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T cells sense biophysical cues using lamellipodia and filopodia to optimize intraluminal path finding† Kwang Hoon Song,‡a Keon Woo Kwon,‡b Jong-Cheol Choi,a JaeHwang Jung,c YongKeun Park,c Kahp-Yang Suhb and Junsang Doh*ad Intraluminal crawling is considered to be important for extravasation of leukocytes in blood vessels, but biochemical/biophysical cues guiding the crawling of leukocytes have not been clearly understood. Here we provide evidence that T cells sense the topography of luminal surfaces and the nuclei of endothelial cells (ECs) using lamellipodia and filopodia, respectively, to optimize path finding during intraluminal crawling. Well-aligned EC layers or replicas of EC layers, which exhibit topography similar to that of EC layers, were fabricated, and flow was applied either parallel or perpendicular to the orientation of EC alignment. T cells crawled along the valleys of the topographical landscapes of the EC layers, while
Received 2nd February 2014, Accepted 19th February 2014
avoiding nuclei of ECs regardless of flow direction. Pharmacological inhibitor treatments revealed that
DOI: 10.1039/c4ib00021h
Lamellipodia or filopodia-inhibited T cells crawled significantly longer distances for extravasation than
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did normal T cells, indicating that sensing biophysical cues are critical for optimizing routes for extravasation.
sensing of topography and nuclei of EC layers was mediated by lamellipodia and filopodia, respectively.
Introduction Leukocytes in the bloodstream undergo a series of adhesion cascade, rolling, firm adhesion, crawling, and transendothelial migration (TEM) in order to infiltrate into tissues for immune surveillance and immune responses.1,2 While it has been frequently observed that leukocytes crawl a considerable distance from their initial adhesion sites to extravasation sites,3–5 whether intraluminal crawling of leukocytes is guided by certain biochemical or biophysical cues or not has not yet been made clear. Considering that crawling leukocytes are under extreme shear stresses that can detach them during crawling,6
a
Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-Gu, Pohang, Gyeongbuk, 790-784, Korea. E-mail: [email protected]; Fax: +82-54-279-3199; Tel: +82-54-279-2189 b School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-742, Korea c Department of Physics, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea d School of Interdisciplinary Bioscience and Bioengineering (I-Bio), Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-Gu, Pohang, Gyeongbuk, 790-784, Korea † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4ib00021h ‡ These authors contributed equally to the work.
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optimal path finding strategies to reach portals for TEM would help their tissue infiltration by minimizing detachment. Intravital imaging of neutrophil crawling has revealed that neutrophils preferentially crawled along the junction of ECs, indicating that the surface of EC layers may contain guiding cues for leukocyte crawling.7 On the other hand, there have been many reports describing that leukocytes crawl either toward8,9 or against4,10,11 the flow direction depending on the types of leukocytes or on the experimental settings, meaning that flow also plays a certain role. While both EC junctions and flows can affect the crawling direction of leukocytes, experimental settings currently widely used do not allow a systematic examination on which factor has predominant roles in determining the intraluminal crawling direction. In vivo, ECs align along the direction of flow so that the EC junction orientation and flow direction are tightly coupled. On the other hand, in vitro experiments were performed by applying flow to cultured monolayers of ECs with random orientation; thus only the effect of flow direction has been considered. To overcome this limitation, we fabricated pre-aligned EC monolayers by culturing ECs on nanogrooved surfaces as previously described,12 and applied flow in either parallel or perpendicular direction of EC orientation. Using this novel experimental setting, we first demonstrated that the crawling direction of T cells was predominantly affected by EC alignment rather than the flow direction. Furthermore, we showed that
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T cells sense the topography of EC layers by lamellipodia to direct their migration toward the junctions, and also they sense nuclei of ECs by filopodia to avoid nuclei of ECs. Considering TEM of leukocytes typically occurs near EC junctions, never through nuclei, the aforementioned strategies would help T cells to efficiently survey luminal surfaces to find sites for TEM.
Results Experimental settings to decouple flow direction and EC orientation In order to identify potential guiding cues for intraluminal crawling of T cells, we first addressed whether the flow direction or EC orientation had a predominant effect on the direction of T cell crawling. To independently control the flow direction and EC orientation, well-aligned EC layers were prepared by culturing bEnd.3 cells, a murine brain endothelial cell line, on surfaces containing nanoscale ridges/grooves (350 nmridges, 700 nm-grooves and 300 nm-heights, shown in Fig. 1A) as previously described (Fig. 1B);12,13 these layers were inserted into a parallel plate flow chamber either parallel or perpendicular to the flow direction (cases are denoted ‘parallel’ and ‘perpendicular’, respectively, in Fig. 1D). Randomly oriented EC layers cultured on flat surfaces were also prepared for control (Fig. 1C, denoted ‘random’ in Fig. 1D). EC monolayers cultured either on nanostructured or flat surfaces were stimulated with tumor necrosis factor a (TNF-a) for 4 h, overlaid with stromal cell-derived factor-1a (SDF-1a) for 10 min, and mounted in a
Fig. 1 Experimental settings using well-aligned/randomly oriented endothelial cell (EC) layers. (A) Scanning electron microscope (SEM) image of a nanostructured (350 nm/700 nm/300 nm, ridge/groove/depth) surface used to align ECs. (B, C) Differential interference contrast (DIC) images of ECs cultured on a nanostructured (B) or a flat surface (C). Scale bar: 50 mm. (D) Schematic illustration of three different configurations of flow experiments. (E) Schematic illustration of experimental procedures and T cell dynamics on EC layers.
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shear chamber (Chamlide CF, Live Cell Instrument, Korea). TNF-a triggers upregulation of adhesion molecules on ECs,1,14 and SDF-1a bound on the surfaces of ECs induces activation of lymphocyte function-associated antigen 1 (LFA-1) of T cells to support T cells to stably crawl on ECs under shear flow.3 Flow assays were performed as schematically shown in Fig. 1E. Briefly, suspension of DO11.10 T cell blasts in the binding media15 was perfused with low shear stress (0.25 dynes cm 2) on a monolayer of ECs for 10 min to accumulate T cells on the ECs. Then, various shear stresses (0, 2, 10 dynes cm 2) were applied to the adhering T cells on the ECs by perfusing binding media into the chamber; the dynamics of the T cells were observed by time-lapse microscopy for 20 min.12 Effects of EC orientation and flow direction on T cell crawling T cells under shear flow exhibited various behaviors as schematically shown in Fig. 1E. The effects of EC alignment on the frequency of each behavior has been reported in the previous study,12 thus here, we primarily focused on the direction of crawling. Only T cells crawling substantial distances (more than 50 mm, typically B70% of T cells in a field of view) were considered. Representative trajectories of crawling T cells under 2 dyne cm 2 of shear stress are plotted in Fig. 2A for all three different configurations considered. When flow and EC orientation were parallel to each other, it was clear that the crawling of T cells was biased toward the direction of the flow/ EC alignment. In contrast, when flow and EC orientation were perpendicular to each other, it appeared that both the flow and the EC-alignment affected the crawling of T cells. To evaluate whether the flow or the EC-alignment had a predominant effect on the crawling of T cells, we quantitatively analyzed T cell crawling as schematically shown in Fig. 2B. The x-axis is assigned parallel to the flow direction; directionality index dx and time average velocity to the x-axis Vx were defined from the displacements. The measured dx and Vx values for various experimental conditions are plotted in Fig. 2C and D, respectively. In the absence of shear stress, T cells crawled along the direction of the EC alignment: the average dx value of T cells crawling on the well-aligned ECs with ECs oriented toward the x-axis (or ‘parallel’) was B0.6, while the average dx value of T cells crawling on the randomly-oriented ECs (or ‘random’) was B0.5 (Fig. 2C). When flow was applied parallel to the EC alignment, the dx values of the T cells increased, indicating that shear flow and EC orientation synergistically guided the crawling of T cells. When flow was applied to randomly-oriented ECs, the dx values of the T cells slightly increased, meaning that T cell crawling was affected by the flow. However, when flow was applied perpendicular to the EC alignment, the average dx values of the T cells remained significantly smaller than 0.5, or decreased even further at the highest shear stress (10 dynes cm 2), suggesting that the orientation of the EC alignment had a more pronounced effect on the crawling of T cells than did the flow direction. A significant reduction of the average dx values of the T cells at the highest shear stress may mean that T cells crawling along the direction of the EC alignment were not detached by high shear stress as much as
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Fig. 2 Crawling direction of T cells is predominantly determined by the direction of EC orientation. (A) Representative trajectories of crawling T cells under shear flow (2 dyne cm 2) with parallel, random, and perpendicular configurations. (B) Definitions of parameters for T cell crawling analysis. (C, D) Effect of experimental configurations and shear stresses on dx (C) and Vx (D) values of crawling T cells on ECs. (E, F) Crawling distance (E) and duration (F) of T cells in the direction of EC alignment and other directions in perpendicular configuration under 10 dyne cm 2 of shear flow. Data are representative of 5 independent experiments (Mann–Whitney U-test, two-tailed, *p o 0.05, **p o 0.01, ***p o 0.001).
