Journal of Immunological Methods 404 (2014) 59–70

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Research paper

Systematic single cell analysis of migration and morphological changes of human neutrophils over stimulus concentration gradients Akira Yamauchi a,⁎, Mikako Degawa-Yamauchi b, Futoshi Kuribayashi a, Shiro Kanegasaki c, Tomoko Tsuchiya c,⁎⁎ a b c

Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan Phoenix Medical Clinic, 3-41-6 Sendagaya, Tokyo 151-0051, Japan YU-ECI Research Center for Medical Science, Yeungnam University, Gyeongsan-City 712-749, Republic of Korea

a r t i c l e

i n f o

Article history: Received 26 August 2013 Received in revised form 1 December 2013 Accepted 10 December 2013 Available online 24 December 2013 Keywords: Chemotaxis Chemoattractants Imaging Polarity Lamellipodia TAXIScan

a b s t r a c t To compare the responses of individual neutrophils to chemoattractants, migration pathway data were obtained using TAXIScan, an optically accessible/horizontal apparatus in which a concentration gradient is established reproducibly for a given stimulus. The observed linear-mode trajectory pattern of neutrophils toward N-formyl-methionyl-leucyl-phenylalanine (fMLP) or Interleukin (IL)-8/CXCL8 was distinguished from random migration patterns toward leukotriene (LT) B4 or platelet activating factor (PAF). The median values of velocity and directionality calculated for individual cells toward fMLP and IL-8 were both relatively similar and high, whereas the values toward LTB4 and PAF were widely dispersed over a lower range of directionality and from low to high ranges of velocity. The different patterns between the groups may be explained by unique morphology with single polarity toward fMLP and IL-8, and unstable morphology with multiple polarities toward LTB4 and PAF. Unique morphologies toward fMLP and IL-8 were not affected by coexisting LTB4 or PAF. On the other hand, the addition of suboptimum concentrations of fMLP or IL-8 to LTB4 or PAF induced a nearly maximum chemotactic response in most cells. These data suggest that exogenous formyl peptides and endogenous chemokines augment neutrophil accumulation at inflammation sites, whereas lipid mediators may play a role in supporting activation of the inflammatory cells for recruitment. © 2013 Elsevier B.V. All rights reserved.

Abbreviations: fMLP, N-formyl-methionyl-leucyl-phenylalanine; IL, interleukin; LT, leukotriene; PAF, platelet activating factor; C5a, complement 5a; CCD, charge-coupled device; HUVEC, human umbilical vein endothelial cells; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PBS, phosphate buffered saline; EDTA, ethylenediaminetetraacetic acid; RBC, red blood cells; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PTX, pertussis toxin; GTP, guanosine triphosphate; GPCR, G-protein coupled receptor. ⁎ Correspondence to: A. Yamauchi, Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan. Tel.: +81 86 462 1111; fax: +81 86 462 1199. ⁎⁎ Correspondence to: T. Tsuchiya, YU-ECI Research Center for Medical Science, Yeungnam University, Gyeognsan, Gyeongbuk 712-749, Republic of Korea. Tel.: +82 53 810 1519; fax: +82 53 810 4761. E-mail addresses: [email protected] (A. Yamauchi), [email protected] (T. Tsuchiya). 0022-1759/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2013.12.005

1. Introduction Chemotaxis is defined as the directional movement of cells along a concentration gradient of attractants and is a highly conserved, fundamental characteristic of cells from primitive organisms to mammals (Bagorda et al., 2006; Dormann and Weijer, 2006). In humans, neutrophils are recruited from peripheral blood to inflammatory or bacterially infected sites where various stimuli are generated. A number of attractants are known to induce neutrophil migration including formyl peptides exogenously generated from bacteria, and proteins and lipid mediators endogenously secreted from cells at the surrounding site (Ward et al., 1968; Showell et al., 1976; Ye et al., 2009). The most popularly known chemoattractants are

