Nanotubes Connect CD4+ T Cells to Airway Smooth Muscle Cells: Novel Mechanism of T Cell Survival Saba Al Heialy, Melissa Zeroual, Soroor Farahnak, Toby McGovern, Paul-André Risse, Mauro Novali, Anne-Marie Lauzon, Horia N. Roman and James G. Martin J Immunol published online 1 May 2015 http://www.jimmunol.org/content/early/2015/05/01/jimmun ol.1401718
Supplementary Material
http://www.jimmunol.org/content/suppl/2015/05/01/jimmunol.140171 8.DCSupplemental.html
Subscriptions
Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions
Permissions Email Alerts
Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc
The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
This information is current as of June 4, 2015.
Published May 1, 2015, doi:10.4049/jimmunol.1401718 The Journal of Immunology
Nanotubes Connect CD4+ T Cells to Airway Smooth Muscle Cells: Novel Mechanism of T Cell Survival Saba Al Heialy, Melissa Zeroual, Soroor Farahnak, Toby McGovern, Paul-Andre´ Risse, Mauro Novali, Anne-Marie Lauzon, Horia N. Roman, and James G. Martin
T
he prevalence of chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and asthma has been increasing worldwide. Profound structural changes occur in the tissues consequent upon chronic inflammation (1). T cells are central to the inflammatory process and have been shown to have trophic properties, affecting tissue repair, and have been suggested to be involved in mediating structural changes (2). Previous studies have shown an intimate relationship between CD4+ T cells and airway smooth muscle (ASM) cells in the tissues (3, 4) and a contact-dependent induction of ASM cell proliferation in vitro. Adherence of T lymphocytes to ASM cells has been shown to be dependent on interactions involving the hyaluronan receptor, CD44, and the integrins ICAM-1 and VCAM-1 (5). Prosurvival mechanisms are most likely involved in these interactions, as a reduction of T cell apoptosis occurred in vitro when T cells were cultured in contact with ASM cells (3). The discovery of thin, actin filament–containing membrane protrusions bridging phaeochromocytoma cells, termed nanotubes, provided a novel mechanism for cell-to-cell communication (6). Subsequently, immune cells such as macrophages, NK cells, and EBV-transformed B cells were described to form a complex netMeakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec H2X 2P2, Canada; and Research Institute, McGill University Health Centre, Montreal, Quebec H2X 2P2, Canada Received for publication July 14, 2014. Accepted for publication April 11, 2015. This work was supported by a Discovery Grant to J.G.M. from the Natural Sciences and Engineering Research Council of Canada. Address correspondence and reprint requests to Prof. James G. Martin, Meakins Christie Laboratories, 1001 Decarie Boulevard, Montreal, QC H4A 3J1, Canada. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: AFM, atomic force microscopy; ASM, airway smooth muscle; DiD, 1,19-dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine, 4-chlorobenzene-sulfonate salt; DiO, N,N-dimethylformamide; PI, propidium iodide; siRNA, small interfering RNA. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401718
work of nanotubes in vitro, often found to extend beyond 140 mm in length. Transfer of functional material via nanotubes was revealed in experiments detailing the transfer of glycophosphatidylinositol conjugated to GFP between EBV-transformed human B cells (7). Nanotube-mediated transfer of bacteria, membrane components, and cytoplasmic components, including the passage of organelles through tubes with diameters as great as 700 nm in macrophages, has been reported (6, 8, 9). Nanotubes have been identified in vivo and, importantly, are implicated in models of immune-mediated injury. Nanotube formation between bone marrow–derived MHC-II+ dendritic cells has been demonstrated in the corneal stroma of mice, and these nanotubes are shown to be more abundant when animals were subjected to injury or treatment with LPS (10). A pathogenic role for nanotubes has been proposed in the transmission of the HIV-1, increasing the efficiency of viral spread between T cells via nanotubes (11) and influenza virus (12). However, studies to date have focused largely on nanotubes connecting immune cells to each other, whereas intercellular communication via nanotubes has been little explored between immune cells and structural cells. We reasoned that contact-dependent mechanisms between T cells and surrounding cells may prolong T cell survival. Although it appears that direct cell-to-cell contact is important for prolonged T cell survival during disease pathogenesis, mechanisms driving these effects are largely unknown. We postulated that structural cells such as ASM cells may favor immune cell survival and that these survival signals may be communicated via nanotubes by the latter cell type. Therefore, the transfer of anti-apoptotic mediators from ASM cells to CD4+ T cells may be an important mechanism mediating survival, expanding the potential functional roles of nanotubes in cell–cell interaction. In this study, we explored communication between CD4+ T cells and ASM cells via nanotube formation extending from CD4+ T cells toward ASM cells. We found that the presence of nanotubes provided a mechanism to account for increased survival of the CD4+ T cell population in contact with ASM cells. We ob-
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
Contact between airway smooth muscle (ASM) cells and activated CD4+ T cells, a key interaction in diseases such as asthma, triggers ASM cell proliferation and enhances T cell survival. We hypothesized that direct contact between ASM and CD4+ T cells facilitated the transfer of anti-apoptotic proteins via nanotubes, resulting in increased survival of activated CD4+ T cells. CD4+ T cells, isolated from PBMCs of healthy subjects, when activated and cocultured with ASM cells for 24 h, formed nanotubes that were visualized by immunofluorescence and atomic force microscopy. Cell-to-cell transfer of the fluorescent dye calcein-AM confirmed cytoplasmic communication via nanotubes. Immunoreactive B cell lymphoma 2 (Bcl-2) and induced myeloid leukemia cell differentiation protein (Mcl-1), two major anti-apoptotic proteins, were present within the nanotubes. Downregulation of Mcl-1 by small interfering RNA in ASM cells significantly increased T cell apoptosis, whereas downregulation of Bcl-2 had no effect. Transfer of GFP-tagged Mcl-1 from ASM cells to CD4+ T cells via the nanotubes confirmed directionality of transfer. In conclusion, activated T cells communicate with ASM cells via nanotube formation. Direct transfer of Mcl-1 from ASM to CD+ T cells via nanotubes is involved in T cell survival. This study provides a novel mechanism of survival of CD4+ T cells that is dependent on interaction with a structural cell. The Journal of Immunology, 2015, 194: 000–000.
2 served that the anti-apoptotic proteins B cell lymphoma-2 (Bcl2) and myeloid leukemia cell differentiation protein (Mcl-1) could be detected within nanotubes connecting CD4+ T cells to ASM cells. Inhibition of Mcl-1 synthesis by ASM cells attenuated the prosurvival effect on CD4+ T cells on coculture of ASM.
Materials and Methods
NANOTUBES ENHANCE T CELL SURVIVAL k = 0.01 N/m were used. In this AFM imaging mode, the tip is brought into stable contact with the sample. The vertical deflection signal from the photodetector is used as feedback to the z-piezo (vertical positioning component of the scanner). In this way, the tip-sample normal force is maintained at a constant value (specified by the set point). Raster scanning with the x- and y-components of the piezo allows for the topographic features of the sample surface (under constant normal force) to be mapped, with the z-piezo control serving as the height signal.
Single-cell intracellular calcium measurement
Peripheral blood was obtained from healthy volunteers, and lower tracheal and proximal bronchial tissues were harvested at the time of lung transplantation. The protocols were approved by institutional review boards, and informed consent was obtained. Approximately 50 ml blood was collected into heparin-coated plastic tubes (Vacutainer; BD Pharmingen, Mississauga, ON, Canada). PBMCs were isolated by Ficoll–Paque separation. Briefly, diluted peripheral blood was pipetted onto Ficoll–Paque (v:v; 3:1) and centrifuged at 1300 rpm for 35 min. Following two washes with PBS, PBMCs were incubated for 24 h to allow the separation of loosely adherent and nonadherent cells. CD4+ T cells were isolated by immunomagnetic negative selection using a CD4+ T cell isolation kit (Miltenyi Biotec, Cambridge, MA). Cells were cultured in RPMI 1640 (Life Technologies, Burlington, ON, Canada) containing 10% FBS (Life Technologies), 1% penicillin/streptomycin/glutamine (Life Technologies), and HEPES (Life Technologies) and activated with 20 ng/ml PMA and 250 nM ionomycin A. The cells were maintained at 37˚C with 5% CO2 for 72 h prior to coculture with ASM cells.
ASM cells were cultured on sterile glass coverslips, disposed in a six-well plate, and grown in 10% FBS-containing medium. Subconfluent cells (∼50% confluency) were starved in 0.1% FBS-containing medium for 48 h. T cells were added for 24 h. To measure calcium signals, cells were incubated in HBSS containing the following in mM: 137 NaCl, 4.2 NaHCO3, 10 glucose, 3 Na2HPO4, 5.4 KCl, 0.4 KH2PO4, 1.3 CaCl2, 0.5 MgCl2, 0.8 MgSO4, and 5 HEPES, at 37˚C with 5 mM ratiometric fluorophore fura 2-AM (Molecular Probes) for 30 min. Coverslips were placed in a Leiden chamber (Medical Systems, Greenville, NY) and placed on an inverted microscope equipped for cell imaging with a 340 oil-immersion objective (Olympus, Tokyo, Japan). Cells were excited at 340 and 380 nm, and the intensity of the emitted fluorescence was measured at 510 nm with image-master software (Photon Technology International, Birmingham, NJ). Histamine (1 mM) was added 20 s after beginning of measurement, and the fluorescence ratio (340/380) was measured in individual cells (13).
ASM isolation and culture
The cells cultured on MatTek dishes (MatTek, Ashland, MA) were imaged using Olympus FluoView FV300 (Olympus America, Melville, NY) CLSM coupled to an Olympus IX71 inverted microscope equipped with a 340 1.4 oil-immersion objective lens. HBSS was used as an imaging medium after washing the cultured dish with PBS twice.
ASM cells were cultured from tracheal and bronchial tissues from healthy donor lungs at transplantation and characterized, as previously described (13). Cells were digested using 0.2% collagenase type IV (Sigma-Aldrich, St. Louis, MO) and 0.05% elastase type IV (Sigma-Aldrich) for 20 min with gentle agitation at 37˚C. Cells were then plated in flasks and cultured in complete DMEM:F12 (Life Technologies) medium supplemented with 10% FBS (Life Technologies), penicillin, streptomycin, and amphotericin B (Life Technologies). Medium was changed every 2–3 d and passaged with 0.25% trypsin when cells reached confluency. Cells used were from passages 1–5 and were cultured on six-well plates at a seeding density of 50,000 cells/well. When cells were 50% confluent, cells were growth arrested in DMEM:F12 containing 0.1% BSA for 48 h.