the T cells crawling in other directions were. Indeed, T cells crawling in the direction of EC alignment crawled a significantly longer distance and duration than T cells crawling in other directions prior to detachment (Fig. 2E and F), confirming this possibility. Average Vx values were close to zero for all three configurations in the absence of flow and slightly decreased when shear stress was applied, meaning that T cells crawled in the direction of the flow. Taken together, the intraluminal crawling of T cells under flow was predominantly guided by EC-alignment, and flow played an ancillary role in regulating the crawling direction of T cells. Relationship between adherens junctions/nuclei of EC layers and crawling trajectories of T cells To gain further insight into the guiding mechanisms of intraluminal crawling of T cells on EC surfaces, the trajectories of crawling T cells were overlaid with adherens junctions (visualized by anti-VE-cadherin-FITC) and the nuclei of ECs (visualized by Hoechst, Fig. 3A). Tracks of crawling T cells in all three
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conditions were carefully examined to identify the rules guiding the crawling direction of T cells. Often, T cells that had landed on ECs first approached the junctions of the ECs; once they encountered the junctions, they tended to crawl along the junctions (Fig. 3B and Movie S1 in ESI†), as previously described with neutrophils in vivo.7 In addition, when T cells encountered nuclei of ECs while crawling, the majority of them made detours rather than crossing the nuclei (Fig. 3C and Movie S2 in ESI†). To dissect the mechanisms underneath these intriguing tendencies of T cell crawling, we set hypotheses and performed experiments to evaluate those hypotheses. First, it is possible that uneven distribution of molecules on the EC layers, potentially near the junctions,16 may bias the crawling of T cells. Adhesion molecules such as ICAM-1 and VCAM-1, and chemokines bound to the apical layers of ECs, are known to be important for T cell crawling.3,10 In the absence of SDF-1a, T cells crawled in the direction of EC alignment (Fig. S1 in ESI†), indicating that chemokines bound to ECs may not be a
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Fig. 3 The majority of T cells appear to crawl along the adherens junctions while avoiding the nuclei of ECs. (A) Overlay of T cells (labeled with CMTMR, red), adherens junctions (stained with anti-VE-cadherin-FITC, green) and nuclei of ECs (stained with Hoechst, blue), trajectories of T cells (white) and initial points of trajectories (white dots) (B) Representative time-lapse images of a T cell crawling along adherens junctions. (C) Representative time-lapse images (DIC/Hoechst overlay) of a T cell avoiding crossing nuclei of ECs. Scale bar: 10 mm. Elapse time: mm:ss.
major guiding factor. In addition, we did not notice any accumulation of ICAM-1 or VCAM-1 around the junctions (Fig. S2 in ESI†). Effect of surface topography of EC layers on the direction of T cell crawling On the other hand, T cells may recognize the topographical structures of EC surfaces and prefer to crawl along the valley, and thus they appeared to crawl along the junctions. To directly test this possibility, we replicated the topography of endothelial layers, uniformly coated the replica with key molecules for crawling, and performed flow assays. First, confluent monolayers of ECs cultured on either nanostructured or flat surfaces were fixed and dehydrated (Fig. 4A(i)). Subsequently, the fixed and dehydrated EC layers were replicated using a UV-curable resin poly(urethane acrylate) (PUA) by capillary force lithography, which allows precise replication with sub-100 nm resolution (Fig. 4A(ii)).17 The inverse replicas of the fixed and dehydrated ECs were extensively rinsed with isopropyl alcohol to remove debris of dehydrated cells and dried by nitrogen blowing. Then, by replicating the inverse EC replicas one more time using PUA by CFL (Fig. 4A(iii)), replicas of the original EC monolayers were obtained. Slight shrinkage and deformation during fixing and dehydration would be inevitable,18 but differential interference contrast (DIC) images of monolayers of the live ECs and EC replicas appeared comparable (Fig. 4B). To quantitatively compare the topography of ECs and EC replicas, ECs and EC replicas were imaged using quantitative phase microscopy based on off-axis Mach–Zehnder interferometry.19,20 Pseudo-color images presenting the topography of
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Fig. 4 Fabrication and characterization of EC replicas. (A) Schematic procedure of replica fabrication of confluent EC layers using the UVcurable resin poly(urethane acrylate) (PUA). (B) Representative DIC images of native ECs and EC replicas. (C) Representative pseudo color topography images of confluent EC layers and EC replicas. Scale bar: 100 mm. (D) Height distribution of ECs and EC replicas. Data are representative of 5 independent experiments.
the ECs and EC replicas were generated from the phase images retrieved from interference images (see Methods for details) and displayed in Fig. 4C. Overall topography of ECs was well conserved in EC replicas, but heights of peaks were significantly
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reduced, suggesting that flattening occurred during fixing, dehydration and replication processes. Height distributions of ECs and EC replicas were extracted from the topographical images and shown in Fig. 4D. While the average height of EC replicas (1.5 0.46 mm) was close to that of ECs (1.6 0.92 mm), height distribution of EC replicas was much narrower than that of ECs, suggesting that flattening and shrinking of monolayers of ECs occurred during the replication processes. With these characteristics of EC replicas, then we assessed crawling of T cells on EC replicas. The EC replicas were coated with ICAM-1and SDF-1a, mounted in the shear chamber, and flow assays were performed as described above. T cells crawling on the EC replicas coated with 1 mg ml 1 of ICAM-1, which had a surface density of ICAM-1 similar to that of TNF-a-treated ECs, as shown in Fig. S3A in ESI,† were traced and dx and Vx values of the T cells were calculated and plotted (Fig. 5A and B).
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It was notable that the crawling patterns of T cells on the ICAM1 coated EC replicas were similar to those of T cells on native EC monolayers: the average dx values of the T cells were significantly greater than 0.5 for parallel cases, which is significantly smaller than 0.5 for perpendicular cases, suggesting that the topography of the monolayers of ECs presenting ICAM-1 and SDF-1a was sufficient to guide T cell crawling to the adherens junctions of the ECs. Average Vx values were close to 0 in the absence of shear, and shifted slightly toward negative values when flow was applied, indicating that the T cells passively drifted with the flow rather than actively responding to the flow. However, when we increased the ICAM-1 concentration, the population of T cells crawling against the flow direction significantly increased (Fig. S3B and C in ESI†), as stated in previous studies,4,10 meaning that the ICAM-1 density or the LFA-1 activity may play an important
Fig. 5 Effect of surface topography of EC layers on the direction of T cell crawling. (A, B) Effect of experimental configurations and shear stresses on dx (A) and Vx (B) values of crawling T cells on EC replicas. (C) Definition of probability to cross nuclei of ECs (Pn), and Pn values of T cells on ECs vs. EC replicas. ECs, n = 104; EC replicas, n = 132 in total. Flow experiments were performed in parallel configuration with 2 dyne cm 2 of shear flow. Data are representative of five independent experiments (A and B) or are pooled from 5 independent experiments (C) (Mann–Whitney U-test, two-tailed, *p o 0.05, **p o 0.01, ***p o 0.001).
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role in T cells crawling against the flow. To test whether crawling T cells avoided crossing the nuclei of ECs due to topographical effects, because the nuclei of ECs are typically located around the local maxima of the topographical landscapes formed by EC monolayers, the probability values of crossing the nuclei of ECs (Pn), defined by the ratio of the number of T cells crossing the nuclei of ECs (Nc) to the number of T cells encountering nuclei (Ne), were measured for T cells on ECs and T cells on EC replicas and these values were compared (Fig. 5C). The average Pn value of the T cells on the EC monolayers was about three times lower than that of the T cells on the EC replicas, meaning that topography might not be the major reason for T cells avoiding the nuclei of ECs. Alternatively, nuclei regions of EC replicas were much flatter than those of ECs, as demonstrated in Fig. 4C, so that T cells on EC replicas could cross nuclei regions more easily than T cells on ECs. Roles of lamellipodia and filopodia of T cells in sensing biophysical cues of EC layers To further identify the molecular players involved in recognizing the topography of EC layers and the nuclei of ECs, we inhibited Arp2/3 complexes, actin binding proteins that nucleate branched actin network formation at the leading edge to generate thin sheet-like protrusion lamellipodia, and cdc42, a small GTPase that regulates the formation of needle-shaped filopodia, using pharmacological inhibitors CK-63621 and ML-141,22 respectively. Indeed, it has been suggested that lamellipodia and filopodia play important roles in sensing microenvironments and in determining the direction of cell migration;23,24 however, their role in leukocyte migration in blood vessels has not been investigated. To test the effect of inhibitors on T cell ultrastructures, scanning electron microscope (SEM) images of T cells on ECs, ICAM-1/SDF-1a-coated flat substrates and EC replicas under 2 dyne cm 2 of shear stress were acquired and analyzed (Fig. 6A and B). CK-636-treated cells did not generate thin and wide sheet-structured lamellipodia at the leading edges, resulting in a narrowing of width compared to the DMSO-treated control T cells. On the other hand, ML-141-treated T cells generated a significantly lower number of filopodia than did DMSO-treated T cells. With these clear effects of inhibitors on lamellipodia and filopodia formation of T cells, we then assessed the roles of lamellipodia and filopodia in T cell crawling by performing flow assays with inhibitor-treated T cells. First, the dx values of T cells treated with inhibitors were compared with those of T cells treated with DMSO. Well-aligned ECs or EC replicas of well-aligned ECs were used. CK-636-treated T cells exhibited much sharper leading edges than DMSO-treated T cells, often crossed ridges of EC layers (Fig. S4, Movie S3 and S4 in ESI†), and dx values were close to 0.5 regardless of ‘parallel’ or ‘perpendicular’ configurations (Fig. 7A), indicating that CK-636 treated T cells were no longer guided by the direction of the EC orientation. In contrast, ML-141-treated T cells crawled along the EC orientation, and their dx values were similar to those of the DMSO-treated T cells (Fig. S5 in ESI†).