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chemokines such as interleukin (IL)-8/CXCL8 (Matsushima et al., 1988; Murphy et al., 2000), and complement components including complement (C)5a (Shin et al., 1968; Ward and Newman, 1969). Lipid mediators like leukotriene (LT) B4 (Ford-Hutchinson et al., 1980; Ford-Hutchinson, 1981) and platelet activating factor (PAF) (Braquet et al., 1987) are also reported to induce chemotaxis of neutrophils. Normally, cells migrate along the concentration gradient of attractants at relatively low concentrations, whereas at higher concentrations these molecules attenuate the migration and often stimulate other cellular functions, e.g., degranulation and generation of superoxide anion in the case of neutrophils. This indicates that an appropriate concentration of chemoattractant is necessary for the directional movement of cells. Although a few technologies have been used to date for chemotaxis and migration assays of individual neutrophils such as Zigmond's and Dunn's chamber methods (Zigmond, 1977; Zicha et al., 1991), such methods are unsuitable for systematic and statistic analysis of cells due to various limitations. To counter this, an optically accessible device named TAXIScan was developed, which circumvents many of the limitations of conventional methods for chemotaxis measurement. Furthermore, it can allow visualization and quantification of dynamic changes of cellular behavior such as chemotaxis, degranulation and other cellular activities (Kanegasaki et al., 2003; Nitta et al., 2007, 2009). The device consists of an etched silicon substrate and a flat glass plate, both of which form horizontal channels each with a micrometer-order depth and two compartments either side of a channel. The substrate and the plate are held together with a stainless steel holder that has holes for injecting cells as well as a stimulus to either compartment (typically 1 μl each). The process of migration or degranulation of cells in each channel (of 6 to 48 channels) is filmed with a charge-coupled device (CCD) camera located beneath the glass and images are taken at time-lapse intervals. About 50 to 100 cells are used for a single assay in a channel. Many parameters can be obtained including velocity, directionality and others (Nitta et al., 2007) together with morphological information, and the device has been used to date in basic and clinical research (Terashima et al., 2005; Nishio et al., 2007; Ishii et al., 2009; Nishikimi et al., 2009; Takamatsu et al., 2010). It is, therefore, interesting to compare the parameters and morphology of neutrophils during migration toward various attractants using TAXIScan, since each attractant should have its own effects, and the different effects among the attractants may reflect their roles in inflammatory diseases. In this paper, we show the results of quantitative analysis of neutrophil migration toward various stimuli and morphological differences during migration that seem to influence the cells' behavior. Information obtained from individual neutrophils within the same population should have important consequences for the function of the entire population and for elucidation of inflammation mechanisms. 2. Materials and methods 2.1. Reagents and buffers Recombinant human IL-8 with 72aa was purchased from Peprotech Inc. (Rocky Hill, NJ, USA) and LTB4, PAF and Dextran

200,000 were from Wako Pure Chemical Industries (Osaka, Japan). Ficoll-Paque was from GE Healthcare Ltd. (Piscataway, NJ, USA) and Bovine serum albumin (fatty acid free grade) was from Nakalai Tesque, Inc. (Kyoto, Japan). Other chemicals were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) unless otherwise stated. 2.2. Cell separation Human granulocytes were isolated from peripheral blood freshly drawn from healthy volunteers (2 males and 2 females, ages 26–45). Five milliliters of peripheral blood containing 5 mM ethylenediaminetetraacetic acid (EDTA) was centrifuged on 5 ml Ficoll-Paque at 400 ×g for 20 min. The granulocyte rich fraction was obtained by sedimentation of RBC with 1.5% Dextran at room temperature (25 °C) for 15 min, followed by hypotonic lysis of remaining RBC. The granulocytes were resuspended in RPMI1640 containing 0.1% BSA (fatty acid free) and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Assay-buffer) at 2 × 106 cells per ml. This granulocyte fraction was used as the source of neutrophils (more than 95% are morphologically neutrophils). 2.3. Cell migration assay by TAXIScan system The cell migration (chemotaxis) assay was performed using TAXIScan (Kanegasaki et al., 2003; Nitta et al., 2007). Briefly, the holder was assembled from an etched silicon substrate (chip), a glass plate and other holder assemblies, and set on the recording module. We used silicon chips with a 5-μm-height terrace, which forms a 5-μm-depth microchannel. After filling with assay-buffer in the common space at the top end of the holes for injection, 1 μl of neutrophil suspension (2 × 106 cells/ml) was injected to one of the 2 compartments, followed by aligning the cells along the start line on the edge of the channel. Cell migration was assayed at room temperature (25 °C), since neutrophils migrate too rapidly and too randomly without exposure to an attractant at 37 °C. After starting the recording of the images, 1 μl of attractant/stimulus solution was injected into the compartment opposite to that containing the cells. Images of migrating neutrophils were recorded with the CCD camera at 30 or 60 second intervals. For higher magnification observation, TAXIScan-FL, a TAXIScan system equipped with high-power magnification lens, was used (Nitta et al., 2009). 2.4. Statistical analysis of neutrophil movement Saved images were analyzed by TAXIScan analyzer 2, an accompanying software package. To show the relationship between velocity and directionality of movement for individual cells, we used a Velocity–Directionality (VD) plot mentioned in the previous report (Nitta et al., 2007), where the vertical axis exhibits the median value of velocity, and the horizontal axis shows the median value of the directionality during the initial 30 min. In some cases, we used the VD–B plot, in which Box plot data are combined with the VD plot and expressed in the corresponding axis of the VD plot. Box plot data were calculated from those of median values of either directionality or velocity of each cell. The direction of cell migration (based on different cell positions in 2 photos taken at 30 or 60 s intervals) was

