by experiments performed with CD8 ⫹ cells, which led to similar results. Neutrophil granulocytes were isolated from the pellet of a density-gradient centrifugation. The pellet containing neutrophil granulocytes and erythrocytes was mixed with platelet-depleted serum. This mixture was diluted 1:1.3 with a high-molecular-weight dextran solution (Makrodex, Fresenius, Bad Homburg, Germany) containing 0.01 M ethylendiaminetetraacetic acid. After 2 h erythrocytes had settled down and the neutrophil granulocyte containing supernatant was separated from the pellet. Contaminating erythrocytes were then removed by hypotonic lysis with 0.3% sodium chloride for 2 min on ice. The obtained purified neutrophil granulocytes contained less than 5% eosinophil granulocytes and lymphocytes, as determined by Pappenheim staining. Neutrophil granulocytes were used immediately after isolation. T lymphocytes were maintained overnight in RPMI supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum (Boehringer Mannheim, Mannheim, Germany), and penicillin (50 U/ml) and streptomycin (50 g/ml; Gibco, EggensteinLeopoldshafen, Germany). Neutrophil granulocytes developed their maximum spontaneous migratory activity directly after isolation, whereas T lymphocytes reached their maximum activity 12 to 24 h after isolation. Thus, the experiments with neutrophil granulocytes and T lymphocytes were performed at different time points in order to get optimal spontaneous migratory activity for both cell types.
MATERIALS AND METHODS
Collagen lattices were generated as previously described (25). In short, 2.5 ⫻ 10 5 cells were mixed with 100 l of buffered collagen solution (pH 7.4) containing 1.67 mg/ml collagen type I in minimal essential Eagle’s medium (Flow, McLean, VA). The collagen was bovine
Cell Isolation and Cultivation Human peripheral CD4 ⫹ and CD8 ⫹ cells were isolated from human blood as previously described (24).
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FIG. 1. Chemotaxis chamber. A central hole, 15 mm in diameter, was drilled into an acrylic glass microscope slide. This hole was covered on both sides with coverslips which were sealed with wax (paraffin:vaseline 1:1). On the upper side a gap was left to fill the chamber. From the anterior side, a 1-mm-wide channel was drilled into the acrylic glass slide. In this channel a glass capillary containing the chemoattractant source was inserted.
dermal collagen (Cohesion, Palo Alto, CA) consisting of 95–98% type I collagen with the remainder being composed of type III collagen. The buffered collagen suspension was filled into self-constructed chemotaxis chambers and allowed to polymerize for 20 min at 37°C and a 5% CO 2 humidified atmosphere. For the direct observation of cellular chemotaxis, i.e., directional migration of individual cells, a novel chamber technique was developed (Fig. 1). A central hole, 15 mm wide, was drilled in an acrylic glass slide. Through one long side of the slide a 1-mm-wide channel was drilled to the edge of the central hole. The upper and lower sides of the central hole were sealed with cover slips, leaving a gap to fill the resulting chamber. The buffered collagen solution containing either T lymphocytes or neutrophil granulocytes was added to the chamber, covering the channel on the edge of the central hole. After polymerization, the chamber was sealed and migration was recorded by time-lapse videomicroscopy for 15 min. Subsequently, PBS as control or SDF-1 (Pharmingen, San Diego, CA) for the induction of T lymphocyte chemotaxis or fMLP (Sigma, Deisenhofen, Germany) for the induction of neutrophil granulocyte chemotaxis was added through the channel and the chemotactic behavior of the cells in the collagen matrix adjacent to the channel was recorded for the next 1 to 2 h. Functional involvement of PTKs and the PKC in the regulation of spontaneous locomotion and chemotaxis of neutrophil granulocytes and T lymphocytes was investigated by addition of the PTK inhibitor tyrphostin A23 and the PKC inhibitor calphostin C (both Calbiochem-Novabiochem, San Diego, CA) to the collagen lattices as described previously (23). Time-Lapse Videomicroscopy and Computer-Assisted Cell Tracking Cell locomotion within the three-dimensional collagen lattices was recorded by time-lapse videomicroscopy using four microscopy and recording devices simultaneously. For data analysis, 30 cells of each sample were randomly selected and two-dimensional