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Fig. 6 Effects of pharmacological inhibitors on T cell ultrastructures. (A) Inhibitor-treated T cells on flat substrates, ECs and EC replicas under a shear stress of 2 dyne cm 2 were fixed and analyzed by SEM. CK-636 (100 mM) was used to inhibit Arp2/3 complexes and ML-141 (20 mM) was used to inhibit cdc42. Scale bar: 2 mm. (B) Quantitative analysis of SEM images. B10 cells per each condition were analyzed. (Mann–Whitney U-test, two-tailed, **p o 0.01, ***p o 0.001).
In addition, the average Pn value of the ML-141-treated T cells on live ECs was significantly higher than that of the DMSO-treated T cells, meaning that filopodia of the T cells played an important role in sensing the nuclei of the underlying ECs (Fig. 7B). T cells on ECs may form invasive filopodia that push the cytoplasm of ECs down several hundreds of nanometers to physically sense the nuclei, and the formation of invasive filopodia depends on shear stresses and chemokine signaling.3 Pn values were two to three times higher in the absence of shear flow or SDF-1a than in the presence of shear flow and SDF-1a (Fig. 7C), suggesting that invasive filopodia formation is important for sensing the nuclei of ECs. In contrast, Pn values of CK-636-treated T cells were comparable to those of DMSOtreated T cells (Fig. S6 in ESI†). Finally, to assess whether sensing the topography of the EC layer and the nuclei of ECs using lamellipodia and filopodia has a prominent impact on the TEM of T cells, the percentage of T cells that underwent TEM among the crawling T cells over a period of 20 min, and the crawling distances of the T cells before they underwent TEM, were measured and results are plotted in Fig. 7D and E, respectively. A significantly higher percentage of DMSO-treated T cells underwent TEM than did inhibitor-treated T cells. More importantly, the DMSO-treated T cells crawled much shorter distances before they underwent TEM than did the inhibitor-treated T cells. Taken together, T cells utilized lamellipodia and filopodia to sense the topography of EC layers and the nuclei of ECs, respectively, to optimize their crawling paths.
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Fig. 7 Roles of lamellipodia and filopodia of T cells in sensing biophysical microenvironments of EC layers. (A) Effect of CK-636 treatment on dx values of crawling T cells on ECs and EC replicas. (B) Effect of ML-141 treatment on Pn values of crawling T cells on ECs. Data are pooled from 5 independent experiments (DMSO: n = 116, ML-141: n = 115). (C) Effect of chemokine (SDF-1a) and shear stresses on Pn values of crawling T cells on ECs. Data are pooled from 5 independent experiments. (control: n = 104; SDF-1a-free: n = 110, shear-free: n = 126) (D) percentage of T cells that underwent TEM among crawling T cells. Data are pooled from 4 independent experiments. (DMSO: n = 176, ML-141: n = 250, CK-636: n = 203 in total). (E) Crawling distances of T cells before they underwent TEM. Data are representative of 5 independent experiments (n = 30 for each condition) (Mann–Whitney U-test, two-tailed, *p o 0.05, **p o 0.01, ***p o 0.001).
Discussion Intraluminal crawling of leukocytes has been considered to be important for tissue infiltration, but guiding cues for crawling have not been completely understood. Among many biophysical factors in blood vessels potentially directing leukocyte crawling, shear flow has been most widely studied up to date. Depending on types of leukocytes and experimental conditions, leukocytes crawled toward8,9 or against4,10,11 the direction of the flow, and even in some cases, leukocytes crawled perpendicular to the direction of the flow.25,26 However, considering complex microenvironments of blood vessels, flow will not be the only regulator for crawling direction. It is likely that leukocytes would sense and integrate various biochemical/biophysical cues to determine crawling directions, similar to other types of cells.27,28 In this study, by independently controlling the flow direction and EC orientation using well aligned EC layers cultured on nanostructured surfaces, we demonstrated that EC orientation predominantly determined crawling direction of T cells. The role of flow in our experimental settings was rather ancillary compared with EC orientation; T cells were either detached or drifted by flow. Detachment by shear flow even further enhanced directionality of crawling to EC orientation because T cells crawling in the direction of EC orientation exhibited better shear resistance than T cells crawling in other directions. We further demonstrated that T cells preferentially crawled along the junctions of ECs while avoiding nuclei of ECs, thus junctions and nuclei of ECs may be two cues directing intraluminal crawling of T cells.
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Neutrophils crawling along the junctions of ECs were previously observed by in vivo intravital imaging,26 but mechanisms underlying such crawling behavior were not elucidated. We hypothesized that topography of ECs could guide the crawling direction of T cells. To directly test this hypothesis, we fabricated EC replicas by replicating monolayers of ECs after fixing and dehydration using PUA-based CFL. Overall, crawling behaviors of T cells on EC replicas were comparable to those of T cells on native ECs. These results may suggest that topography of ECs was a major guiding factor directing crawling of T cells toward the junctions of ECs, but differences between EC replicas and native EC layers should be considered to appropriately interpret data. Of note, topography of EC replicas assessed by quantitative phase microscopy was not exactly identical to that of ECs due to shrinking and flattening of ECs during fixing and dehydration, but overall topography of ECs was still conserved in EC replicas. In addition, EC replicas are significantly stiffer than ECs. Young’s modulus of PUA is 0.3–1.5 GPa29,30 while Young’s modulus of ECs is typically 1–6 KPa,31–33 thus EC replicas are six orders of magnitude stiffer than ECs. We selected PUA for the fabrication of EC replicas because PUA-based CFL allows faithful replication of original features with a sub-100 nm resolution.17,34 Conversely, precise replication of topographical structures using soft materials is technically challenging.17,34 Considering motility and chemotaxis of neutrophils are significantly affected by substrate stiffness,35,36 how substrate stiffness affects topographyguided migration of leukocytes would be an interesting subject for future investigation.
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While EC replicas were flatter and stiffer than native ECs, effects of Arp2/3 complex inhibition on the behaviors of T cells on EC replicas were similar to those of T cells on ECs. Arp2/3 complex-inhibited T cells lacked lamellipodia and the crawling direction was not much influenced by the EC orientation compared with normal T cells. These results mean that lamellipodia formation mediated by Arp2/3 complexes was critical for T cells to crawl along the junctions of ECs, presumably by sensing topography of EC layers. In addition, these results may further confirm that EC replicas might be an appropriate model to assess the role of topography of ECs in the crawling direction of T cells. Recently, it was reported that Arp2/3 complexmediated lamellipodia formation is essential for directed migration via chemotaxis37,38 or haptotaxis,39 but the role of lamellipodia in sensing surface topography to guide cell migration has not been determined. While the detailed mechanisms of topography sensing by lamellipodia need to be determined, F-actin at the leading edges of cells bent by curved surfaces may generate biased protrusions of lamellipodia40 because curvatures in F-actin can bias branching by regulating Arp2/3 complex activity. Actin-rich invasive protrusions formed underneath T cells during intraluminal crawling have been considered to be important for probing sites for TEM,3,41,42 but their exact roles have not been made clear. Herein, we provided evidence that invasive filopodia formation mediated by chemokine signaling and shear flow3,43 was critical for guiding intraluminal crawling by sensing and actively avoiding the nuclei of ECs. Considering inhibition of lamellipodia formation in T cells mostly affected dx values, which represent the global direction of T cells relative to flow or EC orientation, without altering Pn values, which quantify local motion of T cells with respect to nuclei of ECs, while inhibition of filopodia formation in T cells significantly reduced Pn values with minimal effects on dx values, lamellipodia and filopodia appeared to sense biophysical cues in intraluminal spaces independently. The longer the leukocytes crawl in blood vessels, the more likely they are to be detached by disruptive shear flows. Therefore, optimization of the crawling routes would be critical for the successful TEM of leukocytes and their subsequent immune functions in local tissues; however, guiding cues for optimal crawling pathway finding have not been made clear. The TEM of leukocytes can occur either through junctions formed among neighboring ECs, the paracellular route, or through endothelial cell bodies, the transcellular route.1,44,45 Crawling along the local minima of the topographical landscape of EC surfaces, where junctions are typically located and where the cytoplasm of the ECs is very thin, would clearly be beneficial to both transmigration routes. In addition, even transcellular migration occurs through the cytoplasm, not through the nucleus,41 and so avoiding the nuclei of ECs would minimize unnecessary traveling routes. In conclusion, we have shown the unique roles of lamellipodia and filopodia in sensing the biophysical microenvironments of luminal surfaces to optimally guide T cells to explore the most probable places for TEM while efficiently excluding unnecessary places.