Immunofluorescent staining and microscopy CD4+ T cells were stained with the lipophilic probe N,N-dimethylformamide (DiO; Molecular Probes, Burlington, ON, Canada). ASM cells were stained with 1,19-dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine, 4-chlorobenzene-sulfonate salt (DiD; Molecular Probes). Following the manufacturer’s protocol, CD4+ T cells and ASM cells were incubated separately for 20 min at 37˚C in DiO and DiD, respectively, and washed three times with PBS for 10 min. One million CD4+ T cells were added to ASM cells in DMEM:RPMI 1640, supplemented with 0.5% FBS, HEPES, and penicillin/streptomycin/glutamine. Following 24-h coculture, cells were fixed with 4% paraformaldehyde for 12 min and images were taken using a fluorescent microscope. CD4+ T cells were also stained with calcein-AM (Life Technologies), a cytoplasmic marker, for the study of transfer of cytoplasmic contents. Briefly, cells were washed with PBS and resuspended in PBS containing 7 mM calcein-AM and incubated at dark at 37˚C with 5% CO2 for 30 min, followed by three washes with RPMI 1640. Calcein-AM was excited at 488 nm with an argon laser. Cells were allowed to recover for an additional 30 min at 37˚C. Cells were then cocultured for a period of 24 h at a ratio of 1:1. Live-cell imaging was performed with an inverted confocal microscope and glycerol 403 objective lens. Cells were kept in a heated chamber at 37˚C and 5% CO2. Cells were fixed following 24 h of coculture and stained for Bcl-2 and Mcl-1 using anti-human Bcl-2 and Mcl-1 (Abcam, Cambridge, MA), at 0.5 and 2 mg/ml, respectively, for 60 min in a humid chamber following permeabilization with 0.25% Triton X-100 for 10 min and blocking with 1% BSA in PBS for 30 min. Goat anti-rabbit IgG Alexa 488 was used for the secondary Ab.
Atomic force microscopy Images were taken in contact mode with a Bioscope atomic force microscopy (AFM) equipped with a G-type scanner, Nanoscope IIIa control electronics, and software version 4.43r8 (Digital Instruments, Veeco Metrology Group, Santa Barbara, CA). Silicon nitride triangular microlevers (Bruker AFM Probes, Camarillo, CA) with a nominal spring constant of
Confocal microscopy
Inhibition of Bcl-2 and Mcl-1 expression AT101 (Tocris, Burlington, ON, Canada), a BH3 mimetic, was used to inhibit the actions of Bcl-2 and Mcl-1 (14). The drug has also been described to downregulate the expression of Bcl-2 and Mcl-1 (15). It was administered to CD4+ T cells at increasing concentrations of 0.1, 1, and 3 mM for 24 h prior to assessment of apoptosis. Bcl-2 and Mcl-1 small interfering RNA (siRNA) were used to selectively inhibit mRNA expression. Lipofectamine (Invitrogen, Burlington, ON, Canada) and siRNA (Santa Cruz, Dallas, TX) were added to ASM cells at a 1:1 ratio for 6 h. Following transfection, medium was changed and cells were incubated for an additional 48 h in DMEM containing 0.1% FBS and 1% PSA. A MTT assay was used to assess cell viability. Briefly, thiazol blue tetrazolium bromide (5 mg/ml) was added to the culture medium 2 h prior to collection of cells. At time of collection, 800 ml DMSO was added to dissolve the compound, and supernatant was collected and read by spectrophotometer to assess viability of cells. Cells were also collected for RNA extraction. mRNA expression of Bcl-2 and Mcl-1 was measured by real-time quantitative RT-PCR. Following 48 h of culture and coculture with CD4+ T cells, ASM cell RNA was extracted using the RNeasy mini kit, according to the manufacturer’s instructions (Qiagen, Valencia, CA). RNA yield was measured using the NanoDrop (Thermo Scientific, Waltham, MA) system, and RNA quality was assessed with an automated electrophoresis system (Bio-Rad, Hercules, CA). cDNA was prepared using 100 mg/ml total RNA, oligo(dT)12–18 primer, dNTP mix, RNaseOUT, and SuperScript II reverse transcriptase (Invitrogen), according to the manufacturer’s instructions. RT-PCR was performed using the LightCycler (Roche) and QuantiTect SYBR Green PCR kit (Qiagen).
CD44, T cell adhesion, and apoptosis A single-beam laser-trap assay was built using the Laser Tweezers Workstation (Cell Robotics, Albuquerque, NM). Following coculture for 24 h on coverslips, a single CD4+ T cell, visualized in bright field by a charge-coupled device camera (XC-75; Sony of America), was captured in the trap, and its position was recorded at a constant velocity of 5 mm/s. T cells and ASM cells were cocultured both in the presence and absence of anti-CD44, a glycoprotein involved in cell adhesion (1 mg/ml; Abcam) for 24 h. To assess the apoptotic effect of inhibiting the CD44-dependent contact between cocultured cells, activated CD4+ T cells (1 3 106 cell/ml) were either cultured alone or cocultured with 70% confluent ASM cells in the presence or absence of 1 mg/ml anti CD44 Ab for 24 h, and the frequency of apoptotic population of CD4+ T cells was determined by flow cytometry with FITC-annexin V and propidium iodide (PI) staining (FITC annexin V
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
CD4 T cell isolation and culture
The Journal of Immunology
3
apoptosis detection kit; BD Pharmingen). Data were acquired by FACSCalibur (BD Biosciences, San Jose, CA). Additionally, apoptosis was assessed on ASM cultured on glass coverslips in the presence of CD4+ T cells using ApopTag peroxidase in situ apoptosis detection kit (Millipore, Etobicoke, ON, Canada), and cells were imaged using light microscopy.
Plasmid transfections ASM cells were transfected with a Mcl-1-GFP plasmid (Origene, Rockville, MD). Briefly, ASM cells were plated in eight-well chambers at 0.5 mg plasmid in lipofectamine 2000 (Invitrogen) at a ratio of 1:2.5 in serum-free and antibiotic-free medium for 6 h. Following transfection, cells were left to recover in normal growth medium for an additional 48 h. CD4+ T cells were cocultured with transfected ASM cells for 24 h. Cells were fixed with 4% paraformaldehyde, and nuclei were stained with Hoechst (Molecular Probes).
Statistical analysis Data are expressed as the mean 6 SE. When the data were not normally distributed, the data were log transformed. Using GraphPad Prism, oneway ANOVA with a Newman–Keuls post hoc test was applied to assess significance. A p value #0.05 was considered statistically significant.
Results CD4+ T cells and ASM are connected via nanotubes CD4+ T cells were isolated from peripheral blood of healthy subjects, and, following isolation, the cells were activated with PMA and ionomycin A. Forty-eight hours later, we noted the formation of long protrusions from CD4+ T cells (Fig. 1A), consistent with descriptions of nanotubes (16). Nanotubes were absent
FIGURE 2. ASM cells and CD4+ T cells (Tc) were stained with lipophilic dyes, DiO (green) and DiD (red), separately and then cocultured for 24 h. Cells were imaged with a fluorescence microscope.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
FIGURE 1. (A) CD4+ T cells were isolated from PBMCs of healthy subjects and activated with PMA (20 ng/ml) and ionomycin A (250 nM) for 72 h. Following activation, CD4+ T cells were visualized by light microscopy. (B) ASM cells alone and in coculture with activated CD4+ T cells for 24 h were cultured on glass coverslips and fixed with 4% paraformaldehyde. Images were taken by atomic force microscopy (see Materials and Methods for further details).
4
Nanotubes allow transfer of material between cells To determine the extent of membrane exchange between cells, we used lipophilic fluorescent dyes. ASM cells were separately stained with DiO (red), and CD4+ T cells with DiD (green). Subsequently, cells were cocultured for 24 h. We observed both DiO and DiD in both cell types, suggesting bidirectional transfer of cell membrane between cell types (Fig. 2). To confirm that CD4+ T cells were transferring cytoplasmic material directly to ASM cells, we loaded CD4+ T cells with calcein-AM, a cytoplasmic marker. Following 24 h of coculture, we observed calcein-AM in ASM cells (Fig. 3C). Nanotubes transmit calcium signals from ASM to T cells We next sought to determine whether histamine-induced calcium release from ASM cells could affect calcium levels in CD4+ T cells connected to ASM by nanotubes. First, we loaded CD4+ T cells with fura 2-AM and applied histamine in a range of concentrations (1024,25,26 M) and found histamine was unable to induce the release of calcium within activated CD4+ T cells alone in our model (data not shown). As expected, ASM cells in monoculture responded to increasing doses of histamine with release of calcium
FIGURE 3. (A and B) Calcium release was observed in ASM cells cultured alone and stimulated with histamine at increasing concentrations; ASM cells were cocultured for 24 h with CD4+ T cells. Individual CD4+ T cells were observed for Ca2+ release (C1-[11C]). (C) Fluorescent image of cocultured dish of ASM cells labeled with DiD and T cells with calcein-AM. Bright cells are T cells, whereas spreading cells are ASM cells.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
in monocultured ASM. Following confirmation that nanotubes were formed, activated CD4+ T cells were cocultured in the presence of ASM cells for 24 h in low-serum medium. AFM revealed the presence of nanotubes running between activated CD4+ T cells and ASM cells (Fig. 1B). When T cells and ASM cells were cocultured, nanotubes formed preferentially between these cell types and fewer T cell to T cell or nanotubes not in contact with other cells were present. The fraction of T cells that had visible nanotubes over total T cells was 0.19 (6 0.01, 1 SD; n = 4 independent experiments and calculated over 20 high power fields). Laser tweezers were used to exert traction forces on CD4+ T cells that were in close proximity to ASM cells. Firmly adherent cells could not be detached from the ASM cells. However, administration of anti-CD44 Ab following the establishment of the coculture cells appeared to weaken the forces of adhesion, and a substantial proportion of the T cells could be pulled to a distance of several cell diameters from the ASM cell. When the laser traction was released, the CD4+ T cell retracted to its original location on the ASM cell (Supplemental Video 1), indicating a residual physical connection between the structural cell and the inflammatory cell.