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expressed as the angle [radian] toward the concentration gradient, namely π/2 and −π/2, indicating that the cell is migrating toward or against the concentration, respectively. Statistical analysis was performed among groups of data by the non-parametric Kruskal–Wallis test followed by the Dunn's multiple-comparison test.

3. Results 3.1. Comparison of trajectory patterns of neutrophil migration toward fMLP, IL-8, LTB4 and PAF We chose fMLP, IL-8, LTB4 and PAF as attractants representative of peptides, chemokines, arachidonic acid metabolites and active phospholipids, in that order and analyzed neutrophil migration using TAXIScan. As shown in Fig. 1A and Supplemental Fig. 2. (movie), the trajectory pattern of individual neutrophils toward fMLP or IL-8 showed that they migrated along a concentration gradient of these attractants. In contrast, most of the patterns of migration were tortuous in a multi-directional manner upon exposure to LTB4 or PAF, suggesting that each neutrophil migrated rather randomly upon exposure to these stimuli. From the migratory pathway data obtained, we calculated the median values of directionality and velocity of individual cells during migration, which were expressed as VD plots (Nitta et al., 2007), where the vertical and horizontal axes exhibit the values of velocity and directionality of each cell, respectively. As shown in Fig. 1B, the median values of all cells were plotted above or around 1 rad on the horizontal axes when the cells were exposed to fMLP or IL-8, indicating that these attractants induced prominent chemotaxis. The migration rate of individual cells induced by these attractants was relatively similar and their velocity values were plotted within the range of 0.15 and 0.3 μm/s. When the cells were exposed to concentration gradients of LTB4 or PAF, the median values of directionality and velocity for individual cells were much more widely dispersed over a lower range of directionality (0 to 1 or 1.2 rad) and in the low to high range of velocity (0 to 0.25 μm/s). In these cases, a significant number of cells were plotted at less than 0.5 rad, indicating that at least this population of cells does not exhibit directional movement along the concentration gradient. To determine that these differences were not related to the neutrophil source, we performed similar experiments employing cells from 3 additional individuals (another male and 2 females) and compared them with the results shown in Fig. 1 (male). To make the comparison easier, we present the data in a new plot, named “VD–B plot”, in which Box plot data are combined with the VD plot figure (Fig. 2A), and further combined all data from different individuals together in one chart for each stimulus (Fig. 2B). Essentially similar results were obtained, for all samples from different individuals, to those shown in Fig. 1 (these data are included in Fig. 2A as person B for comparison), though the distribution of velocity values for individual cells from each individual was somewhat different. Again, there are a significant number of cells that did not show clear directional movement (plotted at less than 0.5 rad) when LTB4 or PAF was used as the stimulus.