projections of the paths were digitized as x/y-coordinates in 1-min step intervals by computer-assisted cell tracking. From the obtained data migrated distance and direction of migration as well as time-related parameters such as migrated time, duration of migration breaks, and velocity were calculated for each single cell and the whole population. For the analysis of directionality, a modified form of a Markov-chain analysis (26) was established. The angle of each step of the cell paths was analyzed independently and the number of steps toward each quadrant of a coordinate system was counted. Therefore, the starting point of each step was set as the origin of the coordinate system. A random migration would have a 25% probability for the distribution of the steps into each quadrant. The number of steps toward or away from the site of addition of chemoattractants was determined by summing up all steps of the two quadrants adjacent to or opposite the source. The classical application of the Markov-chain analysis assumes that every step has the same length and therefore considers only the angles of the steps. Since the length of steps varies considerably, especially after the addition of fMLP, we added the length of steps directed into each quadrant, resulting in a net movement into each direction. Confocal Laser Scan Microscopy For immunofluorescent staining of the microtubule system, 50 l of a suspension of 3 ⫻ 10 5 neutrophil granulocytes or T lymphocytes in PBS or PBS containing 10 M fMLP or 1000 ng/ml SDF-1, respectively, was mixed with 100 l buffered collagen and the solution was transferred onto a coverslip. After 20 min of polymerization, locomotory activity was recorded for 10 min by time-lapse videomicroscopy and the respective migration paths were digitized as described above to determine the direction of migration prior to fixation. Subsequently, the cells were fixed with 3.7% formaldehyde (15 min, 20°C) and subsequently permeabilized with 0.5% Triton X-100 (10 min, 20°C). Samples were then incubated with 10 g/ml (1 h, 20°C) of mouse anti-␣-tubulin antibody B-5-1-2 (Sigma). After being washed with PBS, samples were incubated (1 h, 20°C) with 10 g/ml of lissamine rhodamine B sulfonylchloride-conjugated Fab fragments, specific for mouse Fc␥ (The Jackson Laboratories, Westgrove, PA). After a further washing step the coverslips were inverted and mounted on slides. Confocal laser scanning microscopy using a Leica TCS 4D microscope was performed as previously described (14, 27). Flow Cytometry Assembled tubulin in the cells was measured as described previously (14). In brief, 1 ⫻ 10 6 neutrophil granulocytes or T lymphocytes were treated for 1 h at
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37°C with 10 nM fMLP or 1000 ng/ml SDF-1, respectively. As positive and negative controls, cells were treated with 15 M Taxol (paclitaxel; Sigma) or 1 g/ml colcemid (Demecolcine; Sigma), respectively. After incubation, the cells were fixed (4% paraformaldehyde, 15 min, 20°C), permeabilized (0.5% Triton X-100, 10 min, 20°C), and immunostained for tubulin using 10 g/ml of mouse anti-␣-tubulin antibody B-5-1-2 as described for confocal laser scan microscopy. The mean fluorescence intensity was a direct measure of the assembled tubulin, since tubulin that was not bound to microtubules was washed out during Triton X-100 permeabilization. Immunoblotting The total amount of cellular ␣-tubulin was analyzed by immunoblotting as described previously (23). Neutrophil granulocytes and T lymphocytes (0.1 ⫻ 10 6 to 2 ⫻ 10 6) were lysed in Laemmli sample buffer (10 min, 95°C). Proteins were separated using polyacrylamide electophoresis according to the method of Laemmli (28) and were transferred to an Immobilion-P membrane (Millipore, Bedford, MA). After blocking of the membranes with 5% dry milk powder (1 h, 20°C), membranes were incubated with 1 g/ml of mouse anti-␣tubulin antibody B-5-1-2 (1 h, 20°C) and subsequently with a peroxidase-linked anti-mouse antibody (1 g/ ml, 1 h, 20°C). After incubation of the membrane with