NANOTUBES ENHANCE T CELL SURVIVAL
The Journal of Immunology from intracellular stores (Fig. 3A). Next, following coculture of CD4+ T cells and ASM cells for 24 h, we observed increased levels of calcium in a proportion of the CD4+ T cells following histamine stimulation (Fig. 3B). The responsiveness of ASM cells to histamine was markedly reduced by coculture with the T cells. In some instances in which CD4+ T cells had high levels of intracellular calcium prior to stimulation, histamine stimulation resulted in a decrease in calcium in these CD4+ T cells. Anti-CD44 Ab increases T cell apoptosis in coculture with ASM cells
FIGURE 4. (A and B) Apoptosis of CD4+ T cells was assessed using Apoptag kit in the absence (c-c) and presence of 1 mg/ml neutralizing Ab to CD44 (c-c + anti-CD44). Pictures were taken to show the presence of nanotubes. (C) Apoptosis of CD4+ T cells following coculture was confirmed using annexin V/PI staining and by flow cytometry in the presence (c-c + anti-CD44) and absence (c-c) of anti-CD44 and in the presence of isotype control (c-c + i.c.). n = 3, *p # 0.05. (D) Apoptosis of CD4+ T cells was assessed using annexin V/PI stain alone or in the presence of anti-CD44 at 1 mg/ml. n = 4, *p # 0.05.
coculture alone and coculture in the presence of the isotype control Ab. Treatment of T cells alone with the same concentration of anti-CD44 (1 mg/ml) did not induce T cell apoptosis (Fig. 4D), although in higher concentration (10 mg/ml), 83.3 6 6.8% (n = 4), was apoptotic at 24 h after Ab treatment. Transfer of anti-apoptotic factors within nanotubes To establish the plausibility that smooth muscle–derived antiapoptotic proteins could participate in T cell survival when in coculture, we assessed whether two major anti-apoptotic proteins, Mcl-1 and Bcl-2, appeared within nanotubes connecting CD4+ T cells to ASM cells (Fig. 5A, 5B). These proteins were visualized by immunofluorescence not only in the cells as expected, but also within the nanotubes. This result suggested that the enhanced T cell survival may be mediated by the transfer of anti-apoptotic material from one cell to another when the T cell may otherwise undergo apoptosis in serum-free medium. We tested next the importance of these anti-apoptotic proteins using a pharmacological approach. AT101, a Bcl-2 family, was administered at increasing concentrations of 0.1, 1, and 3 mM (Fig. 6A). Following AT101 treatment, we observed an increase in T cell apoptosis, as assessed by annexin V/PI stain with increasing concentrations of AT101. In addition, AT101 was successful in downregulating the Bcl-2 and Mcl-1 mRNA expression in CD4+ T cells, as has been previously described in other cells (15). We next evaluated
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
Quantitative analysis of T cell apoptosis was performed using Apoptag staining when cocultured ASM and annexin V/PI were assessed by flow cytometry coculture of ASM cells and CD4+ T cells reduced the percentage of apoptotic CD4+ T cells in a culture medium containing low levels of serum (Fig. 4A–C). Bright field imaging following Apoptag immunostaining was used to assess the presence of nanotubes connecting live CD4+ T cells to ASM cells, and there was a paucity of nanotubes when antiCD44 was administered to cultured cells. Pretreatment of cocultures with an Ab to CD44 resulted in a significantly higher ratio of apoptotic to live T cells. These results were confirmed using annexin V/PI stain and assessed by flow cytometry (Fig. 4C). In the presence of anti-CD44, there was an increase in the number of annexin V–positive and PI-positive cells as compared with the
5
6
NANOTUBES ENHANCE T CELL SURVIVAL Therefore, ASM cells were transfected with Mcl-1-GFP and cocultured with CD4+ T cells for 24 h. Immunofluorescent microscopy revealed the transfer of Mcl-1-GFP toward CD4+ T cells via the nanotubes (Fig. 5C).
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
FIGURE 5. (A and B) Following 24-h coculture of the two types of cells, cells were fixed and stained separately for Bcl-2 and Mcl-1 conjugated to Alexa 488 (green). Immunofluorescent microscopy was used to observe the stain for both proteins. Nuclei were stained with Hoechst (blue). Arrow represents the presence of nanotubes. (C) ASM cells were transfected with GFP-Mcl-1 plasmid. Following transfection, CD4+ T cells were cocultured with transfected ASM cells. After coculture, cells were fixed and imaged for GFP-Mcl-1.
whether Bcl-2 and Mcl-1 expression in ASM cells was directly related to T cell survival using siRNA. Downregulation of Bcl-2 and Mcl-1 had no effect on ASM cell survival per se as assessed by MTT assay (Fig. 6B). However, inhibition of Mcl-1 expression alone in ASM cells increased T cell apoptosis in coculture. Specific inhibition of Bcl-2 expression in ASM cells by siRNA did not alter T cell rates of apoptosis (Fig. 7). Downregulation of both proteins together did not have any further effect. It is also to be noted that Mcl-1 and Bcl-2 downregulation was maintained during the coculture period, as was observed by measuring mRNA expression following coculture (Fig. 6C). Having established that Mcl-1 was involved in T cell survival during coculture with ASM cells, we next wished to verify that Mcl-1 could be transferred from ASM to T cells via nanotubes.
FIGURE 6. (A) Apoptosis of CD4+ T cells was assessed using Apoptag kit. CD4+ T cells were stimulated with AT101 at 0.1, 1, and 3 mM for 24 h in low-serum medium. CD4+ T cells were then coculured with ASM cells following washing of AT101. (B) ASM cell viability was assessed by MTT assay in the presence or absence of siRNA for Mcl-1, Bcl-2, and combination of Bcl-2 and Mcl-1. Control (ctl) siRNA was used to verify the viability of ASM cells. (C) Downregulation of Mcl-1 and Bcl-2 in ASM cells was verified following 24-h coculture with CD4+ T cells. n = 3, ***p # 0.0001.
The Journal of Immunology
7
Discussion In the current study, we identified the formation of cytoplasmic tubular structures that fit the criteria for previously described nanotubes. We postulated that the enhancement of T cell survival when in contact with ASM cells was mediated by the transfer of anti-apoptotic proteins via nanotubes. Using the BH3 mimetic AT101, which inhibits Bcl-2 and Mcl-1, and silencing RNA targeting Bcl-2 and Mcl-1 synthesis by ASM, we demonstrated that Mcl-1, but not Bcl-2, was important for T cell survival. These findings together with the observation of Mcl-1 within the nanotubes support the conclusion that ASM transfers this anti-apoptotic protein to the T cell, affecting its survival. The discovery of nanotubes connecting immune cells has been well documented (7). The presence of these structures between immune and structural cells is less well described, although reports are appearing with increasing frequency indicating that these structures are observed between other cell types, in particular involving mesenchymal stem cells (9, 17–19). In an attempt to elucidate the functional role that nanotubes between T cells and ASM cells serve, we first sought to characterize these structures. Using AFM, we were able to visualize the presence of long protrusions resembling the nanotubes described in previous literature (20). Following confirmation of nanotube formation, we sought to establish a functional role. We hypothesized that these nanotubes
were capable of transferring intracellular material between ASM and T cells. Following 24 h of coculture, we observed bidirectional communication between cells as the flow of fluorescent dyes by immunofluorescence imaging, as previously shown for T cell to T cell communication by nanotubes (11). The flow of cytoplasmic material was confirmed with calcein-AM loaded in CD4+ T cells. Following coculture, calcein-AM was noted also in ASM cells. Moreover, we observed that nanotubes could affect intracellular signaling, as calcium was observed to move from ASM to T cells in response to histamine, whereas T cells alone did not show increases in intracellular calcium on exposure to histamine. The flow of calcium observed could also be bidirectional because some CD4+ T cells exhibited lower concentrations of calcium following histamine stimulation. Taken together, these data suggest the presence of nanotubes connecting T cells to ASM cells with the transfer of material in a bidirectional manner. Recent reports indicate the potential for the movement of organelles such as mitochondria through nanotubes (17, 21). The contact between T cells and ASM cells also appears to have altered the latter cells. The histamine-induced calcium responses were substantially attenuated in the ASM cells in contact with T cells. Activated T cells have been shown to cause ASM cells to proliferate when in coculture. Inhibition of contractile properties may accompany the phenotype change from contractile to secretory/proliferative (22). Previous studies have shown that anti-
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
FIGURE 7. (A) Apoptosis of CD4+ T cells cocultured (c-c) in the presence of ASM cells treated with siRNA for Mcl-1, Bcl-2, and Mcl-1 + Bcl-2; isotype control was assessed by annexin V/PI stain. n = 3, *p # 0.05. (B) Representative dot plots for apoptosis of CD4+ T cells, as assessed by annexin V/PI stain.
8
gether, these results suggest that Mcl-1 may be a critical antiapoptotic protein involved in T cell survival during coculture with ASM cells. It should be noted that Bcl-2–specific staining revealed the presence of Bcl-2 within nanotubes, and therefore it may be of importance and requires further investigations. Moreover, previous studies have shown that Mcl-1 is crucial for the survival of T lymphocytes (32). We wanted to verify that Mcl-1 was in fact transferred from ASM cells to CD4+ T cells and used a GFP-tagged Mcl-1 construct to transfect ASM and detected the fluorophore within the nanotubes. Taken together, these findings suggest that ASM cells and CD4+ T cells communicate via nanotubes. This communication most likely facilitates the transfer of anti-apoptotic material essential for the survival of the cells, but could potentially extend to the transfer of other signals of potential importance in asthma. This finding suggests a novel mechanism for the prolongation of the life of a T cell in contact with structural cells. This study provides insights into the processes by which immune and structural cells communicate. We focused on cells that are in close proximity in airways of asthmatic patients and that contribute to the features of asthma. However, these findings may be extended to many diseases in which different cell types communicate and are found in close proximity. T cells are versatile cells that communicate with many cells such as B cells (33) and dendritic cells (34) for the generation of the immune response. These findings may provide another mechanism of cell-to-cell interaction during the activation of the immune response.
Acknowledgments We gratefully acknowledge the assistance of Dr. Peter Grutter with atomic force microscopy.
Disclosures The authors have no financial conflicts of interest.