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Methods Nanostructured surface fabrication Nanostructured surfaces were fabricated by capillary force lithography (CFA).34 Coverslips of 18 mm diameter (Marienfeld) were coated with an adhesive primer (Minuta Tech., Korea) at 3000 rpm for 15 s and baked at 120 1C for 20 min. Then, 50 ml of poly urethane acrylate (PUA) precursor solution (Minuta Tech., Korea) was overlaid on the primer-coated coverslip and subsequently a PUA mold replicated from its silicon counterpart was placed in conformal contact. The surfaces were exposed to UV for 30 s (l = 250–400 nm, 100 mJ cm 2, Minuta Tech., Korea) and the PUA mold was stripped off, leaving behind the patterned surface consisting of nanoscale ridges/grooves (350 nmridges, 700 nm-grooves and 300 nm-heights, see Fig. 1A). The surfaces of PUA-patterned coverslips were thoroughly rinsed with isopropyl alcohol, dried by nitrogen blowing, and exposed to UV again for 1 h to completely cure residual chemicals. Cell culture PUA substrates were treated with air plasma (200–500 w, Femto Science, Korea) for 1 min and coated with 0.1% gelatin (Sigma) for 30 min at 37 1C. The gelatin-coated substrate was placed in a well of a 12-well plate and 1 ml of 1.5 105 cells per ml bEnd.3 cells (ATTC) suspended in DMEM medium (Invitrogen) containing 10% FBS and 1% penicillin–streptomycin were added to the substrate. In general, bEnd.3 cells formed a confluent monolayer 2 days after seeding. DO11.10 T cell receptor transgenic mice (Jackson Laboratories) were bred in the POSTECH Biotech Center (PBC). All experiments involving mice were approved by the Institutional Animal Care and Use Committee at PBC. DO11.10 blasts were prepared by stimulating cells of the spleen and lymph nodes of DO11.10 transgenic mice with 1 mg ml 1 of OVA323–339 peptide (ISQAVHAAHAEINEAGR, Peptron, Inc. Korea). DO11.10 blasts were grown in RPMI 1640 (Invitrogen) supplemented with 10% FBS (Gibco), 100 U ml 1 penicillin, 100 mg ml 1 streptomycin (Invitrogen), and 0.1% beta-mercaptoethanol (Sigma). On the 2nd day of stimulation, 1–2 U ml 1 of IL-2 was added and cells from the 5th to 7th days were used for the experiments. Replication of EC monolayers Confluent monolayers of bEnd.3 cells cultured either on nanostructured or flat surfaces were fixed with PBS supplemented with 4% paraformaldehyde (Electron Microscopy Sciences) and 2% sucrose (Sigma) for 10 min at 4 1C, washed in PBS and fixed again with 0.1 M cacodylate buffer (Sigma) containing 2.5% glutaraldehyde (Sigma) and 1% sucrose for 10 min at 4 1C. After extensive rinsing with cacodylate buffer, the samples were postfixed with 1% osmium tetroxide in cacodylate buffer for 20 min at room temperature, washed, dehydrated in graded ethanol (30%, 50%, 70%, 80%, 90%, and twice in 99.5% ethanol for 5 min each), and dipped into graded isopentyl acetate (Sigma, at the rate of 1 : 3, 1 : 1, and 3 : 1 with absolute ethanol and pure isopentyl acetate for 10 min each). Then, samples in isopentyl acetate were completely dried using a
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critical point dryer (Hitachi, hcp-2). The PUA precursor solution was applied to the dried samples which were then illuminated with UV light for 30 s through a PET film placed on top of the PUA precursor solution. The PET film attached to the UV-cured PUA, which contained an inverse topographical structure of the EC monolayer, was peeled off, extensively rinsed with isopropyl alcohol and dried by nitrogen blowing. The inverse EC replicas were further exposed to UV for another 1 h to completely cure the chemical residues. Then, the PUA precursor solution was placed onto a primer-coated coverslip, overlaid with the inverse EC replica, and irradiated with UV for 30 s. By peeling off the inverse EC replica, an EC replica of the EC monolayer on the coverslip was obtained. At last, the EC replicas were extensively rinsed with isopropyl alcohol, dried with nitrogen blowing and exposed to UV again for 1 h to cure the resin completely. Fluorescence microscope A modified Zeiss Axio Observer.Z1 epi-fluorescence microscope with 20 (Plan-Neofluar, NA = 0.5) and 40 (Plan-Neofluar, NA = 1.3) objective lenses and a Roper Scientific CoolSnap HQ CCD camera were used for imaging. XBO 75 W/2 Xenon lamp (75 W, Osram) and DAPI (EX. 365, BS 395, EMBP445/50), eGFP (EX BP 470/40, BS 495, EMBP 525/50), Cy3 (EX BP 550/25, BS 570, EMBP 605/70), Cy5 (EX BP 620/60, BS 660, EMBP 770/75) filter sets were used for fluorescence imaging. The microscope was automatically controlled using Axiovision 4.6 (Carl Zeiss); acquired images were analyzed and processed using Methamorph (Universal Imaging, Molecular Devices) or ImageJ. Parallel plate chamber assay A confluent monolayer of bEnd.3 cells was treated with TNF-a (10 ng ml 1) for 4 h, overlaid with SDF-1 (100 ng ml 1, PeproTech) for 10 min, and mounted in a shear chamber (Chamlide CF, Live Cell Instrument, Korea) with channel dimensions of 0.2 mm (height), 2 mm (width) and 17 mm (length). DO11.10 T cells harvested from the culture flasks were resuspended in the binding medium (cation-free Hank’s balanced salt solution containing 10 mM HEPES at pH 7.4 and 2 mg ml 1 of bovine serum albumin supplemented with Ca2+ and Mg2+ at 1 mM each)15 at a concentration of 1.5 106 cells per ml after removing dead cells using Histopaque (Sigma). DO11.10 T cells in the binding medium were perfused over the bEnd.3 cell monolayer using a syringe pump (New Era Pump Systems, US) directly connected to the inlet of the shear chamber. To maintain a constant temperature of 37 1C, inline and stage heaters (Live Cell Instrument, Korea) were used. Initially, DO11.10 blasts in the binding medium were injected at 0.25 dyne cm 2 for 10 min to accumulate DO11.10 blasts on the endothelium. Then, the shear stress was elevated to 2 dyne cm 2 for 20 min by perfusing the binding medium alone. The dynamics of the T cells in the flow chamber was observed using a 20 objective lens by performing time-lapse microscopy at 15 s intervals for 20 min. For pharmacological inhibition, T cells were treated with 20 mM ML-141 (R&D systems), a cdc42 inhibitor, or 100 mM CK-636 (Sigma), an Arp2/3 complex inhibitor, for 20 min prior to the parallel plate flow assays.
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Live cell fluorescence imaging of T cells, nuclei and adherens junctions of ECs Parallel plate chamber assays were performed with DO11.10 T cell blasts labeled with CellTrackert orange CMTMR (1 mM, Invitrogen); DIC and red fluorescence images were sequentially acquired by time-lapse imaging. Then, cells were fixed and stained while they were mounted on the microscope stage. Nuclei and adherens junctions of the bEnd.3 cells were stained with Hoechst 33342 (5 mg ml 1, Invitrogen) and anti-VEcadherin-FITC (clone: eBioBV13, eBioscience), respectively; they were then imaged at the same position as that used for the parallel plate chamber assays, and were overlaid with timelapse fluorescence images of T cells using Methamorph. Quantitative phase microscopy A monolayer of live ECs or an EC replica immersed in DMEM medium was sandwiched between two coverslips with a 0.6 mm thick PDMS spacer. Then, the quantitative phase images of the samples were acquired using quantitative phase microscopy based on off-axis Mach–Zehnder interferometry.19,20 A Diodepumped solid-state (DPSS) laser (l = 532 nm, SambaTM, Cobalt Inc., Sweden) was used as a light source for illumination. A home-built inverted microscope equipped with a 20 objective (0.5 NA, UPLFLN20X, Olympus) was utilized, and the total magnification of the imaging system with additional optics was 16. A scientific CMOS camera (Neo sCMOS, Andor) was used to record interferograms. The phase delay images were then retrieved from the measured interferograms using a phase retrieval algorithm developed previously.46 The height of sample h(x, y) was obtained from the retrieved phase delay images f(x, y) via h(x, y) = [l/(2pDn)]f(x, y) where Dn is the refractive index difference between the sample and medium. As described in previous reports, refractive indices of live cells, EC replicas (PUA), and the medium are 1.38,47,48 1.49,29 and 1.334,49 respectively. Further detailed information about quantitative phase microscopy can be found elsewhere.50,51 Statistical analysis The statistical significance was tested using the Mann–Whitney U-test. For bar graphs, average values with a standard error of mean (s.e.m.) are presented.
Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (JD, Grant No. 2012-004146).