chemiluminescence substrate (Boehringer Mannheim; 2 min, 20°C) the chemiluminescence signal was detected by exposure to a Kodak X-OMAT AR film sheet (Sigma). RESULTS The Migratory Response of T Lymphocytes to SDF-1 and of Neutrophil Granulocytes to fMLP Was Chemotactic After placing 1000 ng/ml SDF-1 into the capillary of the chemotaxis chamber, T lymphocytes showed a chemotactic response (Fig. 2A). Modified Markov-chain analysis revealed that within the first 15 min 70.5% of the steps, with the length of the steps taken into account, were directed toward the chemoattractant source, whereas only 29.5% were orientated in the opposite direction. For the following 15 min only 53.3% of the migrated steps were oriented toward the source (Fig. 2C), probably because the gradient had disappeared due to diffusion. The addition of 10 nM fMLP to collagen lattices with neutrophil granulocytes resulted in a pronounced chemotactic response also. Within the first 15 min after addition of fMLP 77.8% of all steps were directed toward the source and only 22.2% were oriented to the opposite direction (Fig. 2B). Again the fMLP gradient probably disappeared due to diffusion, resulting in a nearly randomly distributed migration of
FIG. 2. Vector diagrams of chemotactic response. The migration paths of T lymphocytes in response to SDF-1 (A and C) and of neutrophil granulocytes in response to fMLP (B and D) were divided into 15-min intervals. Starting points of each interval of the paths were moved into the center and the number of steps toward the chemoattractant source and away from it was analyzed, taking the varying length of steps into consideration. The arrows indicate the direction from which 1000 ng/ml SDF-1 (A) and 10 nM fMLP (C) were added. Thirty cells were analyzed for each vector diagram. Data shown are representative of three independent experiments performed with neutrophil granulocytes and T lymphocytes from different donors.
the neutrophil granulocytes in the following 15 min (Fig. 2D). Only 53.4% of the steps were directed toward the source, whereas 46.6% were aimed in the opposite direction. The Locomotory Behavior of T Lymphocytes and of Neutrophil Granulocytes Characteristically Changes after Induction of Chemotaxis After incorporation within the three-dimensional collagen matrix both T lymphocytes and neutrophil granulocytes developed spontaneous locomotory activity (Table 1). After the induction of chemotaxis of T lymphocytes with 1000 ng/ml SDF-1, the locomotory activity significantly and dose-dependently increased from 19.5 to 64.6% of the cell population. In neutrophil granulocytes, induction of chemotaxis with 10 nM fMLP significantly increased the locomotory activity from 13.7% spontaneously migrating cells to 67.0% of the population. The induction of chemotaxis in neutrophil granulocytes was accompanied by a significant increase of the velocity (actual mean speed during migratory activity excluding breaks) from 3.9 to 12.7 m/ min, whereas the velocity of T lymphocytes remained nearly unchanged at around 9 m/min after induction of chemotaxis (Table 1). Both cell types changed their migratory behavior concerning the frequency and duration of breaks (Table 1). Migrating cells were not permanently locomotory active. Spontaneously migrating neutrophil granulocytes performed on average 4.5 breaks per h, each break lasting 9.9 min. After induc-
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TABLE 1 Differences in Dynamic Parameters of Spontaneous Locomotion and Migration in Response to Chemoattractants of T Lymphocytes and Neutrophil Granulocytes Velocity (m/min)
Locomoting cells (%)
T lymphocytes (n ⫽ 5) Neutrophil granulocytes (n ⫽ 6)
Spontaneous
Induced
Spontaneous
Induced
19.5 (⫾14.2) 13.7 (⫾10.6)
64.6 (⫾24.3)* 67.0 (⫾18.2)‡
8.2 (⫾0.6) 3.9 (⫾2.3)
9.5 (⫾1.4) 12.7 (⫾3.1)‡
Frequency of breaks (h ⫺1)
T lymphocytes (n ⫽ 5) Neutrophil granulocytes (n ⫽ 7)
Duration of breaks (min)
Spontaneous
Induced
Spontaneous
Induced
3.0 (⫾1.0) 4.5 (⫾1.0)
2.8 (⫾0.3) 2.4 (⫾1.1)†
13.0 (⫾3.0) 9.9 (⫾3.1)
8.8 (⫾1.6)* 6.8 (⫾3.7)
Note. n, number of independent experiments. Thirty cells were analyzed per experiment. Statistical significance of changes was calculated using Student’s t test. * P ⬍ 0.05. † P ⬍ 0.005. ‡ P ⬍ 0.0005.