References 1. Pare´, P. D., T. R. Bai, and C. R. Roberts. 1997. The structural and functional consequences of chronic allergic inflammation of the airways. Ciba Found. Symp. 206: 71–86, discussion 86–89, 106–110. 2. Koohsari, H., M. Tamaoka, H. R. Campbell, and J. G. Martin. 2007. The role of gamma delta T cells in airway epithelial injury and bronchial responsiveness after chlorine gas exposure in mice. Respir. Res. 8: 21. 3. Ramos-Barbo´n, D., J. F. Presley, Q. A. Hamid, E. D. Fixman, and J. G. Martin. 2005. Antigen-specific CD4+ T cells drive airway smooth muscle remodeling in experimental asthma. J. Clin. Invest. 115: 1580–1589. 4. Ramos-Barbo´n, D., R. Fraga-Iriso, N. S. Brienza, C. Montero-Martı´nez, H. Verea-Hernando, R. Olivenstein, C. Lemiere, P. Ernst, Q. A. Hamid, and J. G. Martin. 2010. T cells localize with proliferating smooth muscle alpha-actin + cell compartments in asthma. Am. J. Respir. Crit. Care Med. 182: 317–324. 5. Lazaar, A. L., S. M. Albelda, J. M. Pilewski, B. Brennan, E. Pure´, and R. A. Panettieri, Jr. 1994. T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis. J. Exp. Med. 180: 807–816. 6. Rustom, A., R. Saffrich, I. Markovic, P. Walther, and H. H. Gerdes. 2004. Nanotubular highways for intercellular organelle transport. Science 303: 1007– 1010. 7. Onfelt, B., S. Nedvetzki, K. Yanagi, and D. M. Davis. 2004. Cutting edge: membrane nanotubes connect immune cells. J. Immunol. 173: 1511–1513. 8. Arkwright, P. D., F. Luchetti, J. Tour, C. Roberts, R. Ayub, A. P. Morales, J. J. Rodrı´guez, A. Gilmore, B. Canonico, S. Papa, and M. D. Esposti. 2010. Fas stimulation of T lymphocytes promotes rapid intercellular exchange of death signals via membrane nanotubes. Cell Res. 20: 72–88. 9. Lou, E., S. Fujisawa, A. Morozov, A. Barlas, Y. Romin, Y. Dogan, S. Gholami, A. L. Moreira, K. Manova-Todorova, and M. A. Moore. 2012. Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma. PLoS One 7: e33093. 10. Chinnery, H. R., E. Pearlman, and P. G. McMenamin. 2008. Cutting edge: membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea. J. Immunol. 180: 5779–5783. 11. Sowinski, S., C. Jolly, O. Berninghausen, M. A. Purbhoo, A. Chauveau, K. Ko¨hler, S. Oddos, P. Eissmann, F. M. Brodsky, C. Hopkins, et al. 2008. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat. Cell Biol. 10: 211–219.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
CD3–activated T cells upregulate a variety of adhesion molecules and adhere to the surface of naive human ASM cells (23). Contractile responsiveness to acetylcholine is increased, whereas b-adrenergic receptor–mediated relaxation is reduced in isolated rabbit ASM tissues. Additionally, coincubation of CD4 T cells with human ASM cells pulsed with staphylococcal enterotoxin A has been reported to lead to the formation of an immunological synapse, upregulating mRNA expression and release of IL-13 (24). The latter cytokine increases intracellular calcium signals in ASM cells (25). The explanation for the discrepancy in the effects of T cells on ASM calcium signals in the current experiments and previous results may lie in the differences in the source of the tissues (rabbit airway versus human cells) or relate to the fact that we have used cultured human cells and not fresh tissue. After the removal of mitogenic stimuli, T cells rapidly undergo cell death, but have been reported to be rescued in coculture with fibroblasts (26). However, the mechanism remains unknown. We found previously that coculture of rat CD4+ T cells and ASM cells enhanced T cell survival (3), findings that were confirmed for human cells in the current study. When evaluating apoptotic T cells, we noted that live CD4+ T cells were connected to ASM cells via nanotubes, suggesting an anti-apoptotic role of nanotubes. This finding was supported by observations of the effects of an Ab targeted to CD44. Blocking CD44 weakens the attachment of T cells to ASM and resulted in an increase in the number of apoptotic CD4+ T cells. It has been suggested that CD44 plays a role in T cell spreading (27). Our results suggest that CD44 may also be involved in the formation of nanotubes and that interference with the adhesion of the cells via CD44 may affect the formation of nanotubes between ASM cells and CD4+ T cells and consequently reduce CD4+ T cell survival. However, such a conclusion remains speculative because we do not know whether CD44 has a role in the formation of nanotubes. Indeed, in the presence of anti-CD44, there was a residual connection of CD4+ T cells to ASM cells, as seen by the return of T cells to their site of adhesion to ASM following their displacement with laser tweezers. Perhaps once the connection between T cell and ASM has been established, neutralizing CD44 does not necessarily lead to the dissolution of the connection. Further experimentation is required to clarify the link between CD44 and nanotube formation. The Bcl-2 family of proteins play a major role in cell viability (28). Using immunofluorescence microscopy, we detected the presence of Bcl-2 and Mcl-1 within the nanotubes. Mcl-1 is a 37.3-kDa protein that belongs to the family of anti-apoptotic Bcl-2 proteins along with Bcl-2 and Bcl-xL (29). Similar to Bcl-2, Mcl-1 can prolong the survival of cells deprived of required growth factors or that have been exposed to cytotoxic agents such as radiation (28, 30). We found that inhibiting these anti-apoptotic proteins in ASM cells with AT101 did not affect ASM cells, but led to an increase in CD4+ T cell apoptosis, as assessed by annexin V/PI staining. This suggests differential sensitivity of ASM and T cells to inhibition of Bcl-2 and Mcl-1. However, AT101 is not specific and inhibits a number of anti-apoptotic proteins simultaneously, and also causes apoptosis by several mechanisms, including the binding to Bcl-2 and Mcl-1 as BH3 mimetics, the downregulation of Bcl-2 and Mcl-1, and the generation of oxidative stress (14, 15, 31). Therefore, we wished to more directly address the roles of Bcl-2 and Mcl-1 synthesis by ASM. To assess the specific role of Mcl-1, we used siRNA to downregulate Mcl-1 in ASM cells and found siRNA targeted against Mcl-1 resulted in a substantial increase in the number of apoptotic CD4+ T cells. In contrast, ASM cells transfected with siRNA targeted against Bcl-2 did not affect the number of apoptotic CD4+ T cells. Taken to-
NANOTUBES ENHANCE T CELL SURVIVAL
The Journal of Immunology
24.
25.
26.
27.
28.
29.
30. 31.
32.
33. 34.
T lymphocytes: role in induction of altered airway responsiveness. J. Immunol. 166: 293–303. Veler, H., A. Hu, S. Fatma, J. S. Grunstein, C. M. DeStephan, D. Campbell, J. S. Orange, and M. M. Grunstein. 2007. Superantigen presentation by airway smooth muscle to CD4+ T lymphocytes elicits reciprocal proasthmatic changes in airway function. J. Immunol. 178: 3627–3636. Deshpande, D. A., S. Dogan, T. F. Walseth, S. M. Miller, Y. Amrani, R. A. Panettieri, and M. S. Kannan. 2004. Modulation of calcium signaling by interleukin-13 in human airway smooth muscle: role of CD38/cyclic adenosine diphosphate ribose pathway. Am. J. Respir. Cell Mol. Biol. 31: 36–42. Gombert, W., N. J. Borthwick, D. L. Wallace, H. Hyde, M. Bofill, D. Pilling, P. C. Beverley, G. Janossy, M. Salmon, and A. N. Akbar. 1996. Fibroblasts prevent apoptosis of IL-2-deprived T cells without inducing proliferation: a selective effect on Bcl-XL expression. Immunology 89: 397–404. Wong, N. K., J. C. Lai, N. Maeshima, and P. Johnson. 2011. CD44-mediated elongated T cell spreading requires Pyk2 activation by Src family kinases, extracellular calcium, phospholipase C and phosphatidylinositol-3 kinase. Cell. Signal. 23: 812–819. Hockenbery, D., G. Nun˜ez, C. Milliman, R. D. Schreiber, and S. J. Korsmeyer. 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348: 334–336. Kozopas, K. M., T. Yang, H. L. Buchan, P. Zhou, and R. W. Craig. 1993. MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc. Natl. Acad. Sci. USA 90: 3516–3520. Miyashita, T., and J. C. Reed. 1993. Bcl-2 oncoprotein blocks chemotherapyinduced apoptosis in a human leukemia cell line. Blood 81: 151–157. Barba-Barajas, M., G. Herna´ndez-Flores, J. M. Lerma-Dı´az, P. C. OrtizLazareno, J. R. Domı´nguez-Rodrı´guez, L. Barba-Barajas, R. de Celis, L. F. JaveSuarez, A. C. Aguilar-Lemarroy, M. G. Guevara-Barraza, and A. Bravo-Cuellar. 2009. Gossypol induced apoptosis of polymorphonuclear leukocytes and monocytes: involvement of mitochondrial pathway and reactive oxygen species. Immunopharmacol. Immunotoxicol. 31: 320–330. Dzhagalov, I., A. Dunkle, and Y. W. He. 2008. The anti-apoptotic Bcl-2 family member Mcl-1 promotes T lymphocyte survival at multiple stages. J. Immunol. 181: 521–528. Singer, A., and R. J. Hodes. 1983. Mechanisms of T cell-B cell interaction. Annu. Rev. Immunol. 1: 211–241. Celli, S., Z. Garcia, H. Beuneu, and P. Bousso. 2008. Decoding the dynamics of T cell-dendritic cell interactions in vivo. Immunol. Rev. 221: 182–187.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
12. Roberts, K. L., B. Manicassamy, and R. A. Lamb. 2015. Influenza A virus uses intercellular connections to spread to neighboring cells. J. Virol. 89: 1537– 1549. 13. Risse, P. A., T. Jo, F. Suarez, N. Hirota, B. Tolloczko, P. Ferraro, P. Grutter, and J. G. Martin. 2011. Interleukin-13 inhibits proliferation and enhances contractility of human airway smooth muscle cells without change in contractile phenotype. Am. J. Physiol. Lung Cell. Mol. Physiol. 300: L958–L966. 14. Balakrishnan, K., W. G. Wierda, M. J. Keating, and V. Gandhi. 2008. Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells. Blood 112: 1971–1980. 15. Paulus, A., K. Chitta, S. Akhtar, D. Personett, K. C. Miller, K. J. Thompson, J. Carr, S. Kumar, V. Roy, S. M. Ansell, et al. 2014. AT-101 downregulates BCL2 and MCL1 and potentiates the cytotoxic effects of lenalidomide and dexamethasone in preclinical models of multiple myeloma and Waldenstro¨m macroglobulinaemia. Br. J. Haematol. 164: 352–365. 16. Rechavi, O., I. Goldstein, and Y. Kloog. 2009. Intercellular exchange of proteins: the immune cell habit of sharing. FEBS Lett. 583: 1792–1799. 17. Li, X., Y. Zhang, S. C. Yeung, Y. Liang, X. Liang, Y. Ding, M. S. Ip, H. F. Tse, J. C. Mak, and Q. Lian. 2014. Mitochondrial transfer of induced pluripotent stem cells-derived MSCs to airway epithelial cells attenuates cigarette smoke-induced damage. Am. J. Respir. Cell Mol. Biol. 51: 455–465. 18. Lehmann, T. P., K. Filipiak, W. Juzwa, P. Sujka-Kordowska, P. P. Jagodzinski, M. Zabel, J. Głowacki, E. Misterska, M. Walczak, and M. Głowacki. 2014. Co‑culture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes. Mol. Med. Rep. 9: 574–582. 19. Tavi, P., T. Korhonen, S. L. Ha¨nninen, J. D. Bruton, S. Lo¨o¨f, A. Simon, and H. Westerblad. 2010. Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes. J. Cell. Physiol. 223: 376–383. 20. Watkins, S. C., and R. D. Salter. 2005. Functional connectivity between immune cells mediated by tunneling nanotubules. Immunity 23: 309–318. 21. Plotnikov, E. Y., T. G. Khryapenkova, A. K. Vasileva, M. V. Marey, S. I. Galkina, N. K. Isaev, E. V. Sheval, V. Y. Polyakov, G. T. Sukhikh, and D. B. Zorov. 2008. Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture. J. Cell. Mol. Med. 12: 1622–1631. 22. Halayko, A. J., H. Salari, X. Ma, and N. L. Stephens. 1996. Markers of airway smooth muscle cell phenotype. Am. J. Physiol. 270: L1040–L1051. 23. Hakonarson, H., C. Kim, R. Whelan, D. Campbell, and M. M. Grunstein. 2001. Bi-directional activation between human airway smooth muscle cells and
9
Supplementary Material
http://www.jimmunol.org/content/suppl/2015/05/01/jimmunol.140171 8.DCSupplemental.html
Subscriptions
Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions
Permissions Email Alerts
Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc
The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
This information is current as of June 4, 2015.