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T cells sense biophysical cues using lamellipodia and filopodia to optimize intraluminal path finding† Kwang Hoon Song,‡a Keon Woo Kwon,‡b Jong-Cheol Choi,a JaeHwang Jung,c YongKeun Park,c Kahp-Yang Suhb and Junsang Doh*ad Intraluminal crawling is considered to be important for extravasation of leukocytes in blood vessels, but biochemical/biophysical cues guiding the crawling of leukocytes have not been clearly understood. Here we provide evidence that T cells sense the topography of luminal surfaces and the nuclei of endothelial cells (ECs) using lamellipodia and filopodia, respectively, to optimize path finding during intraluminal crawling. Well-aligned EC layers or replicas of EC layers, which exhibit topography similar to that of EC layers, were fabricated, and flow was applied either parallel or perpendicular to the orientation of EC alignment. T cells crawled along the valleys of the topographical landscapes of the EC layers, while
Received 2nd February 2014, Accepted 19th February 2014
avoiding nuclei of ECs regardless of flow direction. Pharmacological inhibitor treatments revealed that
DOI: 10.1039/c4ib00021h
Lamellipodia or filopodia-inhibited T cells crawled significantly longer distances for extravasation than
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did normal T cells, indicating that sensing biophysical cues are critical for optimizing routes for extravasation.
sensing of topography and nuclei of EC layers was mediated by lamellipodia and filopodia, respectively.
Introduction Leukocytes in the bloodstream undergo a series of adhesion cascade, rolling, firm adhesion, crawling, and transendothelial migration (TEM) in order to infiltrate into tissues for immune surveillance and immune responses.1,2 While it has been frequently observed that leukocytes crawl a considerable distance from their initial adhesion sites to extravasation sites,3–5 whether intraluminal crawling of leukocytes is guided by certain biochemical or biophysical cues or not has not yet been made clear. Considering that crawling leukocytes are under extreme shear stresses that can detach them during crawling,6
a
Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-Gu, Pohang, Gyeongbuk, 790-784, Korea. E-mail: [email protected]; Fax: +82-54-279-3199; Tel: +82-54-279-2189 b School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-742, Korea c Department of Physics, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea d School of Interdisciplinary Bioscience and Bioengineering (I-Bio), Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-Gu, Pohang, Gyeongbuk, 790-784, Korea † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4ib00021h ‡ These authors contributed equally to the work.
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optimal path finding strategies to reach portals for TEM would help their tissue infiltration by minimizing detachment. Intravital imaging of neutrophil crawling has revealed that neutrophils preferentially crawled along the junction of ECs, indicating that the surface of EC layers may contain guiding cues for leukocyte crawling.7 On the other hand, there have been many reports describing that leukocytes crawl either toward8,9 or against4,10,11 the flow direction depending on the types of leukocytes or on the experimental settings, meaning that flow also plays a certain role. While both EC junctions and flows can affect the crawling direction of leukocytes, experimental settings currently widely used do not allow a systematic examination on which factor has predominant roles in determining the intraluminal crawling direction. In vivo, ECs align along the direction of flow so that the EC junction orientation and flow direction are tightly coupled. On the other hand, in vitro experiments were performed by applying flow to cultured monolayers of ECs with random orientation; thus only the effect of flow direction has been considered. To overcome this limitation, we fabricated pre-aligned EC monolayers by culturing ECs on nanogrooved surfaces as previously described,12 and applied flow in either parallel or perpendicular direction of EC orientation. Using this novel experimental setting, we first demonstrated that the crawling direction of T cells was predominantly affected by EC alignment rather than the flow direction. Furthermore, we showed that
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T cells sense the topography of EC layers by lamellipodia to direct their migration toward the junctions, and also they sense nuclei of ECs by filopodia to avoid nuclei of ECs. Considering TEM of leukocytes typically occurs near EC junctions, never through nuclei, the aforementioned strategies would help T cells to efficiently survey luminal surfaces to find sites for TEM.
Results Experimental settings to decouple flow direction and EC orientation In order to identify potential guiding cues for intraluminal crawling of T cells, we first addressed whether the flow direction or EC orientation had a predominant effect on the direction of T cell crawling. To independently control the flow direction and EC orientation, well-aligned EC layers were prepared by culturing bEnd.3 cells, a murine brain endothelial cell line, on surfaces containing nanoscale ridges/grooves (350 nmridges, 700 nm-grooves and 300 nm-heights, shown in Fig. 1A) as previously described (Fig. 1B);12,13 these layers were inserted into a parallel plate flow chamber either parallel or perpendicular to the flow direction (cases are denoted ‘parallel’ and ‘perpendicular’, respectively, in Fig. 1D). Randomly oriented EC layers cultured on flat surfaces were also prepared for control (Fig. 1C, denoted ‘random’ in Fig. 1D). EC monolayers cultured either on nanostructured or flat surfaces were stimulated with tumor necrosis factor a (TNF-a) for 4 h, overlaid with stromal cell-derived factor-1a (SDF-1a) for 10 min, and mounted in a
Fig. 1 Experimental settings using well-aligned/randomly oriented endothelial cell (EC) layers. (A) Scanning electron microscope (SEM) image of a nanostructured (350 nm/700 nm/300 nm, ridge/groove/depth) surface used to align ECs. (B, C) Differential interference contrast (DIC) images of ECs cultured on a nanostructured (B) or a flat surface (C). Scale bar: 50 mm. (D) Schematic illustration of three different configurations of flow experiments. (E) Schematic illustration of experimental procedures and T cell dynamics on EC layers.
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shear chamber (Chamlide CF, Live Cell Instrument, Korea). TNF-a triggers upregulation of adhesion molecules on ECs,1,14 and SDF-1a bound on the surfaces of ECs induces activation of lymphocyte function-associated antigen 1 (LFA-1) of T cells to support T cells to stably crawl on ECs under shear flow.3 Flow assays were performed as schematically shown in Fig. 1E. Briefly, suspension of DO11.10 T cell blasts in the binding media15 was perfused with low shear stress (0.25 dynes cm 2) on a monolayer of ECs for 10 min to accumulate T cells on the ECs. Then, various shear stresses (0, 2, 10 dynes cm 2) were applied to the adhering T cells on the ECs by perfusing binding media into the chamber; the dynamics of the T cells were observed by time-lapse microscopy for 20 min.12 Effects of EC orientation and flow direction on T cell crawling T cells under shear flow exhibited various behaviors as schematically shown in Fig. 1E. The effects of EC alignment on the frequency of each behavior has been reported in the previous study,12 thus here, we primarily focused on the direction of crawling. Only T cells crawling substantial distances (more than 50 mm, typically B70% of T cells in a field of view) were considered. Representative trajectories of crawling T cells under 2 dyne cm 2 of shear stress are plotted in Fig. 2A for all three different configurations considered. When flow and EC orientation were parallel to each other, it was clear that the crawling of T cells was biased toward the direction of the flow/ EC alignment. In contrast, when flow and EC orientation were perpendicular to each other, it appeared that both the flow and the EC-alignment affected the crawling of T cells. To evaluate whether the flow or the EC-alignment had a predominant effect on the crawling of T cells, we quantitatively analyzed T cell crawling as schematically shown in Fig. 2B. The x-axis is assigned parallel to the flow direction; directionality index dx and time average velocity to the x-axis Vx were defined from the displacements. The measured dx and Vx values for various experimental conditions are plotted in Fig. 2C and D, respectively. In the absence of shear stress, T cells crawled along the direction of the EC alignment: the average dx value of T cells crawling on the well-aligned ECs with ECs oriented toward the x-axis (or ‘parallel’) was B0.6, while the average dx value of T cells crawling on the randomly-oriented ECs (or ‘random’) was B0.5 (Fig. 2C). When flow was applied parallel to the EC alignment, the dx values of the T cells increased, indicating that shear flow and EC orientation synergistically guided the crawling of T cells. When flow was applied to randomly-oriented ECs, the dx values of the T cells slightly increased, meaning that T cell crawling was affected by the flow. However, when flow was applied perpendicular to the EC alignment, the average dx values of the T cells remained significantly smaller than 0.5, or decreased even further at the highest shear stress (10 dynes cm 2), suggesting that the orientation of the EC alignment had a more pronounced effect on the crawling of T cells than did the flow direction. A significant reduction of the average dx values of the T cells at the highest shear stress may mean that T cells crawling along the direction of the EC alignment were not detached by high shear stress as much as
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Fig. 2 Crawling direction of T cells is predominantly determined by the direction of EC orientation. (A) Representative trajectories of crawling T cells under shear flow (2 dyne cm 2) with parallel, random, and perpendicular configurations. (B) Definitions of parameters for T cell crawling analysis. (C, D) Effect of experimental configurations and shear stresses on dx (C) and Vx (D) values of crawling T cells on ECs. (E, F) Crawling distance (E) and duration (F) of T cells in the direction of EC alignment and other directions in perpendicular configuration under 10 dyne cm 2 of shear flow. Data are representative of 5 independent experiments (Mann–Whitney U-test, two-tailed, *p o 0.05, **p o 0.01, ***p o 0.001).