tion of chemotaxis, the frequency of breaks was significantly reduced to 2.4 per h, whereas the decrease of the duration of breaks to 6.8 min was not a statistically significant change. T lymphocytes revealed a distinct pattern of changes. During spontaneous locomotory activity, the cells performed 3.0 breaks per h, each lasting an average of 13.0 min. After the induction of chemotaxis using SDF-1, the frequency of breaks was unchanged (2.8 per h), but the duration of breaks was significantly reduced to 8.8 min (Table 1). Spontaneous Migration and Chemotaxis of T Lymphocytes and Neutrophil Granulocytes Are Differentially Regulated by PTKs and the PKC We have reported previously that spontaneous migration of T lymphocytes depends on PTK activity, but not on PKC activity, as was investigated by inhibition of enzyme activity using the specific inhibitors tyrphostin A23 and calphostin C, respectively (23). In contrast to spontaneous locomotion, SDF-1-induced locomotion of T lymphocytes was dependent on both PTK and PKC activity (Figs. 3A and 3C). The inhibition of PTK activity led to a substantial reduction in the number of cells that migrated. However, the migration dynamics concerning velocity as well as frequency and duration of breaks were similar to spontaneous locomotion. The SDF-1-induced migratory activity of T lymphocytes was reduced to spontaneous migratory activity after inhibition of the PKC and was decreased to 10% after inhibition of PTK activity. Similar results were obtained using the CXC chemokine interleukin-8 and the class C chemokine lymphotactin (data not shown). The herein used concentrations of the PKC inhibitor calphostin C (20 nM) and of the PTK inhibitor tyrphostin A23 (100 M) were shown to be the most effective
concentrations in that they did not influence cell viability (23). These concentrations were also used to investigate the regulation of neutrophil granulocytes (Figs. 3B and 3D). Tyrphostin A23 reduced fMLP-induced locomotion of neutrophil granulocytes from nearly 100 to 80%, but did not influence spontaneous migration of these cells (Fig. 3B). On the other hand, calphostin C nearly completely abolished fMLP-induced locomotion, but had only a little effect on spontaneous locomotion (Fig. 3D). The effects of both kinase inhibitors on the migration of neutrophil granulocytes were not due to cell death, as was assesssed by propidium iodide staining and flow cytometry 24 h after the end of the migration experiments. Interestingly, although the chemotactic response of neutrophil granulocytes to fMLP and of T lymphocytes to SDF-1 disappeared after 15 min (Fig. 2), the migratory activity of the cells remained constant on a high level of activity of nearly 100% (neutrophil granulocytes) and over 60% (T lymphocytes) for the following h (Fig. 3). Therefore, the ability of chemokines to induce migratory activity can occur independently from the property to guide the directionality of cell movement. The Organization of the Microtubule System Does Not Change after the Induction of Chemotaxis in T Lymphocytes and Neutrophil Granulocytes In spherical, nonlocomoting cells the microtubule organizing center (MTOC) was located at the periphery of the cell with the mictotubules surrounding the nucleus (Fig. 4A). In migrating T lymphocytes, the MTOC was located in the trailing uropod with microtubules radiating into the cell body (Fig. 4B). Occasionally, the MTOC was found at the leading part of the locomoting cell (Fig. 4C). After treatment with SDF-1, no changes
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FIG. 3. Involvement of PTKs and the PKC in spontaneous locomotion (white symbols) and in migration in response to chemoattractans (black symbols). SDF-1 (1000 ng/ml) (T lymphocytes; A and C) and fMLP (10 nM) (neutrophil granulocytes; B and D) were used as chemoattractants. In A and B 100 M of the PTK-inhibitor tyrphostin A23 (TA) was added, in C and D 20 nM of the PKC-inhibitor calphostin C was added (CC). Graphs represent mean values of three independent experiments (90 cells were analyzed) with neutrophil granulocytes and T lymphocytes from different donors.