Published May 1, 2015, doi:10.4049/jimmunol.1401718 The Journal of Immunology
Nanotubes Connect CD4+ T Cells to Airway Smooth Muscle Cells: Novel Mechanism of T Cell Survival Saba Al Heialy, Melissa Zeroual, Soroor Farahnak, Toby McGovern, Paul-Andre´ Risse, Mauro Novali, Anne-Marie Lauzon, Horia N. Roman, and James G. Martin
T
he prevalence of chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and asthma has been increasing worldwide. Profound structural changes occur in the tissues consequent upon chronic inflammation (1). T cells are central to the inflammatory process and have been shown to have trophic properties, affecting tissue repair, and have been suggested to be involved in mediating structural changes (2). Previous studies have shown an intimate relationship between CD4+ T cells and airway smooth muscle (ASM) cells in the tissues (3, 4) and a contact-dependent induction of ASM cell proliferation in vitro. Adherence of T lymphocytes to ASM cells has been shown to be dependent on interactions involving the hyaluronan receptor, CD44, and the integrins ICAM-1 and VCAM-1 (5). Prosurvival mechanisms are most likely involved in these interactions, as a reduction of T cell apoptosis occurred in vitro when T cells were cultured in contact with ASM cells (3). The discovery of thin, actin filament–containing membrane protrusions bridging phaeochromocytoma cells, termed nanotubes, provided a novel mechanism for cell-to-cell communication (6). Subsequently, immune cells such as macrophages, NK cells, and EBV-transformed B cells were described to form a complex netMeakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec H2X 2P2, Canada; and Research Institute, McGill University Health Centre, Montreal, Quebec H2X 2P2, Canada Received for publication July 14, 2014. Accepted for publication April 11, 2015. This work was supported by a Discovery Grant to J.G.M. from the Natural Sciences and Engineering Research Council of Canada. Address correspondence and reprint requests to Prof. James G. Martin, Meakins Christie Laboratories, 1001 Decarie Boulevard, Montreal, QC H4A 3J1, Canada. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: AFM, atomic force microscopy; ASM, airway smooth muscle; DiD, 1,19-dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine, 4-chlorobenzene-sulfonate salt; DiO, N,N-dimethylformamide; PI, propidium iodide; siRNA, small interfering RNA. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401718
work of nanotubes in vitro, often found to extend beyond 140 mm in length. Transfer of functional material via nanotubes was revealed in experiments detailing the transfer of glycophosphatidylinositol conjugated to GFP between EBV-transformed human B cells (7). Nanotube-mediated transfer of bacteria, membrane components, and cytoplasmic components, including the passage of organelles through tubes with diameters as great as 700 nm in macrophages, has been reported (6, 8, 9). Nanotubes have been identified in vivo and, importantly, are implicated in models of immune-mediated injury. Nanotube formation between bone marrow–derived MHC-II+ dendritic cells has been demonstrated in the corneal stroma of mice, and these nanotubes are shown to be more abundant when animals were subjected to injury or treatment with LPS (10). A pathogenic role for nanotubes has been proposed in the transmission of the HIV-1, increasing the efficiency of viral spread between T cells via nanotubes (11) and influenza virus (12). However, studies to date have focused largely on nanotubes connecting immune cells to each other, whereas intercellular communication via nanotubes has been little explored between immune cells and structural cells. We reasoned that contact-dependent mechanisms between T cells and surrounding cells may prolong T cell survival. Although it appears that direct cell-to-cell contact is important for prolonged T cell survival during disease pathogenesis, mechanisms driving these effects are largely unknown. We postulated that structural cells such as ASM cells may favor immune cell survival and that these survival signals may be communicated via nanotubes by the latter cell type. Therefore, the transfer of anti-apoptotic mediators from ASM cells to CD4+ T cells may be an important mechanism mediating survival, expanding the potential functional roles of nanotubes in cell–cell interaction. In this study, we explored communication between CD4+ T cells and ASM cells via nanotube formation extending from CD4+ T cells toward ASM cells. We found that the presence of nanotubes provided a mechanism to account for increased survival of the CD4+ T cell population in contact with ASM cells. We ob-
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
Contact between airway smooth muscle (ASM) cells and activated CD4+ T cells, a key interaction in diseases such as asthma, triggers ASM cell proliferation and enhances T cell survival. We hypothesized that direct contact between ASM and CD4+ T cells facilitated the transfer of anti-apoptotic proteins via nanotubes, resulting in increased survival of activated CD4+ T cells. CD4+ T cells, isolated from PBMCs of healthy subjects, when activated and cocultured with ASM cells for 24 h, formed nanotubes that were visualized by immunofluorescence and atomic force microscopy. Cell-to-cell transfer of the fluorescent dye calcein-AM confirmed cytoplasmic communication via nanotubes. Immunoreactive B cell lymphoma 2 (Bcl-2) and induced myeloid leukemia cell differentiation protein (Mcl-1), two major anti-apoptotic proteins, were present within the nanotubes. Downregulation of Mcl-1 by small interfering RNA in ASM cells significantly increased T cell apoptosis, whereas downregulation of Bcl-2 had no effect. Transfer of GFP-tagged Mcl-1 from ASM cells to CD4+ T cells via the nanotubes confirmed directionality of transfer. In conclusion, activated T cells communicate with ASM cells via nanotube formation. Direct transfer of Mcl-1 from ASM to CD+ T cells via nanotubes is involved in T cell survival. This study provides a novel mechanism of survival of CD4+ T cells that is dependent on interaction with a structural cell. The Journal of Immunology, 2015, 194: 000–000.
2 served that the anti-apoptotic proteins B cell lymphoma-2 (Bcl2) and myeloid leukemia cell differentiation protein (Mcl-1) could be detected within nanotubes connecting CD4+ T cells to ASM cells. Inhibition of Mcl-1 synthesis by ASM cells attenuated the prosurvival effect on CD4+ T cells on coculture of ASM.
Materials and Methods
NANOTUBES ENHANCE T CELL SURVIVAL k = 0.01 N/m were used. In this AFM imaging mode, the tip is brought into stable contact with the sample. The vertical deflection signal from the photodetector is used as feedback to the z-piezo (vertical positioning component of the scanner). In this way, the tip-sample normal force is maintained at a constant value (specified by the set point). Raster scanning with the x- and y-components of the piezo allows for the topographic features of the sample surface (under constant normal force) to be mapped, with the z-piezo control serving as the height signal.
Single-cell intracellular calcium measurement
Peripheral blood was obtained from healthy volunteers, and lower tracheal and proximal bronchial tissues were harvested at the time of lung transplantation. The protocols were approved by institutional review boards, and informed consent was obtained. Approximately 50 ml blood was collected into heparin-coated plastic tubes (Vacutainer; BD Pharmingen, Mississauga, ON, Canada). PBMCs were isolated by Ficoll–Paque separation. Briefly, diluted peripheral blood was pipetted onto Ficoll–Paque (v:v; 3:1) and centrifuged at 1300 rpm for 35 min. Following two washes with PBS, PBMCs were incubated for 24 h to allow the separation of loosely adherent and nonadherent cells. CD4+ T cells were isolated by immunomagnetic negative selection using a CD4+ T cell isolation kit (Miltenyi Biotec, Cambridge, MA). Cells were cultured in RPMI 1640 (Life Technologies, Burlington, ON, Canada) containing 10% FBS (Life Technologies), 1% penicillin/streptomycin/glutamine (Life Technologies), and HEPES (Life Technologies) and activated with 20 ng/ml PMA and 250 nM ionomycin A. The cells were maintained at 37˚C with 5% CO2 for 72 h prior to coculture with ASM cells.
ASM cells were cultured on sterile glass coverslips, disposed in a six-well plate, and grown in 10% FBS-containing medium. Subconfluent cells (∼50% confluency) were starved in 0.1% FBS-containing medium for 48 h. T cells were added for 24 h. To measure calcium signals, cells were incubated in HBSS containing the following in mM: 137 NaCl, 4.2 NaHCO3, 10 glucose, 3 Na2HPO4, 5.4 KCl, 0.4 KH2PO4, 1.3 CaCl2, 0.5 MgCl2, 0.8 MgSO4, and 5 HEPES, at 37˚C with 5 mM ratiometric fluorophore fura 2-AM (Molecular Probes) for 30 min. Coverslips were placed in a Leiden chamber (Medical Systems, Greenville, NY) and placed on an inverted microscope equipped for cell imaging with a 340 oil-immersion objective (Olympus, Tokyo, Japan). Cells were excited at 340 and 380 nm, and the intensity of the emitted fluorescence was measured at 510 nm with image-master software (Photon Technology International, Birmingham, NJ). Histamine (1 mM) was added 20 s after beginning of measurement, and the fluorescence ratio (340/380) was measured in individual cells (13).
ASM isolation and culture
The cells cultured on MatTek dishes (MatTek, Ashland, MA) were imaged using Olympus FluoView FV300 (Olympus America, Melville, NY) CLSM coupled to an Olympus IX71 inverted microscope equipped with a 340 1.4 oil-immersion objective lens. HBSS was used as an imaging medium after washing the cultured dish with PBS twice.