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3.2. Statistical analysis of migration pattern of neutrophils exposed to fMLP and IL-8 From the data obtained, we suspected that there might be some difference in the distribution pattern of neutrophil migration between fMLP and IL-8 attractants. We chose one individual as the source of neutrophils and made a statistical analysis by increasing the number of cells examined. The following data were obtained on the same day using the same neutrophil suspension. Since the optimum concentration of these attractants seems to be between 10 and 100 nM in TAXIScan (Supplemental Fig. 1), these concentrations were employed for comparison. Table 1 shows the number of cells in each channel used for statistical analysis when 10 nM of fMLP or IL-8 was used as the attractants. After calculation of the median values of directionally and velocity for individual cells in all 6 channels for each attractant, all data were expressed as a single VD–B plot (Fig. 3). The total number of cells was 556 and 437 for fMLP and IL-8, respectively. The distribution of the values of cells exposed to fMLP was narrower (see B plot) and significantly shifted to the upper and right sides of the VD plot as compared to those for IL-8. The results indicate that neutrophils from this individual showed a better response to fMLP than to IL-8 as a population (p b 0.0001), likely due to the presence of cells within this population that responded feebly to IL-8. When the cells were exposed to 100 nM each of fMLP and IL-8, no such statistical difference was found (data not shown). To determine how few cells can show these minor differences, statistical analyses were performed after reducing the number of cells. For this purpose, we used data from 5, 4, 3, 2 and 1 channels. In any combination of data from 5 or 4 channels among 6 channels (total 36 or 225 combinations, respectively), the difference between fMLP and IL-8 was significant (p b 0.0001). The probability was decreased 10-fold (p b 0.001) when data from 3 channels were combined (a total of 400 combinations were examined: minimum number of cells = 249 and 196 for fMLP and IL-8, respectively). In the case of 2 channels or 1 channel combinations, 205 out of 225 combinations or 29 out of 36 combinations, respectively, gave a probability in the range of 0.05 and 0.001, but for the rest the difference was not significant. These results indicate that a different response between fMLP and IL-8 can reproducibly be shown if 200 or more cells in 3 channels are employed under the experimental conditions. 3.3. Morphological difference of neutrophils during migration toward various chemoattractants Changes in neutrophil morphology during migration toward various attractants/stimuli were captured using a high magnification lens. As shown in Fig. 4 and Supplemental Fig. 3 (movie), when neutrophils migrate along a concentration gradient of fMLP, every cell has a widely-spread lamellipodium, opened like a fan, at the leading edge followed by a compact main body and a very short tail. When the cells migrate along a concentration gradient of IL-8, the lamellipodium is spread much less widely than for fMLP followed by a longer main body and a longer tail. Occasionally a delayed main body movement was observed that pinched out the lamellipodium-leading and remaining main body parts like an hourglass (Fig. 4 and

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Supplemental Fig. 3). On the other hand, when neutrophils were exposed to a concentration gradient of LTB4 or PAF, multiple leading edges were formed and the cell movements unsettled.

3.4. Effect of additional attractant/stimulus on migration of neutrophils toward fMLP, IL-8, LTB4 and PAF At inflammation sites, various kinds of attractants and stimuli are generated and present in situ. To determine how another attractant/stimulus might influence the migration of neutrophils toward fMLP, IL-8, LTB4 or PAF, different concentrations of attractants were mixed and neutrophil behavior in TAXIScan was examined (Fig. 5). When 1 to 100 nM of another attractant/stimulus was mixed with 100 nM fMLP or 100 nM IL-8, the influence was limited in directionality (Fig. 5A and B, left) especially in the case of fMLP, and the values were plotted over a similar range around 1.2 rad. When 100 nM of LTB4 or 100 nM PAF was used as the basic stimulus, the addition of 1 nM of fMLP resulted in a dramatic change in the distribution pattern of directionality values (Fig. 5C and D, left, 3rd column). The plots were clustered (much more concentrated) in a small range and shifted to a higher range as compared to 100 nM of LTB4 or PAF alone (Fig. 5C and D, left, 2nd column). When this concentration of fMLP alone was used as the attractant, the plots were more widely distributed over a lower range (Supplemental Fig. 1, 3rd column), indicating that there is a positive effect on neutrophil chemotaxis by the combination of low concentration fMLP and either LTB4 or PAF. With the addition of a higher concentration of fMLP to each basic stimulus, directionality and velocity values were shifted to a lower range in a concentration dependent manner, though the values were still significantly higher than those with each basic stimulus or with fMLP alone. Similar enhancements were also observed when 1 to 100 nM IL-8 was added to each basic stimulus. Although when 1 nM IL-8 was the stimulus, directionality plots were widely distributed over a lower range (Supplemental Fig. 1B, left, 3rd column), combination with 100 nM LTB4 or PAF resulted in clustered distribution of directionality (Fig. 5C or D, left, 6th column, respectively) as compared to 100 nM LTB4 or PAF alone (Fig. 5C or D, left, 2nd column, respectively). For the combination of LTB4 and PAF, neutrophils acquired enhanced directionality values (Fig. 5C and D, left, 9th–11th columns) in a concentration dependent manner as compared to LTB4 or PAF alone (Supplemental Fig. 1C, 2nd–4th columns, or D, 3rd–5th columns, respectively). Morphological changes observed with 100 nM fMLP, with widely spread lamellipodia at the leading edge followed by a compact main body were not affected by the addition of