in the microtubule organization were observed (Fig. 4D equal with 4B). In contrast, the microtubule system of neutrophil granulocytes was not as well organized as was that of T lymphocytes (Figs. 4E– 4G). Tubulin revealed a clustered, polarized distribution during nonlocomoting (Fig. 4E) and in spontaneously migrating neutrophil granulocytes (Fig. 4F). After induction of locomotion using 10 nM fMLP no changes in the tubulin distribution were observed (Fig. 4G). In accordance with the findings of Chiplonkar et al. (29) the microtubule system of neutrophil granulocytes was predominantly located in the leading front of the migrating cells. In accordance with our observations made by confocal microscopy, we did not detect any significant changes in the microtubule cytoskeleton assembly analyzed by flow cytometry (Fig. 5). The mean fluorescence intensity of anti-tubulin antibody bound was not changed after SDF-1 treatment of T lymphocytes (Fig. 5A) or fMLP-treatment of neutrophil granulocytes (Fig. 5B) compared to PBS-treated control cells. Colcemid and Taxol served as negative and positive controls
in order to avoid the possibility of technical errors. Whereas ␣-tubulin assembly significantly changed after treatment of T lymphocytes with colcemid or Taxol, only a tendency for changes in assembly was observed in neutrophil granulocytes. The statistically nonsignificant effect of colcemid or Taxol on the tubulin assembly of neutrophil granulocytes (Fig. 5B) compared to T lymphocytes (Fig. 5A) results from the lower overall tubulin content of neutrophils (Fig. 5C) as was assessed by immunoblotting. DISCUSSION The migratory behavior of both T lymphocytes and neutrophil granulocytes changes upon treatment with chemoattractant substances in terms of migratory activity of the population, velocity of migration, and frequency and duration of breaks. These distinct types of locomotion (spontaneous, matrix-induced locomotion vs chemokine-induced migration) are based on distinct molecular signaling states in both cell types. Chemotaxis depends on the activity of the PKC, whereas
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FIG. 4. Distribution of tubulin in T lymphocytes (A to D) and neutrophil granulocytes (E to G). The cells were incubated within collagen matrices for 30 min with or without a chemoattractant. Subsequently the cells were fixed, permeabilized, and immunostained with the anti-tubulin antibody B-5-1-2. Three-channel confocal microscopy was performed for reflection contrast (upper), fluorescence image (middle), and transmission light (lower). A: Nonlocomoting T lymphocyte; B and C: spontaneously locomoting T lymphocytes; D: migratory activity of
REGULATION OF LEUKOCYTE MIGRATION
FIG. 5. Flow cytometric analysis of assembled tubulin in T lymphocytes and neutrophil granulocytes. Changes in cellular tubulin content were assessed 1 h after incubation with 1000 ng/ml SDF-1 (T lymphocytes; A) and 10 nM fMLP (neutrophil granulocytes; B). The mean fluorescence intensity of the control (black bars) was adjusted close to 250 in the setup procedure prior to acquisition of data. Taxol (15 M) and colcemid (1 g/ml) were used as positive and negative controls, respectively. Bars are mean values and standard deviation of three independent experiments with cells from different donors. Statistically significant changes are marked by asterisks (P ⬍ 0.05) as was calculated by Student’s t test. C: Total amount of cellular ␣-tubulin of neutrophil granulocytes and T lymphocytes was compared by immunoblotting. As indicated, four different cell concentrations (0.1 to 2 ⫻ 10 6) of each cell type were applied per lane.