ASM cells were cultured from tracheal and bronchial tissues from healthy donor lungs at transplantation and characterized, as previously described (13). Cells were digested using 0.2% collagenase type IV (Sigma-Aldrich, St. Louis, MO) and 0.05% elastase type IV (Sigma-Aldrich) for 20 min with gentle agitation at 37˚C. Cells were then plated in flasks and cultured in complete DMEM:F12 (Life Technologies) medium supplemented with 10% FBS (Life Technologies), penicillin, streptomycin, and amphotericin B (Life Technologies). Medium was changed every 2–3 d and passaged with 0.25% trypsin when cells reached confluency. Cells used were from passages 1–5 and were cultured on six-well plates at a seeding density of 50,000 cells/well. When cells were 50% confluent, cells were growth arrested in DMEM:F12 containing 0.1% BSA for 48 h.
Immunofluorescent staining and microscopy CD4+ T cells were stained with the lipophilic probe N,N-dimethylformamide (DiO; Molecular Probes, Burlington, ON, Canada). ASM cells were stained with 1,19-dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine, 4-chlorobenzene-sulfonate salt (DiD; Molecular Probes). Following the manufacturer’s protocol, CD4+ T cells and ASM cells were incubated separately for 20 min at 37˚C in DiO and DiD, respectively, and washed three times with PBS for 10 min. One million CD4+ T cells were added to ASM cells in DMEM:RPMI 1640, supplemented with 0.5% FBS, HEPES, and penicillin/streptomycin/glutamine. Following 24-h coculture, cells were fixed with 4% paraformaldehyde for 12 min and images were taken using a fluorescent microscope. CD4+ T cells were also stained with calcein-AM (Life Technologies), a cytoplasmic marker, for the study of transfer of cytoplasmic contents. Briefly, cells were washed with PBS and resuspended in PBS containing 7 mM calcein-AM and incubated at dark at 37˚C with 5% CO2 for 30 min, followed by three washes with RPMI 1640. Calcein-AM was excited at 488 nm with an argon laser. Cells were allowed to recover for an additional 30 min at 37˚C. Cells were then cocultured for a period of 24 h at a ratio of 1:1. Live-cell imaging was performed with an inverted confocal microscope and glycerol 403 objective lens. Cells were kept in a heated chamber at 37˚C and 5% CO2. Cells were fixed following 24 h of coculture and stained for Bcl-2 and Mcl-1 using anti-human Bcl-2 and Mcl-1 (Abcam, Cambridge, MA), at 0.5 and 2 mg/ml, respectively, for 60 min in a humid chamber following permeabilization with 0.25% Triton X-100 for 10 min and blocking with 1% BSA in PBS for 30 min. Goat anti-rabbit IgG Alexa 488 was used for the secondary Ab.
Atomic force microscopy Images were taken in contact mode with a Bioscope atomic force microscopy (AFM) equipped with a G-type scanner, Nanoscope IIIa control electronics, and software version 4.43r8 (Digital Instruments, Veeco Metrology Group, Santa Barbara, CA). Silicon nitride triangular microlevers (Bruker AFM Probes, Camarillo, CA) with a nominal spring constant of
Confocal microscopy
Inhibition of Bcl-2 and Mcl-1 expression AT101 (Tocris, Burlington, ON, Canada), a BH3 mimetic, was used to inhibit the actions of Bcl-2 and Mcl-1 (14). The drug has also been described to downregulate the expression of Bcl-2 and Mcl-1 (15). It was administered to CD4+ T cells at increasing concentrations of 0.1, 1, and 3 mM for 24 h prior to assessment of apoptosis. Bcl-2 and Mcl-1 small interfering RNA (siRNA) were used to selectively inhibit mRNA expression. Lipofectamine (Invitrogen, Burlington, ON, Canada) and siRNA (Santa Cruz, Dallas, TX) were added to ASM cells at a 1:1 ratio for 6 h. Following transfection, medium was changed and cells were incubated for an additional 48 h in DMEM containing 0.1% FBS and 1% PSA. A MTT assay was used to assess cell viability. Briefly, thiazol blue tetrazolium bromide (5 mg/ml) was added to the culture medium 2 h prior to collection of cells. At time of collection, 800 ml DMSO was added to dissolve the compound, and supernatant was collected and read by spectrophotometer to assess viability of cells. Cells were also collected for RNA extraction. mRNA expression of Bcl-2 and Mcl-1 was measured by real-time quantitative RT-PCR. Following 48 h of culture and coculture with CD4+ T cells, ASM cell RNA was extracted using the RNeasy mini kit, according to the manufacturer’s instructions (Qiagen, Valencia, CA). RNA yield was measured using the NanoDrop (Thermo Scientific, Waltham, MA) system, and RNA quality was assessed with an automated electrophoresis system (Bio-Rad, Hercules, CA). cDNA was prepared using 100 mg/ml total RNA, oligo(dT)12–18 primer, dNTP mix, RNaseOUT, and SuperScript II reverse transcriptase (Invitrogen), according to the manufacturer’s instructions. RT-PCR was performed using the LightCycler (Roche) and QuantiTect SYBR Green PCR kit (Qiagen).
CD44, T cell adhesion, and apoptosis A single-beam laser-trap assay was built using the Laser Tweezers Workstation (Cell Robotics, Albuquerque, NM). Following coculture for 24 h on coverslips, a single CD4+ T cell, visualized in bright field by a charge-coupled device camera (XC-75; Sony of America), was captured in the trap, and its position was recorded at a constant velocity of 5 mm/s. T cells and ASM cells were cocultured both in the presence and absence of anti-CD44, a glycoprotein involved in cell adhesion (1 mg/ml; Abcam) for 24 h. To assess the apoptotic effect of inhibiting the CD44-dependent contact between cocultured cells, activated CD4+ T cells (1 3 106 cell/ml) were either cultured alone or cocultured with 70% confluent ASM cells in the presence or absence of 1 mg/ml anti CD44 Ab for 24 h, and the frequency of apoptotic population of CD4+ T cells was determined by flow cytometry with FITC-annexin V and propidium iodide (PI) staining (FITC annexin V
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
CD4 T cell isolation and culture
The Journal of Immunology
3
apoptosis detection kit; BD Pharmingen). Data were acquired by FACSCalibur (BD Biosciences, San Jose, CA). Additionally, apoptosis was assessed on ASM cultured on glass coverslips in the presence of CD4+ T cells using ApopTag peroxidase in situ apoptosis detection kit (Millipore, Etobicoke, ON, Canada), and cells were imaged using light microscopy.
Plasmid transfections ASM cells were transfected with a Mcl-1-GFP plasmid (Origene, Rockville, MD). Briefly, ASM cells were plated in eight-well chambers at 0.5 mg plasmid in lipofectamine 2000 (Invitrogen) at a ratio of 1:2.5 in serum-free and antibiotic-free medium for 6 h. Following transfection, cells were left to recover in normal growth medium for an additional 48 h. CD4+ T cells were cocultured with transfected ASM cells for 24 h. Cells were fixed with 4% paraformaldehyde, and nuclei were stained with Hoechst (Molecular Probes).
Statistical analysis Data are expressed as the mean 6 SE. When the data were not normally distributed, the data were log transformed. Using GraphPad Prism, oneway ANOVA with a Newman–Keuls post hoc test was applied to assess significance. A p value #0.05 was considered statistically significant.
Results CD4+ T cells and ASM are connected via nanotubes CD4+ T cells were isolated from peripheral blood of healthy subjects, and, following isolation, the cells were activated with PMA and ionomycin A. Forty-eight hours later, we noted the formation of long protrusions from CD4+ T cells (Fig. 1A), consistent with descriptions of nanotubes (16). Nanotubes were absent
FIGURE 2. ASM cells and CD4+ T cells (Tc) were stained with lipophilic dyes, DiO (green) and DiD (red), separately and then cocultured for 24 h. Cells were imaged with a fluorescence microscope.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
FIGURE 1. (A) CD4+ T cells were isolated from PBMCs of healthy subjects and activated with PMA (20 ng/ml) and ionomycin A (250 nM) for 72 h. Following activation, CD4+ T cells were visualized by light microscopy. (B) ASM cells alone and in coculture with activated CD4+ T cells for 24 h were cultured on glass coverslips and fixed with 4% paraformaldehyde. Images were taken by atomic force microscopy (see Materials and Methods for further details).
4
Nanotubes allow transfer of material between cells To determine the extent of membrane exchange between cells, we used lipophilic fluorescent dyes. ASM cells were separately stained with DiO (red), and CD4+ T cells with DiD (green). Subsequently, cells were cocultured for 24 h. We observed both DiO and DiD in both cell types, suggesting bidirectional transfer of cell membrane between cell types (Fig. 2). To confirm that CD4+ T cells were transferring cytoplasmic material directly to ASM cells, we loaded CD4+ T cells with calcein-AM, a cytoplasmic marker. Following 24 h of coculture, we observed calcein-AM in ASM cells (Fig. 3C). Nanotubes transmit calcium signals from ASM to T cells We next sought to determine whether histamine-induced calcium release from ASM cells could affect calcium levels in CD4+ T cells connected to ASM by nanotubes. First, we loaded CD4+ T cells with fura 2-AM and applied histamine in a range of concentrations (1024,25,26 M) and found histamine was unable to induce the release of calcium within activated CD4+ T cells alone in our model (data not shown). As expected, ASM cells in monoculture responded to increasing doses of histamine with release of calcium
FIGURE 3. (A and B) Calcium release was observed in ASM cells cultured alone and stimulated with histamine at increasing concentrations; ASM cells were cocultured for 24 h with CD4+ T cells. Individual CD4+ T cells were observed for Ca2+ release (C1-[11C]). (C) Fluorescent image of cocultured dish of ASM cells labeled with DiD and T cells with calcein-AM. Bright cells are T cells, whereas spreading cells are ASM cells.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
in monocultured ASM. Following confirmation that nanotubes were formed, activated CD4+ T cells were cocultured in the presence of ASM cells for 24 h in low-serum medium. AFM revealed the presence of nanotubes running between activated CD4+ T cells and ASM cells (Fig. 1B). When T cells and ASM cells were cocultured, nanotubes formed preferentially between these cell types and fewer T cell to T cell or nanotubes not in contact with other cells were present. The fraction of T cells that had visible nanotubes over total T cells was 0.19 (6 0.01, 1 SD; n = 4 independent experiments and calculated over 20 high power fields). Laser tweezers were used to exert traction forces on CD4+ T cells that were in close proximity to ASM cells. Firmly adherent cells could not be detached from the ASM cells. However, administration of anti-CD44 Ab following the establishment of the coculture cells appeared to weaken the forces of adhesion, and a substantial proportion of the T cells could be pulled to a distance of several cell diameters from the ASM cell. When the laser traction was released, the CD4+ T cell retracted to its original location on the ASM cell (Supplemental Video 1), indicating a residual physical connection between the structural cell and the inflammatory cell.