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1–100 nM of IL-8, LTB4 or PAF (Supplemental Fig. 4, top row panels). Similarly, the effects observed with 100 nM IL-8 were not affected by the addition of 1–10 nM fMLP, 1–100 nM of LTB4 or PAF (Supplemental Fig. 4, 2nd row). However, with the addition of 100 nM fMLP, the cells showed a similar morphology to that observed with fMLP alone. On the contrary, when 1–100 nM fMLP or IL-8 was mixed with 100 nM LTB4 or 100 nM PAF, the morphological changes of the neutrophils were similar to those observed with fMLP or IL-8 alone (Supplemental Fig. 4, 3rd and 4th rows). To confirm the effect on neutrophil chemotaxis caused by the combination of low concentration fMLP or IL-8 and either LTB4 or PAF, additional experiments were performed and the results were expressed as VD–B plot. As shown in Fig. 6A, plots distributed a lower range of directionality when 1 nM fMLP alone or 100 nM LTB4 alone was the stimulus. When these stimuli combined, plots were clustered in a small range of directionality and shifted to a higher range. The similar directionality shift was observed when 1 nM fMLP was combined with 100 nM PAF (Fig. 6B). The results of combination of 1 nM IL-8 and 100 nM LTB4 or 100 nM PAF also showed enhanced chemotactic response of neutrophils as compared with those to each stimulus (Fig. 6C and D).

4. Discussion In this paper, we show that the migration behavior of human neutrophils toward 4 representative stimuli fall into 2 characteristic categories. In the case of fMLP and IL-8, each cell has a large and relatively similar median value of directionality and velocity, and the migration track is close to linear. On the other hand, in the case of LTB4 and PAF, the cells had smaller and more dispersed values for directionality, and the VD plots of each cell distributed widely over both axes. These differences are not attributable to the nature of the lipid stimuli having affected the establishment of the gradient in the channel. It has been shown that mouse bone marrow neutrophils and polymorphonuclear leukocytes (Carbo et al., 2010; Simarro et al., 2010; Kubota et al., in press) migrated toward LTB4, and human eosinophils toward prostaglandin D2 (Nitta et al., 2007), all showing migration tracks close to linear in a TAXIScan device like the cases of fMLP and IL-8 in the present investigation. This indicates that these lipid stimuli, at least, form concentration gradients reproducibly in the device. All experiments were performed at 25 °C, because we found that isolated neutrophils migrated too rapidly and too randomly at 37 °C even without exposure to an attractant, and that the high background due to this rapid random migration often obscures directional movement toward an attractant, making it difficult to analyze statistically. The present results

Fig. 1. A: The trajectory patterns of neutrophil migration toward various stimuli. Images of migrating cells in TAXIScan channels were filmed and the paths of migrating cells were traced. Each arrow shows the direction and the distance of each cell in 30 s, from which median values of directionality and velocity were calculated. The concentration of injected stimuli and their gradient are schematically shown in the left axis. One representative tracing of cell migration with each stimulus (among 4 independent experiments with 4 different individuals) is shown. The distance between the start line (bottom) and the finish line (top) of each channel is 260 μm. Scale bar represents 40 μm length. B: Velocity–Directionality (VD) plots of neutrophil migration. Each dot corresponds to a cell. The method for calculation of each value is described in the Materials and methods section. The concentrations of the stimuli are; fMLP: 100 nM, IL-8: 100 nM, LTB4: 100 nM, and PAF: 100 nM. One representative VD plot obtained from the traces mentioned in A is shown. Note: Cells migrating along the concentration gradient of a stimulus have a high directionality value and are plotted to the right most area of the graph.