the T cells crawling in other directions were. Indeed, T cells crawling in the direction of EC alignment crawled a significantly longer distance and duration than T cells crawling in other directions prior to detachment (Fig. 2E and F), confirming this possibility. Average Vx values were close to zero for all three configurations in the absence of flow and slightly decreased when shear stress was applied, meaning that T cells crawled in the direction of the flow. Taken together, the intraluminal crawling of T cells under flow was predominantly guided by EC-alignment, and flow played an ancillary role in regulating the crawling direction of T cells. Relationship between adherens junctions/nuclei of EC layers and crawling trajectories of T cells To gain further insight into the guiding mechanisms of intraluminal crawling of T cells on EC surfaces, the trajectories of crawling T cells were overlaid with adherens junctions (visualized by anti-VE-cadherin-FITC) and the nuclei of ECs (visualized by Hoechst, Fig. 3A). Tracks of crawling T cells in all three
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conditions were carefully examined to identify the rules guiding the crawling direction of T cells. Often, T cells that had landed on ECs first approached the junctions of the ECs; once they encountered the junctions, they tended to crawl along the junctions (Fig. 3B and Movie S1 in ESI†), as previously described with neutrophils in vivo.7 In addition, when T cells encountered nuclei of ECs while crawling, the majority of them made detours rather than crossing the nuclei (Fig. 3C and Movie S2 in ESI†). To dissect the mechanisms underneath these intriguing tendencies of T cell crawling, we set hypotheses and performed experiments to evaluate those hypotheses. First, it is possible that uneven distribution of molecules on the EC layers, potentially near the junctions,16 may bias the crawling of T cells. Adhesion molecules such as ICAM-1 and VCAM-1, and chemokines bound to the apical layers of ECs, are known to be important for T cell crawling.3,10 In the absence of SDF-1a, T cells crawled in the direction of EC alignment (Fig. S1 in ESI†), indicating that chemokines bound to ECs may not be a
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Fig. 3 The majority of T cells appear to crawl along the adherens junctions while avoiding the nuclei of ECs. (A) Overlay of T cells (labeled with CMTMR, red), adherens junctions (stained with anti-VE-cadherin-FITC, green) and nuclei of ECs (stained with Hoechst, blue), trajectories of T cells (white) and initial points of trajectories (white dots) (B) Representative time-lapse images of a T cell crawling along adherens junctions. (C) Representative time-lapse images (DIC/Hoechst overlay) of a T cell avoiding crossing nuclei of ECs. Scale bar: 10 mm. Elapse time: mm:ss.
major guiding factor. In addition, we did not notice any accumulation of ICAM-1 or VCAM-1 around the junctions (Fig. S2 in ESI†). Effect of surface topography of EC layers on the direction of T cell crawling On the other hand, T cells may recognize the topographical structures of EC surfaces and prefer to crawl along the valley, and thus they appeared to crawl along the junctions. To directly test this possibility, we replicated the topography of endothelial layers, uniformly coated the replica with key molecules for crawling, and performed flow assays. First, confluent monolayers of ECs cultured on either nanostructured or flat surfaces were fixed and dehydrated (Fig. 4A(i)). Subsequently, the fixed and dehydrated EC layers were replicated using a UV-curable resin poly(urethane acrylate) (PUA) by capillary force lithography, which allows precise replication with sub-100 nm resolution (Fig. 4A(ii)).17 The inverse replicas of the fixed and dehydrated ECs were extensively rinsed with isopropyl alcohol to remove debris of dehydrated cells and dried by nitrogen blowing. Then, by replicating the inverse EC replicas one more time using PUA by CFL (Fig. 4A(iii)), replicas of the original EC monolayers were obtained. Slight shrinkage and deformation during fixing and dehydration would be inevitable,18 but differential interference contrast (DIC) images of monolayers of the live ECs and EC replicas appeared comparable (Fig. 4B). To quantitatively compare the topography of ECs and EC replicas, ECs and EC replicas were imaged using quantitative phase microscopy based on off-axis Mach–Zehnder interferometry.19,20 Pseudo-color images presenting the topography of
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Fig. 4 Fabrication and characterization of EC replicas. (A) Schematic procedure of replica fabrication of confluent EC layers using the UVcurable resin poly(urethane acrylate) (PUA). (B) Representative DIC images of native ECs and EC replicas. (C) Representative pseudo color topography images of confluent EC layers and EC replicas. Scale bar: 100 mm. (D) Height distribution of ECs and EC replicas. Data are representative of 5 independent experiments.
the ECs and EC replicas were generated from the phase images retrieved from interference images (see Methods for details) and displayed in Fig. 4C. Overall topography of ECs was well conserved in EC replicas, but heights of peaks were significantly
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reduced, suggesting that flattening occurred during fixing, dehydration and replication processes. Height distributions of ECs and EC replicas were extracted from the topographical images and shown in Fig. 4D. While the average height of EC replicas (1.5 0.46 mm) was close to that of ECs (1.6 0.92 mm), height distribution of EC replicas was much narrower than that of ECs, suggesting that flattening and shrinking of monolayers of ECs occurred during the replication processes. With these characteristics of EC replicas, then we assessed crawling of T cells on EC replicas. The EC replicas were coated with ICAM-1and SDF-1a, mounted in the shear chamber, and flow assays were performed as described above. T cells crawling on the EC replicas coated with 1 mg ml 1 of ICAM-1, which had a surface density of ICAM-1 similar to that of TNF-a-treated ECs, as shown in Fig. S3A in ESI,† were traced and dx and Vx values of the T cells were calculated and plotted (Fig. 5A and B).
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It was notable that the crawling patterns of T cells on the ICAM1 coated EC replicas were similar to those of T cells on native EC monolayers: the average dx values of the T cells were significantly greater than 0.5 for parallel cases, which is significantly smaller than 0.5 for perpendicular cases, suggesting that the topography of the monolayers of ECs presenting ICAM-1 and SDF-1a was sufficient to guide T cell crawling to the adherens junctions of the ECs. Average Vx values were close to 0 in the absence of shear, and shifted slightly toward negative values when flow was applied, indicating that the T cells passively drifted with the flow rather than actively responding to the flow. However, when we increased the ICAM-1 concentration, the population of T cells crawling against the flow direction significantly increased (Fig. S3B and C in ESI†), as stated in previous studies,4,10 meaning that the ICAM-1 density or the LFA-1 activity may play an important
Fig. 5 Effect of surface topography of EC layers on the direction of T cell crawling. (A, B) Effect of experimental configurations and shear stresses on dx (A) and Vx (B) values of crawling T cells on EC replicas. (C) Definition of probability to cross nuclei of ECs (Pn), and Pn values of T cells on ECs vs. EC replicas. ECs, n = 104; EC replicas, n = 132 in total. Flow experiments were performed in parallel configuration with 2 dyne cm 2 of shear flow. Data are representative of five independent experiments (A and B) or are pooled from 5 independent experiments (C) (Mann–Whitney U-test, two-tailed, *p o 0.05, **p o 0.01, ***p o 0.001).
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role in T cells crawling against the flow. To test whether crawling T cells avoided crossing the nuclei of ECs due to topographical effects, because the nuclei of ECs are typically located around the local maxima of the topographical landscapes formed by EC monolayers, the probability values of crossing the nuclei of ECs (Pn), defined by the ratio of the number of T cells crossing the nuclei of ECs (Nc) to the number of T cells encountering nuclei (Ne), were measured for T cells on ECs and T cells on EC replicas and these values were compared (Fig. 5C). The average Pn value of the T cells on the EC monolayers was about three times lower than that of the T cells on the EC replicas, meaning that topography might not be the major reason for T cells avoiding the nuclei of ECs. Alternatively, nuclei regions of EC replicas were much flatter than those of ECs, as demonstrated in Fig. 4C, so that T cells on EC replicas could cross nuclei regions more easily than T cells on ECs. Roles of lamellipodia and filopodia of T cells in sensing biophysical cues of EC layers To further identify the molecular players involved in recognizing the topography of EC layers and the nuclei of ECs, we inhibited Arp2/3 complexes, actin binding proteins that nucleate branched actin network formation at the leading edge to generate thin sheet-like protrusion lamellipodia, and cdc42, a small GTPase that regulates the formation of needle-shaped filopodia, using pharmacological inhibitors CK-63621 and ML-141,22 respectively. Indeed, it has been suggested that lamellipodia and filopodia play important roles in sensing microenvironments and in determining the direction of cell migration;23,24 however, their role in leukocyte migration in blood vessels has not been investigated. To test the effect of inhibitors on T cell ultrastructures, scanning electron microscope (SEM) images of T cells on ECs, ICAM-1/SDF-1a-coated flat substrates and EC replicas under 2 dyne cm 2 of shear stress were acquired and analyzed (Fig. 6A and B). CK-636-treated cells did not generate thin and wide sheet-structured lamellipodia at the leading edges, resulting in a narrowing of width compared to the DMSO-treated control T cells. On the other hand, ML-141-treated T cells generated a significantly lower number of filopodia than did DMSO-treated T cells. With these clear effects of inhibitors on lamellipodia and filopodia formation of T cells, we then assessed the roles of lamellipodia and filopodia in T cell crawling by performing flow assays with inhibitor-treated T cells. First, the dx values of T cells treated with inhibitors were compared with those of T cells treated with DMSO. Well-aligned ECs or EC replicas of well-aligned ECs were used. CK-636-treated T cells exhibited much sharper leading edges than DMSO-treated T cells, often crossed ridges of EC layers (Fig. S4, Movie S3 and S4 in ESI†), and dx values were close to 0.5 regardless of ‘parallel’ or ‘perpendicular’ configurations (Fig. 7A), indicating that CK-636 treated T cells were no longer guided by the direction of the EC orientation. In contrast, ML-141-treated T cells crawled along the EC orientation, and their dx values were similar to those of the DMSO-treated T cells (Fig. S5 in ESI†).