NANOTUBES ENHANCE T CELL SURVIVAL
The Journal of Immunology from intracellular stores (Fig. 3A). Next, following coculture of CD4+ T cells and ASM cells for 24 h, we observed increased levels of calcium in a proportion of the CD4+ T cells following histamine stimulation (Fig. 3B). The responsiveness of ASM cells to histamine was markedly reduced by coculture with the T cells. In some instances in which CD4+ T cells had high levels of intracellular calcium prior to stimulation, histamine stimulation resulted in a decrease in calcium in these CD4+ T cells. Anti-CD44 Ab increases T cell apoptosis in coculture with ASM cells
FIGURE 4. (A and B) Apoptosis of CD4+ T cells was assessed using Apoptag kit in the absence (c-c) and presence of 1 mg/ml neutralizing Ab to CD44 (c-c + anti-CD44). Pictures were taken to show the presence of nanotubes. (C) Apoptosis of CD4+ T cells following coculture was confirmed using annexin V/PI staining and by flow cytometry in the presence (c-c + anti-CD44) and absence (c-c) of anti-CD44 and in the presence of isotype control (c-c + i.c.). n = 3, *p # 0.05. (D) Apoptosis of CD4+ T cells was assessed using annexin V/PI stain alone or in the presence of anti-CD44 at 1 mg/ml. n = 4, *p # 0.05.
coculture alone and coculture in the presence of the isotype control Ab. Treatment of T cells alone with the same concentration of anti-CD44 (1 mg/ml) did not induce T cell apoptosis (Fig. 4D), although in higher concentration (10 mg/ml), 83.3 6 6.8% (n = 4), was apoptotic at 24 h after Ab treatment. Transfer of anti-apoptotic factors within nanotubes To establish the plausibility that smooth muscle–derived antiapoptotic proteins could participate in T cell survival when in coculture, we assessed whether two major anti-apoptotic proteins, Mcl-1 and Bcl-2, appeared within nanotubes connecting CD4+ T cells to ASM cells (Fig. 5A, 5B). These proteins were visualized by immunofluorescence not only in the cells as expected, but also within the nanotubes. This result suggested that the enhanced T cell survival may be mediated by the transfer of anti-apoptotic material from one cell to another when the T cell may otherwise undergo apoptosis in serum-free medium. We tested next the importance of these anti-apoptotic proteins using a pharmacological approach. AT101, a Bcl-2 family, was administered at increasing concentrations of 0.1, 1, and 3 mM (Fig. 6A). Following AT101 treatment, we observed an increase in T cell apoptosis, as assessed by annexin V/PI stain with increasing concentrations of AT101. In addition, AT101 was successful in downregulating the Bcl-2 and Mcl-1 mRNA expression in CD4+ T cells, as has been previously described in other cells (15). We next evaluated
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
Quantitative analysis of T cell apoptosis was performed using Apoptag staining when cocultured ASM and annexin V/PI were assessed by flow cytometry coculture of ASM cells and CD4+ T cells reduced the percentage of apoptotic CD4+ T cells in a culture medium containing low levels of serum (Fig. 4A–C). Bright field imaging following Apoptag immunostaining was used to assess the presence of nanotubes connecting live CD4+ T cells to ASM cells, and there was a paucity of nanotubes when antiCD44 was administered to cultured cells. Pretreatment of cocultures with an Ab to CD44 resulted in a significantly higher ratio of apoptotic to live T cells. These results were confirmed using annexin V/PI stain and assessed by flow cytometry (Fig. 4C). In the presence of anti-CD44, there was an increase in the number of annexin V–positive and PI-positive cells as compared with the
5
6
NANOTUBES ENHANCE T CELL SURVIVAL Therefore, ASM cells were transfected with Mcl-1-GFP and cocultured with CD4+ T cells for 24 h. Immunofluorescent microscopy revealed the transfer of Mcl-1-GFP toward CD4+ T cells via the nanotubes (Fig. 5C).
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
FIGURE 5. (A and B) Following 24-h coculture of the two types of cells, cells were fixed and stained separately for Bcl-2 and Mcl-1 conjugated to Alexa 488 (green). Immunofluorescent microscopy was used to observe the stain for both proteins. Nuclei were stained with Hoechst (blue). Arrow represents the presence of nanotubes. (C) ASM cells were transfected with GFP-Mcl-1 plasmid. Following transfection, CD4+ T cells were cocultured with transfected ASM cells. After coculture, cells were fixed and imaged for GFP-Mcl-1.
whether Bcl-2 and Mcl-1 expression in ASM cells was directly related to T cell survival using siRNA. Downregulation of Bcl-2 and Mcl-1 had no effect on ASM cell survival per se as assessed by MTT assay (Fig. 6B). However, inhibition of Mcl-1 expression alone in ASM cells increased T cell apoptosis in coculture. Specific inhibition of Bcl-2 expression in ASM cells by siRNA did not alter T cell rates of apoptosis (Fig. 7). Downregulation of both proteins together did not have any further effect. It is also to be noted that Mcl-1 and Bcl-2 downregulation was maintained during the coculture period, as was observed by measuring mRNA expression following coculture (Fig. 6C). Having established that Mcl-1 was involved in T cell survival during coculture with ASM cells, we next wished to verify that Mcl-1 could be transferred from ASM to T cells via nanotubes.
FIGURE 6. (A) Apoptosis of CD4+ T cells was assessed using Apoptag kit. CD4+ T cells were stimulated with AT101 at 0.1, 1, and 3 mM for 24 h in low-serum medium. CD4+ T cells were then coculured with ASM cells following washing of AT101. (B) ASM cell viability was assessed by MTT assay in the presence or absence of siRNA for Mcl-1, Bcl-2, and combination of Bcl-2 and Mcl-1. Control (ctl) siRNA was used to verify the viability of ASM cells. (C) Downregulation of Mcl-1 and Bcl-2 in ASM cells was verified following 24-h coculture with CD4+ T cells. n = 3, ***p # 0.0001.
The Journal of Immunology
7
Discussion In the current study, we identified the formation of cytoplasmic tubular structures that fit the criteria for previously described nanotubes. We postulated that the enhancement of T cell survival when in contact with ASM cells was mediated by the transfer of anti-apoptotic proteins via nanotubes. Using the BH3 mimetic AT101, which inhibits Bcl-2 and Mcl-1, and silencing RNA targeting Bcl-2 and Mcl-1 synthesis by ASM, we demonstrated that Mcl-1, but not Bcl-2, was important for T cell survival. These findings together with the observation of Mcl-1 within the nanotubes support the conclusion that ASM transfers this anti-apoptotic protein to the T cell, affecting its survival. The discovery of nanotubes connecting immune cells has been well documented (7). The presence of these structures between immune and structural cells is less well described, although reports are appearing with increasing frequency indicating that these structures are observed between other cell types, in particular involving mesenchymal stem cells (9, 17–19). In an attempt to elucidate the functional role that nanotubes between T cells and ASM cells serve, we first sought to characterize these structures. Using AFM, we were able to visualize the presence of long protrusions resembling the nanotubes described in previous literature (20). Following confirmation of nanotube formation, we sought to establish a functional role. We hypothesized that these nanotubes
were capable of transferring intracellular material between ASM and T cells. Following 24 h of coculture, we observed bidirectional communication between cells as the flow of fluorescent dyes by immunofluorescence imaging, as previously shown for T cell to T cell communication by nanotubes (11). The flow of cytoplasmic material was confirmed with calcein-AM loaded in CD4+ T cells. Following coculture, calcein-AM was noted also in ASM cells. Moreover, we observed that nanotubes could affect intracellular signaling, as calcium was observed to move from ASM to T cells in response to histamine, whereas T cells alone did not show increases in intracellular calcium on exposure to histamine. The flow of calcium observed could also be bidirectional because some CD4+ T cells exhibited lower concentrations of calcium following histamine stimulation. Taken together, these data suggest the presence of nanotubes connecting T cells to ASM cells with the transfer of material in a bidirectional manner. Recent reports indicate the potential for the movement of organelles such as mitochondria through nanotubes (17, 21). The contact between T cells and ASM cells also appears to have altered the latter cells. The histamine-induced calcium responses were substantially attenuated in the ASM cells in contact with T cells. Activated T cells have been shown to cause ASM cells to proliferate when in coculture. Inhibition of contractile properties may accompany the phenotype change from contractile to secretory/proliferative (22). Previous studies have shown that anti-
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
FIGURE 7. (A) Apoptosis of CD4+ T cells cocultured (c-c) in the presence of ASM cells treated with siRNA for Mcl-1, Bcl-2, and Mcl-1 + Bcl-2; isotype control was assessed by annexin V/PI stain. n = 3, *p # 0.05. (B) Representative dot plots for apoptosis of CD4+ T cells, as assessed by annexin V/PI stain.
8
gether, these results suggest that Mcl-1 may be a critical antiapoptotic protein involved in T cell survival during coculture with ASM cells. It should be noted that Bcl-2–specific staining revealed the presence of Bcl-2 within nanotubes, and therefore it may be of importance and requires further investigations. Moreover, previous studies have shown that Mcl-1 is crucial for the survival of T lymphocytes (32). We wanted to verify that Mcl-1 was in fact transferred from ASM cells to CD4+ T cells and used a GFP-tagged Mcl-1 construct to transfect ASM and detected the fluorophore within the nanotubes. Taken together, these findings suggest that ASM cells and CD4+ T cells communicate via nanotubes. This communication most likely facilitates the transfer of anti-apoptotic material essential for the survival of the cells, but could potentially extend to the transfer of other signals of potential importance in asthma. This finding suggests a novel mechanism for the prolongation of the life of a T cell in contact with structural cells. This study provides insights into the processes by which immune and structural cells communicate. We focused on cells that are in close proximity in airways of asthmatic patients and that contribute to the features of asthma. However, these findings may be extended to many diseases in which different cell types communicate and are found in close proximity. T cells are versatile cells that communicate with many cells such as B cells (33) and dendritic cells (34) for the generation of the immune response. These findings may provide another mechanism of cell-to-cell interaction during the activation of the immune response.
Acknowledgments We gratefully acknowledge the assistance of Dr. Peter Grutter with atomic force microscopy.