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A. Yamauchi et al. / Journal of Immunological Methods 404 (2014) 59–70 Table 1 Number of cells in a channel for statistical analysis. Experiment Channel number

Chemoattractant Concentration No. of cells in channel

1 1 1 1 1 1 2 2 2 2 2 2

fMLP fMLP fMLP IL-8 IL-8 IL-8 IL-8 IL-8 IL-8 fMLP fMLP fMLP

Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6

10 10 10 10 10 10 10 10 10 10 10 10

nM nM nM nM nM nM nM nM nM nM nM nM

110 100 93 74 64 66 98 66 69 89 67 97

The data were obtained on the same day using the same neutrophil suspension. Data from all cells in different channels (total number of cells; 556 and 437 for fMLP and IL-8, respectively) were combined and expressed as a VD–B plot in Fig. 3.

do not necessarily reflect the in vivo situation, especially the conditions in the body core where the temperature is 37 °C. Conditions at the extremities, however, vary considerably. For example, it must be noted that fingertip temperature even at 23 °C ambient temperature can be near 25 °C and much lower when ambient temperature was reduced further (Montgomery and Williams, 1976). Every cell exposed to a concentration gradient of fMLP or IL-8, respectively, showed a widely-spread lamellipodium with a compact body or a narrower lamellipodium spreading plus a longer main body and tail. It is possible that these morphological differences influenced the slight difference in directionality and velocity of the neutrophil population toward fMLP and IL-8. In contrast, when the cells were exposed to LTB4 or PAF, multiple leading edges were formed in most of these cells, if not all of them, resulting in quite different trajectory patterns as compared with fMLP and IL-8, namely winding and twisting tracks in most cases. It is plausible, however, that some cells migrated randomly whereas the others migrated along the concentration gradient of the attractants with low to high velocity. It is difficult to define at present, however, whether or not each cell itself, which had a somewhat larger median value of directionality, can recognize the concentration gradient and whether the corresponding value reflects bona fide directional movement or chemotaxis. It is possible that such larger values were obtained merely by chance. A new definition of chemotaxis, therefore, may be necessary by taking into account cell polarity and manner of lamellipodium spreading in the concentration gradient of chemoattractants. During chemotaxis, each cell must sense the concentration gradient of an attractant along its external surface, inducing