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Fig. 6 Effects of pharmacological inhibitors on T cell ultrastructures. (A) Inhibitor-treated T cells on flat substrates, ECs and EC replicas under a shear stress of 2 dyne cm 2 were fixed and analyzed by SEM. CK-636 (100 mM) was used to inhibit Arp2/3 complexes and ML-141 (20 mM) was used to inhibit cdc42. Scale bar: 2 mm. (B) Quantitative analysis of SEM images. B10 cells per each condition were analyzed. (Mann–Whitney U-test, two-tailed, **p o 0.01, ***p o 0.001).
In addition, the average Pn value of the ML-141-treated T cells on live ECs was significantly higher than that of the DMSO-treated T cells, meaning that filopodia of the T cells played an important role in sensing the nuclei of the underlying ECs (Fig. 7B). T cells on ECs may form invasive filopodia that push the cytoplasm of ECs down several hundreds of nanometers to physically sense the nuclei, and the formation of invasive filopodia depends on shear stresses and chemokine signaling.3 Pn values were two to three times higher in the absence of shear flow or SDF-1a than in the presence of shear flow and SDF-1a (Fig. 7C), suggesting that invasive filopodia formation is important for sensing the nuclei of ECs. In contrast, Pn values of CK-636-treated T cells were comparable to those of DMSOtreated T cells (Fig. S6 in ESI†). Finally, to assess whether sensing the topography of the EC layer and the nuclei of ECs using lamellipodia and filopodia has a prominent impact on the TEM of T cells, the percentage of T cells that underwent TEM among the crawling T cells over a period of 20 min, and the crawling distances of the T cells before they underwent TEM, were measured and results are plotted in Fig. 7D and E, respectively. A significantly higher percentage of DMSO-treated T cells underwent TEM than did inhibitor-treated T cells. More importantly, the DMSO-treated T cells crawled much shorter distances before they underwent TEM than did the inhibitor-treated T cells. Taken together, T cells utilized lamellipodia and filopodia to sense the topography of EC layers and the nuclei of ECs, respectively, to optimize their crawling paths.
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Fig. 7 Roles of lamellipodia and filopodia of T cells in sensing biophysical microenvironments of EC layers. (A) Effect of CK-636 treatment on dx values of crawling T cells on ECs and EC replicas. (B) Effect of ML-141 treatment on Pn values of crawling T cells on ECs. Data are pooled from 5 independent experiments (DMSO: n = 116, ML-141: n = 115). (C) Effect of chemokine (SDF-1a) and shear stresses on Pn values of crawling T cells on ECs. Data are pooled from 5 independent experiments. (control: n = 104; SDF-1a-free: n = 110, shear-free: n = 126) (D) percentage of T cells that underwent TEM among crawling T cells. Data are pooled from 4 independent experiments. (DMSO: n = 176, ML-141: n = 250, CK-636: n = 203 in total). (E) Crawling distances of T cells before they underwent TEM. Data are representative of 5 independent experiments (n = 30 for each condition) (Mann–Whitney U-test, two-tailed, *p o 0.05, **p o 0.01, ***p o 0.001).
Discussion Intraluminal crawling of leukocytes has been considered to be important for tissue infiltration, but guiding cues for crawling have not been completely understood. Among many biophysical factors in blood vessels potentially directing leukocyte crawling, shear flow has been most widely studied up to date. Depending on types of leukocytes and experimental conditions, leukocytes crawled toward8,9 or against4,10,11 the direction of the flow, and even in some cases, leukocytes crawled perpendicular to the direction of the flow.25,26 However, considering complex microenvironments of blood vessels, flow will not be the only regulator for crawling direction. It is likely that leukocytes would sense and integrate various biochemical/biophysical cues to determine crawling directions, similar to other types of cells.27,28 In this study, by independently controlling the flow direction and EC orientation using well aligned EC layers cultured on nanostructured surfaces, we demonstrated that EC orientation predominantly determined crawling direction of T cells. The role of flow in our experimental settings was rather ancillary compared with EC orientation; T cells were either detached or drifted by flow. Detachment by shear flow even further enhanced directionality of crawling to EC orientation because T cells crawling in the direction of EC orientation exhibited better shear resistance than T cells crawling in other directions. We further demonstrated that T cells preferentially crawled along the junctions of ECs while avoiding nuclei of ECs, thus junctions and nuclei of ECs may be two cues directing intraluminal crawling of T cells.
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Neutrophils crawling along the junctions of ECs were previously observed by in vivo intravital imaging,26 but mechanisms underlying such crawling behavior were not elucidated. We hypothesized that topography of ECs could guide the crawling direction of T cells. To directly test this hypothesis, we fabricated EC replicas by replicating monolayers of ECs after fixing and dehydration using PUA-based CFL. Overall, crawling behaviors of T cells on EC replicas were comparable to those of T cells on native ECs. These results may suggest that topography of ECs was a major guiding factor directing crawling of T cells toward the junctions of ECs, but differences between EC replicas and native EC layers should be considered to appropriately interpret data. Of note, topography of EC replicas assessed by quantitative phase microscopy was not exactly identical to that of ECs due to shrinking and flattening of ECs during fixing and dehydration, but overall topography of ECs was still conserved in EC replicas. In addition, EC replicas are significantly stiffer than ECs. Young’s modulus of PUA is 0.3–1.5 GPa29,30 while Young’s modulus of ECs is typically 1–6 KPa,31–33 thus EC replicas are six orders of magnitude stiffer than ECs. We selected PUA for the fabrication of EC replicas because PUA-based CFL allows faithful replication of original features with a sub-100 nm resolution.17,34 Conversely, precise replication of topographical structures using soft materials is technically challenging.17,34 Considering motility and chemotaxis of neutrophils are significantly affected by substrate stiffness,35,36 how substrate stiffness affects topographyguided migration of leukocytes would be an interesting subject for future investigation.
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While EC replicas were flatter and stiffer than native ECs, effects of Arp2/3 complex inhibition on the behaviors of T cells on EC replicas were similar to those of T cells on ECs. Arp2/3 complex-inhibited T cells lacked lamellipodia and the crawling direction was not much influenced by the EC orientation compared with normal T cells. These results mean that lamellipodia formation mediated by Arp2/3 complexes was critical for T cells to crawl along the junctions of ECs, presumably by sensing topography of EC layers. In addition, these results may further confirm that EC replicas might be an appropriate model to assess the role of topography of ECs in the crawling direction of T cells. Recently, it was reported that Arp2/3 complexmediated lamellipodia formation is essential for directed migration via chemotaxis37,38 or haptotaxis,39 but the role of lamellipodia in sensing surface topography to guide cell migration has not been determined. While the detailed mechanisms of topography sensing by lamellipodia need to be determined, F-actin at the leading edges of cells bent by curved surfaces may generate biased protrusions of lamellipodia40 because curvatures in F-actin can bias branching by regulating Arp2/3 complex activity. Actin-rich invasive protrusions formed underneath T cells during intraluminal crawling have been considered to be important for probing sites for TEM,3,41,42 but their exact roles have not been made clear. Herein, we provided evidence that invasive filopodia formation mediated by chemokine signaling and shear flow3,43 was critical for guiding intraluminal crawling by sensing and actively avoiding the nuclei of ECs. Considering inhibition of lamellipodia formation in T cells mostly affected dx values, which represent the global direction of T cells relative to flow or EC orientation, without altering Pn values, which quantify local motion of T cells with respect to nuclei of ECs, while inhibition of filopodia formation in T cells significantly reduced Pn values with minimal effects on dx values, lamellipodia and filopodia appeared to sense biophysical cues in intraluminal spaces independently. The longer the leukocytes crawl in blood vessels, the more likely they are to be detached by disruptive shear flows. Therefore, optimization of the crawling routes would be critical for the successful TEM of leukocytes and their subsequent immune functions in local tissues; however, guiding cues for optimal crawling pathway finding have not been made clear. The TEM of leukocytes can occur either through junctions formed among neighboring ECs, the paracellular route, or through endothelial cell bodies, the transcellular route.1,44,45 Crawling along the local minima of the topographical landscape of EC surfaces, where junctions are typically located and where the cytoplasm of the ECs is very thin, would clearly be beneficial to both transmigration routes. In addition, even transcellular migration occurs through the cytoplasm, not through the nucleus,41 and so avoiding the nuclei of ECs would minimize unnecessary traveling routes. In conclusion, we have shown the unique roles of lamellipodia and filopodia in sensing the biophysical microenvironments of luminal surfaces to optimally guide T cells to explore the most probable places for TEM while efficiently excluding unnecessary places.