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spontaneous locomotion is PKC-independent. The G-protein-mediated engagement of phospholipases and downstream activation of the PKC is supposed to be a pathway commonly used by chemokines (30). Concerning PTK activity, neutrophil granulocytes and T lymphocytes are regulated in different ways. Both types of locomotory activity (spontaneous locomotion and chemotaxis) of T lymphocytes are PTK dependent. We have previously reported on the crucial role of PTK activity in T lymphocyte locomotion (23). Spontaneous locomotion of neutrophil granulocytes is independent of this group of kinases. Evidence for an involvement of PTK activity in fMLP-mediated signals was provided by Ptasznik et al. (31), who showed that the Shc adaptor protein was phosphorylated on tyrosine by the Lyn tyrosine kinase in response to chemoattractant receptor activation. However, PTK-independent pathways must also contribute to the regulation of fMLP-induced migration of neutrophil granulocytes, since inhibition of PTK activity did only partially inhibit this type of locomotion in our migration assay. The tubulin cytoskeleton of migrating T lymphocytes consists of an MTOC in the uropod with microtubules radiating into the cell body (32). In accordance with findings of Volkov et al. (33) we found the PKC in colocalization with the MTOC (23). After treatment with the chemokine SDF-1 no changes of the microtubule cytoskeleton formation were observed by confocal laser scan microscopy and flow cytometry, and the velocity of migration was only marginally increased. On the other hand, colcemid treatment leads to a detectable reduction of microtubules in migrating T lymphocytes accompanied by a highly significant increase in velocity (14). The authors discuss that an increase in velocity using colcemid might be due to a signal provided by tubulin disassembly rather than an increased motility of the cells reached by a breakdown of the microtubule cytoskeleton in spontaneous, matrix-induced migration. In monocytes, tubulin disassembly leads to PTK-mediated signaling on the actin cytoskeleton (17), which could have hinted at an involvement of tubulin-mediated PTK-activity in the regulation of both types of T lymphocyte locomotion or induced locomotion of neutrophil granulocytes. But since the assembly of the microtubule cytoskeleton did not change after induction of chemotaxis in both cell types, we exclude an involvement of this signaling in the regulation of locomotory activity induced by chemoattractants. We therefore follow the argumentation of Volkov et al. (33) that the PKC might be involved in modulations of the actin cytoskeleton, as was shown in T
T lymphocytes was induced using 1000 ng/ml SDF-1; E: nonlocomoting neutrophil granulocyte; F: spontaneously migrating neutrophil granulocyte; G: migratory activity of neutrophil granulocytes was induced using 10 nM fMLP. Arrows indicate the direction of migration prior to fixation as was assessed by time-lapse videomicroscopy and digitization of the individual paths. Size of images is 33 ⫻ 33 m 2.
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lymphocytes as well as neutrophil granulocytes (34, 35), but we do not have any evidence that the PKC regulates chemotaxis via effects on the microtubule cytoskeleton. Volkov et al. (33) deduced from their results that the PKC might be involved in the regulation of microtubule-dependent motility. This discrepancy of Volkov and colleagues’ results (33) might be explained by different experimental setups that were chosen to observe cell polarisation. Volkov et al. (33) engaged integrin signaling by crosslinking the leukocyte function-associated antigen-1, whereas we showed that migration of T lymphocytes within a three-dimensional collagen matrix occurs independently of  1 integrins (27). Thus, engaging integrin signaling by receptor crosslinking induces an additional signal transduction pathway that is not needed for the migration of T lymphocytes within three-dimensional collagen matrices. As discussed above, the presented flow cytometric analyses of microtubule formations do not suggest a direct involvement of tubulin polymerization/depolymerization in the regulation of migration. However, tubulin undergoes numerous posttranslational modifications (36), which might lead to changes in the binding capacity of tubulin-associated proteins. The -chain-associated protein (ZAP-70) binds to tubulin in Jurkat T cells (37) and was shown to be required for leukocyte function-associated antigen-1-mediated migration of a T cell hybridoma (38). ZAP-70 was found to be associated to the focal adhesion kinase and paxillin in migrating T cells (39), which are important regulatory elements of actin-based migration. Thus, the microtubule cytoskeleton is associated to the migratory machinery, but we have no evidence for functional involvement so far. In this context it might be noteworthy that ZAP-70 binds to the CD3 -chain of the T cell receptor complex and CD3 crosslinking leads to a pronounced induction of T cell migration (40). Therefore, T cell receptor-mediated signaling might have influence on both the actin cytoskeleton in terms of migration and the microtubule cytoskeleton in terms of proliferation. For the fMLP-induced locomotion of neutrophil granulocytes we hypothesize the following signal transduction pathway: fMLP-mediated activation of heterotrimeric G proteins leads to activation of the phospholipases C  1 and  2 and thereby to generation of diacylglycerol and inositol-1,4,5-triphosphate. Inositol1,4,5-triphosphate induces a calcium release from intracellular stores, whereas diacylglycerol activates the PKC. Preliminary results of our group show that the PKC is located in the anterior part of migrating neutrophil granulocytes and that the intracellular calcium concentration is increased in this part of the cell compared to the posterior part. Both activation of the PKC and increased calcium lead to release of actin filaments from the myristoylated alanine-rich C kinase substrate
and from gelsolin resulting in increased actin polymerization, which would be the motivating force for migration of neutrophil granulocytes. Occasionally, we observed T lymphocytes with a MTOC at the leading edge of the migrating cells instead of the usual distribution in the uropod. We presume that these cells are undergoing a directional change, but we do not know whether this altered localization causes a change of directionality or is a consequence of it. It might well be that reorientation of the MTOC is involved in changes in the direction of migration in T lymphocytes. Compared to T lymphocytes the microtubule cytoskeleton of migrating neutrophil granulocytes is less well organized. Tubulin was found in a grainy distribution at the leading part of both spontaneously and fMLP-induced locomoting neutrophil granulocytes. T lymphocytes probably need the microtubule cytoskeleton for stabilizing the directionality of migration and, since these cells are still able to proliferate after activation via the T cell receptor, they need the microtubule cytoskeleton to build the mitotic spindle during mitosis. Neutrophil granulocytes are postmitotic cells that are not able to undergo mitosis. They do not need mitotic machinery for their function as host defense. The advantage of this minor microtubule architecture in combination with the segmented nucleus might be a high cellular flexibility, which enables the neutrophil granulocytes to migrate faster than other leukocytes do, e.g., T lymphocytes, and to infiltrate even dense tissues due to their high morphological dynamic. Both cell types increase locomotory activity after treatment with chemoattractants in a characteristic, distinct manner. While T lymphocytes significantly reduce the duration of their migration breaks, neutrophil granulocytes significantly reduce the frequency of migration breaks, thus belonging to different locomotory phenotypes. These different locomotory phenotypes might be due to the differences shown in the regulatory signal transduction of T lymphocytes and neutrophil granulocytes and additionally to differences in cell– matrix interactions in the process of migration. A strong argument for differences in cell–matrix interactions is given by differences in integrin dependence of migration. The spontaneous migration of T lymphocytes within three-dimensional collagen lattices has been shown to be independent of  1 integrins (27). Furthermore, SDF-1-induced migration of T cell hybridomas through intercellular adhesion molecule1-coated filters was independent of the leukocyte function-associated antigen-1 at 100 ng/ml of this chemokine (41). On the other hand, fMLP leads to  1-integrin-mediated adherence of neutrophil granulocytes to fibrin (42, 43) and blocking of  1 integrins inhibited migration of neutrophil granulocytes into extravascular tissue, as was investigated by Werr et al. (44) using intravital time-lapse videomicroscopy.
REGULATION OF LEUKOCYTE MIGRATION
In summary, spontaneous migration of T lymphocytes and neutrophil granulocytes is different in regulatory signal transduction. This difference is reflected in distinct locomotory phenotypes of the cells. Furthermore, both cell types characteristically change locomotory behavior and intracellular regulatory signaling after the induction of chemotaxis. As we have proposed before (45), neutrophil granulocytes and T lymphocytes have at least two distinct molecular signaling states: a matrix-induced, spontaneous type of locomotion and a type of migration in response to chemoattractants. Alterations between spontaneous locomotion and chemotaxis do not involve changes in the microtubule cytoskeleton. These findings, described here for T lymphocytes and neutrophil granulocytes, reflect the different roles of these cells in the inflammatory response.
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ACKNOWLEDGMENTS We thank Britta Reubke-Gothe and Ilka Stechert for expert technical assistance. This work was supported by the Fritz-Bender-Foundation, Munich, Germany.
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