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consistent morphological change in the cell. We have shown in this study using fMLP and IL-8 as typical examples, the formation of a single lamellipodium followed by the cell body and tail. If a cell cannot sense the chemoattractant gradient, migration must be via chemokinesis, though in some cases there might be only a portion of a cell population that can sense the concentration gradient. Since we observed a single lamellipodium formation against a concentration gradient of certain attractants in eosinophils (Nitta et al., 2007), basophils, monocytes and lymphocytes in addition to neutrophils (unpublished observation), the definition can be applicable at least to various leukocytes and related cell lines. However, this definition may not be directly applicable to cells other than leukocytes (e.g. cell lines such as HT1080 fibrosarcoma cells, MDAHB231 breast cancer cells and CCR2 expressing Chinese hamster ovary cells), since we observed that the moving pattern and morphological change of these cells during migration toward a certain attractant are somewhat different from leukocytes: the leading edge with branching ends is followed by a long body in the concentration gradient of the attractant (Terashima et al., 2005 and unpublished observation). In any case, this is a new aspect of cellular chemotaxis that may be adopted to leukocytes at least, and may be useful to determine whether a stimulus is chemoattractant or not. It is important to elucidate the molecular mechanisms of morphological changes of individual cells that lead to the distinct trajectory pattern of neutrophil migration. High and low affinity receptors are known to exist for fMLP (FPR1 and FPR2), IL-8 (CXCR1 and CXCR2) and LTB4 (BLT1 and BLT2). These and PAF receptors are expressed in human neutrophils and all are known to be G-protein coupled receptors (GPCR). In general, the signals from these receptors are transmitted through the trimeric G-proteins and then through the downstream signaling molecules commonly seen in GPCR signaling such as p38 mitogen-activated protein kinase (MAPK), p42/44 extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC) phospholipase C (PLC) and small GTPases (Roberts et al., 1999; Ambruso et al., 2000; Honda et al., 2002; Yamauchi et al., 2005; Stillie et al., 2009; Ye et al., 2009; Bäck et al., 2011). Of these, a small GTPase Rac2 was shown to play an important role in neutrophil chemotaxis toward fMLP (Roberts et al., 1999; Ambruso et al., 2000; Yamauchi et al., 2005). Nick et al. (1997) reported that both MAP ERK kinase kinase-1 (MEKK-1) and a serine/ threonine kinase Raf were activated strongly by fMLP but minimally by PAF. Also, M'Rabet et al. (1999) showed that a small guanosine triphosphatase (GTPase) Ral was activated differently, where fMLP and PAF, respectively, activated Ral through the pertussis toxin (PTX) dependent and independent pathways. It was suggested that subtypes of the fMLP receptor induce a similar endpoint event through different

Fig. 2. A: Velocity–Directionality–Box (VD–B) plots of migrating neutrophils. In the VD–B plot, Box plot data of directionality and velocity for each cell are shown in the corresponding axis of VD plot. Neutrophils were isolated from 4 different individuals (A, B, C and D) and the median values of directionality and velocity were calculated from the image data of the initial 30 min. The concentrations of the stimuli are; fMLP: 100 nM, IL-8: 100 nM, LTB4: 100 nM, and PAF: 100 nM. B: Combined VD–B plots of migrating neutrophils from 4 different individuals shown by different colors. Data in A were combined and shown as single VD–B plots for each stimulus. Statistical analysis was performed among each plot with the non-parametric Kruskal–Wallis test followed by Dunn's multiple comparison test. Markings indicate statistically significant (**p b 0.01 in comparison with fMLP, ##p b 0.01 in comparison with IL-8).

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Fig. 3. VD–B plots of a large number of migrating neutrophils toward fMLP or IL-8. Data were obtained on the same day using the same neutrophil suspension. All data obtained from 6 channels of 2 experiments for each attractant were combined and expressed in a single VD–B plot. Statistical analysis was performed among each plot with the non-parametric Kruskal–Wallis test followed by Dunn's multiple comparison test. Asterisks indicate statistically significant (**p b 0.01).

signal-transduction intermediates and that one type becomes desensitized in a concentration gradient (Paclet et al., 2004). These reports suggest that different pathways are used when the cells are stimulated by fMLP or PAF, or even by fMLP receptor subtypes. More precise investigation, therefore, is necessary to elucidate the signals that lead to the morphological changes shown in this paper. We found that a relatively low concentration of fMLP or IL-8 induced enhanced chemotactic response in neutrophils if LTB4 or PAF was present. The enhancement by the combination of typical chemoattractants such as fMLP and IL-8 and stimuli like LTB4 and PAF, may be important at the inflammation site. Although each peptide and lipid were added simultaneously in the present experiments, neutrophils can be primed by a high concentration of PAF or LTB4 during the very early stage of migration. PAF priming using

a minuscule concentration was reported to be maximal within 5 min even when assayed by superoxide generation (Vercellotti et al., 1988 — note that the chemotaxis response is much more sensitive than superoxide generation and that very low concentrations of agonist are enough to induce chemotaxis). It is therefore likely that cells migrating in the microchannel shown here in the presence of PAF (or LTB4) were primed within a few minute of incubation. On the other hand, no significant change was observed in directionality when 100 nM fMLP and 1 to 100 nM IL-8 were combined. By the addition of 100 nM fMLP (but not by lower concentrations) to 100 nM IL-8, almost all neutrophils showed a similar morphology to those observed with fMLP alone. These results indicate that if the concentrations of fMLP and IL-8 are similar, each neutrophil responds more robustly to fMLP than to IL-8. There are many reports showing that certain attractants are dominant over others, especially concerning preferential