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Methods Nanostructured surface fabrication Nanostructured surfaces were fabricated by capillary force lithography (CFA).34 Coverslips of 18 mm diameter (Marienfeld) were coated with an adhesive primer (Minuta Tech., Korea) at 3000 rpm for 15 s and baked at 120 1C for 20 min. Then, 50 ml of poly urethane acrylate (PUA) precursor solution (Minuta Tech., Korea) was overlaid on the primer-coated coverslip and subsequently a PUA mold replicated from its silicon counterpart was placed in conformal contact. The surfaces were exposed to UV for 30 s (l = 250–400 nm, 100 mJ cm 2, Minuta Tech., Korea) and the PUA mold was stripped off, leaving behind the patterned surface consisting of nanoscale ridges/grooves (350 nmridges, 700 nm-grooves and 300 nm-heights, see Fig. 1A). The surfaces of PUA-patterned coverslips were thoroughly rinsed with isopropyl alcohol, dried by nitrogen blowing, and exposed to UV again for 1 h to completely cure residual chemicals. Cell culture PUA substrates were treated with air plasma (200–500 w, Femto Science, Korea) for 1 min and coated with 0.1% gelatin (Sigma) for 30 min at 37 1C. The gelatin-coated substrate was placed in a well of a 12-well plate and 1 ml of 1.5 105 cells per ml bEnd.3 cells (ATTC) suspended in DMEM medium (Invitrogen) containing 10% FBS and 1% penicillin–streptomycin were added to the substrate. In general, bEnd.3 cells formed a confluent monolayer 2 days after seeding. DO11.10 T cell receptor transgenic mice (Jackson Laboratories) were bred in the POSTECH Biotech Center (PBC). All experiments involving mice were approved by the Institutional Animal Care and Use Committee at PBC. DO11.10 blasts were prepared by stimulating cells of the spleen and lymph nodes of DO11.10 transgenic mice with 1 mg ml 1 of OVA323–339 peptide (ISQAVHAAHAEINEAGR, Peptron, Inc. Korea). DO11.10 blasts were grown in RPMI 1640 (Invitrogen) supplemented with 10% FBS (Gibco), 100 U ml 1 penicillin, 100 mg ml 1 streptomycin (Invitrogen), and 0.1% beta-mercaptoethanol (Sigma). On the 2nd day of stimulation, 1–2 U ml 1 of IL-2 was added and cells from the 5th to 7th days were used for the experiments. Replication of EC monolayers Confluent monolayers of bEnd.3 cells cultured either on nanostructured or flat surfaces were fixed with PBS supplemented with 4% paraformaldehyde (Electron Microscopy Sciences) and 2% sucrose (Sigma) for 10 min at 4 1C, washed in PBS and fixed again with 0.1 M cacodylate buffer (Sigma) containing 2.5% glutaraldehyde (Sigma) and 1% sucrose for 10 min at 4 1C. After extensive rinsing with cacodylate buffer, the samples were postfixed with 1% osmium tetroxide in cacodylate buffer for 20 min at room temperature, washed, dehydrated in graded ethanol (30%, 50%, 70%, 80%, 90%, and twice in 99.5% ethanol for 5 min each), and dipped into graded isopentyl acetate (Sigma, at the rate of 1 : 3, 1 : 1, and 3 : 1 with absolute ethanol and pure isopentyl acetate for 10 min each). Then, samples in isopentyl acetate were completely dried using a
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critical point dryer (Hitachi, hcp-2). The PUA precursor solution was applied to the dried samples which were then illuminated with UV light for 30 s through a PET film placed on top of the PUA precursor solution. The PET film attached to the UV-cured PUA, which contained an inverse topographical structure of the EC monolayer, was peeled off, extensively rinsed with isopropyl alcohol and dried by nitrogen blowing. The inverse EC replicas were further exposed to UV for another 1 h to completely cure the chemical residues. Then, the PUA precursor solution was placed onto a primer-coated coverslip, overlaid with the inverse EC replica, and irradiated with UV for 30 s. By peeling off the inverse EC replica, an EC replica of the EC monolayer on the coverslip was obtained. At last, the EC replicas were extensively rinsed with isopropyl alcohol, dried with nitrogen blowing and exposed to UV again for 1 h to cure the resin completely. Fluorescence microscope A modified Zeiss Axio Observer.Z1 epi-fluorescence microscope with 20 (Plan-Neofluar, NA = 0.5) and 40 (Plan-Neofluar, NA = 1.3) objective lenses and a Roper Scientific CoolSnap HQ CCD camera were used for imaging. XBO 75 W/2 Xenon lamp (75 W, Osram) and DAPI (EX. 365, BS 395, EMBP445/50), eGFP (EX BP 470/40, BS 495, EMBP 525/50), Cy3 (EX BP 550/25, BS 570, EMBP 605/70), Cy5 (EX BP 620/60, BS 660, EMBP 770/75) filter sets were used for fluorescence imaging. The microscope was automatically controlled using Axiovision 4.6 (Carl Zeiss); acquired images were analyzed and processed using Methamorph (Universal Imaging, Molecular Devices) or ImageJ. Parallel plate chamber assay A confluent monolayer of bEnd.3 cells was treated with TNF-a (10 ng ml 1) for 4 h, overlaid with SDF-1 (100 ng ml 1, PeproTech) for 10 min, and mounted in a shear chamber (Chamlide CF, Live Cell Instrument, Korea) with channel dimensions of 0.2 mm (height), 2 mm (width) and 17 mm (length). DO11.10 T cells harvested from the culture flasks were resuspended in the binding medium (cation-free Hank’s balanced salt solution containing 10 mM HEPES at pH 7.4 and 2 mg ml 1 of bovine serum albumin supplemented with Ca2+ and Mg2+ at 1 mM each)15 at a concentration of 1.5 106 cells per ml after removing dead cells using Histopaque (Sigma). DO11.10 T cells in the binding medium were perfused over the bEnd.3 cell monolayer using a syringe pump (New Era Pump Systems, US) directly connected to the inlet of the shear chamber. To maintain a constant temperature of 37 1C, inline and stage heaters (Live Cell Instrument, Korea) were used. Initially, DO11.10 blasts in the binding medium were injected at 0.25 dyne cm 2 for 10 min to accumulate DO11.10 blasts on the endothelium. Then, the shear stress was elevated to 2 dyne cm 2 for 20 min by perfusing the binding medium alone. The dynamics of the T cells in the flow chamber was observed using a 20 objective lens by performing time-lapse microscopy at 15 s intervals for 20 min. For pharmacological inhibition, T cells were treated with 20 mM ML-141 (R&D systems), a cdc42 inhibitor, or 100 mM CK-636 (Sigma), an Arp2/3 complex inhibitor, for 20 min prior to the parallel plate flow assays.
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Live cell fluorescence imaging of T cells, nuclei and adherens junctions of ECs Parallel plate chamber assays were performed with DO11.10 T cell blasts labeled with CellTrackert orange CMTMR (1 mM, Invitrogen); DIC and red fluorescence images were sequentially acquired by time-lapse imaging. Then, cells were fixed and stained while they were mounted on the microscope stage. Nuclei and adherens junctions of the bEnd.3 cells were stained with Hoechst 33342 (5 mg ml 1, Invitrogen) and anti-VEcadherin-FITC (clone: eBioBV13, eBioscience), respectively; they were then imaged at the same position as that used for the parallel plate chamber assays, and were overlaid with timelapse fluorescence images of T cells using Methamorph. Quantitative phase microscopy A monolayer of live ECs or an EC replica immersed in DMEM medium was sandwiched between two coverslips with a 0.6 mm thick PDMS spacer. Then, the quantitative phase images of the samples were acquired using quantitative phase microscopy based on off-axis Mach–Zehnder interferometry.19,20 A Diodepumped solid-state (DPSS) laser (l = 532 nm, SambaTM, Cobalt Inc., Sweden) was used as a light source for illumination. A home-built inverted microscope equipped with a 20 objective (0.5 NA, UPLFLN20X, Olympus) was utilized, and the total magnification of the imaging system with additional optics was 16. A scientific CMOS camera (Neo sCMOS, Andor) was used to record interferograms. The phase delay images were then retrieved from the measured interferograms using a phase retrieval algorithm developed previously.46 The height of sample h(x, y) was obtained from the retrieved phase delay images f(x, y) via h(x, y) = [l/(2pDn)]f(x, y) where Dn is the refractive index difference between the sample and medium. As described in previous reports, refractive indices of live cells, EC replicas (PUA), and the medium are 1.38,47,48 1.49,29 and 1.334,49 respectively. Further detailed information about quantitative phase microscopy can be found elsewhere.50,51 Statistical analysis The statistical significance was tested using the Mann–Whitney U-test. For bar graphs, average values with a standard error of mean (s.e.m.) are presented.
Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (JD, Grant No. 2012-004146).
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Integrative Biology
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