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Fig. 4. High magnification views of migrating neutrophils toward four different stimuli. Arcs indicate widely spread lamellipodia and arrowheads show narrow lamellipodia. Scale bar represents 10 μm in length. Concentration gradients are schematically shown on the left side of the photographs. The concentrations of the stimuli are; fMLP: 100 nM, IL-8: 100 nM, LTB4: 10 nM, and PAF: 100 nM. The numbers of lamellipodia formed toward fMLP, IL-8, LTB4 or PAF, in the same order, were 0 (0, 34, 12, 7%), 1 (100, 66, 22, 40%), 2 (0, 0. 44, 46%), and more than 3 (0, 0, 22, 7%). The original images of these figures were used for counting. The numbers of lamellipodia change from time to time during migration, especially with exposure to LTB4 and PAF.

migration toward fMLP despite the presence of high concentrations of IL-8. For example, Kitayama et al. (1997) showed, using a modified Boyden chamber method and human umbilical vein endothelial cell (HUVEC) layer to exclude random migration, that fMLP abrogated neutrophil transmigration toward IL-8 even at lower concentrations while IL-8 did not significantly inhibit transmigration toward fMLP. Using the agarose-plate method, Heit et al. (2002) reported that there is a hierarchy of signal transduction among fMLP, C5a, IL-8 and LTB4. We believe that most of the results previously obtained based on endpoint analysis especially using inhibitors need to be reevaluated based on the real-time kinetic analysis shown in the current paper. In fact, in this study we observed that fMLP at lower concentrations did not influence morphological changes of the cells toward 100 nM IL-8.

In conclusion, the real-time kinetic and statistical data on migration of individual cells and morphological information during migration that can be obtained using TAXIScan should provide a new way for analyzing cellular chemotaxis, the mechanisms of cell migration and the precise role of chemotaxis in inflammation and various disorders.

Authorship Contribution: AY, SK and TT designed, analyzed and interpreted data, and wrote the manuscript, AY and MDY performed data collection, and FK provided intellectual input, helped in editing the manuscript and obtained funding. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jim.2013.12.005.

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Fig. 5. Box plot of each neutrophil migration upon exposure to mixed stimuli. Two different stimuli were mixed and injected at the same time and values of directionality and velocity for each cell were calculated from the image data during initial 30 min. Data of one representative from 3 independent experiments are shown.

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Fig. 6. VD–B plots of migrating neutrophils upon exposure to single stimulus or their mixture. A: Plots were obtained using 1 nM fMLP alone, 100 nM LTB4 alone or their combination. B: Plots were obtained using 1 nM fMLP alone, 100 nM PAF alone or their combination. C: Plots were obtained using 1 nM IL-8 alone, 100 nM LTB4 alone or their combination. D: Plots were obtained using 1 nM IL-8 alone, 100 nM PAF alone or their combination. Values of directionality and velocity for each cell were calculated from the image data obtained during initial 30 min Statistical analysis of box plots data was performed among each plot with the non-parametric Kruskal–Wallis test followed by Dunn's multiple comparison test. Asterisks indicate statistically significant (***p b 0.001, *p b 0.05).

Acknowledgments We thank Ms Koyako Kobiki for acquiring data and Drs. Gary Quinn and James E. Pease for critical reading of the manuscript. We also thank Dr. Sung Soo Han's support for YU-ECI Research Center. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (C-24591473) and the Research Fund of Kawasaki Medical School (to AY). References Ambruso, D.R., Knall, C., Abell, A.N., Panepinto, J., Kurkchubasche, A., Thurman, G., Gonzalez-Aller, C., Hiester, A., deBoer, M., Harbeck, R.J., Oyer, R., Johnson, G.L.,

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Systematic single cell analysis of migration and morphological changes of human neutrophils over stimulus concentration gradients.

To compare the responses of individual neutrophils to chemoattractants, migration pathway data were obtained using TAXIScan, an optically accessible/h...
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