Disclosures The authors have no financial conflicts of interest.
References 1. Pare´, P. D., T. R. Bai, and C. R. Roberts. 1997. The structural and functional consequences of chronic allergic inflammation of the airways. Ciba Found. Symp. 206: 71–86, discussion 86–89, 106–110. 2. Koohsari, H., M. Tamaoka, H. R. Campbell, and J. G. Martin. 2007. The role of gamma delta T cells in airway epithelial injury and bronchial responsiveness after chlorine gas exposure in mice. Respir. Res. 8: 21. 3. Ramos-Barbo´n, D., J. F. Presley, Q. A. Hamid, E. D. Fixman, and J. G. Martin. 2005. Antigen-specific CD4+ T cells drive airway smooth muscle remodeling in experimental asthma. J. Clin. Invest. 115: 1580–1589. 4. Ramos-Barbo´n, D., R. Fraga-Iriso, N. S. Brienza, C. Montero-Martı´nez, H. Verea-Hernando, R. Olivenstein, C. Lemiere, P. Ernst, Q. A. Hamid, and J. G. Martin. 2010. T cells localize with proliferating smooth muscle alpha-actin + cell compartments in asthma. Am. J. Respir. Crit. Care Med. 182: 317–324. 5. Lazaar, A. L., S. M. Albelda, J. M. Pilewski, B. Brennan, E. Pure´, and R. A. Panettieri, Jr. 1994. T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis. J. Exp. Med. 180: 807–816. 6. Rustom, A., R. Saffrich, I. Markovic, P. Walther, and H. H. Gerdes. 2004. Nanotubular highways for intercellular organelle transport. Science 303: 1007– 1010. 7. Onfelt, B., S. Nedvetzki, K. Yanagi, and D. M. Davis. 2004. Cutting edge: membrane nanotubes connect immune cells. J. Immunol. 173: 1511–1513. 8. Arkwright, P. D., F. Luchetti, J. Tour, C. Roberts, R. Ayub, A. P. Morales, J. J. Rodrı´guez, A. Gilmore, B. Canonico, S. Papa, and M. D. Esposti. 2010. Fas stimulation of T lymphocytes promotes rapid intercellular exchange of death signals via membrane nanotubes. Cell Res. 20: 72–88. 9. Lou, E., S. Fujisawa, A. Morozov, A. Barlas, Y. Romin, Y. Dogan, S. Gholami, A. L. Moreira, K. Manova-Todorova, and M. A. Moore. 2012. Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma. PLoS One 7: e33093. 10. Chinnery, H. R., E. Pearlman, and P. G. McMenamin. 2008. Cutting edge: membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea. J. Immunol. 180: 5779–5783. 11. Sowinski, S., C. Jolly, O. Berninghausen, M. A. Purbhoo, A. Chauveau, K. Ko¨hler, S. Oddos, P. Eissmann, F. M. Brodsky, C. Hopkins, et al. 2008. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat. Cell Biol. 10: 211–219.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
CD3–activated T cells upregulate a variety of adhesion molecules and adhere to the surface of naive human ASM cells (23). Contractile responsiveness to acetylcholine is increased, whereas b-adrenergic receptor–mediated relaxation is reduced in isolated rabbit ASM tissues. Additionally, coincubation of CD4 T cells with human ASM cells pulsed with staphylococcal enterotoxin A has been reported to lead to the formation of an immunological synapse, upregulating mRNA expression and release of IL-13 (24). The latter cytokine increases intracellular calcium signals in ASM cells (25). The explanation for the discrepancy in the effects of T cells on ASM calcium signals in the current experiments and previous results may lie in the differences in the source of the tissues (rabbit airway versus human cells) or relate to the fact that we have used cultured human cells and not fresh tissue. After the removal of mitogenic stimuli, T cells rapidly undergo cell death, but have been reported to be rescued in coculture with fibroblasts (26). However, the mechanism remains unknown. We found previously that coculture of rat CD4+ T cells and ASM cells enhanced T cell survival (3), findings that were confirmed for human cells in the current study. When evaluating apoptotic T cells, we noted that live CD4+ T cells were connected to ASM cells via nanotubes, suggesting an anti-apoptotic role of nanotubes. This finding was supported by observations of the effects of an Ab targeted to CD44. Blocking CD44 weakens the attachment of T cells to ASM and resulted in an increase in the number of apoptotic CD4+ T cells. It has been suggested that CD44 plays a role in T cell spreading (27). Our results suggest that CD44 may also be involved in the formation of nanotubes and that interference with the adhesion of the cells via CD44 may affect the formation of nanotubes between ASM cells and CD4+ T cells and consequently reduce CD4+ T cell survival. However, such a conclusion remains speculative because we do not know whether CD44 has a role in the formation of nanotubes. Indeed, in the presence of anti-CD44, there was a residual connection of CD4+ T cells to ASM cells, as seen by the return of T cells to their site of adhesion to ASM following their displacement with laser tweezers. Perhaps once the connection between T cell and ASM has been established, neutralizing CD44 does not necessarily lead to the dissolution of the connection. Further experimentation is required to clarify the link between CD44 and nanotube formation. The Bcl-2 family of proteins play a major role in cell viability (28). Using immunofluorescence microscopy, we detected the presence of Bcl-2 and Mcl-1 within the nanotubes. Mcl-1 is a 37.3-kDa protein that belongs to the family of anti-apoptotic Bcl-2 proteins along with Bcl-2 and Bcl-xL (29). Similar to Bcl-2, Mcl-1 can prolong the survival of cells deprived of required growth factors or that have been exposed to cytotoxic agents such as radiation (28, 30). We found that inhibiting these anti-apoptotic proteins in ASM cells with AT101 did not affect ASM cells, but led to an increase in CD4+ T cell apoptosis, as assessed by annexin V/PI staining. This suggests differential sensitivity of ASM and T cells to inhibition of Bcl-2 and Mcl-1. However, AT101 is not specific and inhibits a number of anti-apoptotic proteins simultaneously, and also causes apoptosis by several mechanisms, including the binding to Bcl-2 and Mcl-1 as BH3 mimetics, the downregulation of Bcl-2 and Mcl-1, and the generation of oxidative stress (14, 15, 31). Therefore, we wished to more directly address the roles of Bcl-2 and Mcl-1 synthesis by ASM. To assess the specific role of Mcl-1, we used siRNA to downregulate Mcl-1 in ASM cells and found siRNA targeted against Mcl-1 resulted in a substantial increase in the number of apoptotic CD4+ T cells. In contrast, ASM cells transfected with siRNA targeted against Bcl-2 did not affect the number of apoptotic CD4+ T cells. Taken to-
NANOTUBES ENHANCE T CELL SURVIVAL
The Journal of Immunology
24.
25.
26.
27.
28.
29.
30. 31.
32.
33. 34.
T lymphocytes: role in induction of altered airway responsiveness. J. Immunol. 166: 293–303. Veler, H., A. Hu, S. Fatma, J. S. Grunstein, C. M. DeStephan, D. Campbell, J. S. Orange, and M. M. Grunstein. 2007. Superantigen presentation by airway smooth muscle to CD4+ T lymphocytes elicits reciprocal proasthmatic changes in airway function. J. Immunol. 178: 3627–3636. Deshpande, D. A., S. Dogan, T. F. Walseth, S. M. Miller, Y. Amrani, R. A. Panettieri, and M. S. Kannan. 2004. Modulation of calcium signaling by interleukin-13 in human airway smooth muscle: role of CD38/cyclic adenosine diphosphate ribose pathway. Am. J. Respir. Cell Mol. Biol. 31: 36–42. Gombert, W., N. J. Borthwick, D. L. Wallace, H. Hyde, M. Bofill, D. Pilling, P. C. Beverley, G. Janossy, M. Salmon, and A. N. Akbar. 1996. Fibroblasts prevent apoptosis of IL-2-deprived T cells without inducing proliferation: a selective effect on Bcl-XL expression. Immunology 89: 397–404. Wong, N. K., J. C. Lai, N. Maeshima, and P. Johnson. 2011. CD44-mediated elongated T cell spreading requires Pyk2 activation by Src family kinases, extracellular calcium, phospholipase C and phosphatidylinositol-3 kinase. Cell. Signal. 23: 812–819. Hockenbery, D., G. Nun˜ez, C. Milliman, R. D. Schreiber, and S. J. Korsmeyer. 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348: 334–336. Kozopas, K. M., T. Yang, H. L. Buchan, P. Zhou, and R. W. Craig. 1993. MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc. Natl. Acad. Sci. USA 90: 3516–3520. Miyashita, T., and J. C. Reed. 1993. Bcl-2 oncoprotein blocks chemotherapyinduced apoptosis in a human leukemia cell line. Blood 81: 151–157. Barba-Barajas, M., G. Herna´ndez-Flores, J. M. Lerma-Dı´az, P. C. OrtizLazareno, J. R. Domı´nguez-Rodrı´guez, L. Barba-Barajas, R. de Celis, L. F. JaveSuarez, A. C. Aguilar-Lemarroy, M. G. Guevara-Barraza, and A. Bravo-Cuellar. 2009. Gossypol induced apoptosis of polymorphonuclear leukocytes and monocytes: involvement of mitochondrial pathway and reactive oxygen species. Immunopharmacol. Immunotoxicol. 31: 320–330. Dzhagalov, I., A. Dunkle, and Y. W. He. 2008. The anti-apoptotic Bcl-2 family member Mcl-1 promotes T lymphocyte survival at multiple stages. J. Immunol. 181: 521–528. Singer, A., and R. J. Hodes. 1983. Mechanisms of T cell-B cell interaction. Annu. Rev. Immunol. 1: 211–241. Celli, S., Z. Garcia, H. Beuneu, and P. Bousso. 2008. Decoding the dynamics of T cell-dendritic cell interactions in vivo. Immunol. Rev. 221: 182–187.
Downloaded from http://www.jimmunol.org/ at Florida International University, Medical Library on June 4, 2015
12. Roberts, K. L., B. Manicassamy, and R. A. Lamb. 2015. Influenza A virus uses intercellular connections to spread to neighboring cells. J. Virol. 89: 1537– 1549. 13. Risse, P. A., T. Jo, F. Suarez, N. Hirota, B. Tolloczko, P. Ferraro, P. Grutter, and J. G. Martin. 2011. Interleukin-13 inhibits proliferation and enhances contractility of human airway smooth muscle cells without change in contractile phenotype. Am. J. Physiol. Lung Cell. Mol. Physiol. 300: L958–L966. 14. Balakrishnan, K., W. G. Wierda, M. J. Keating, and V. Gandhi. 2008. Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells. Blood 112: 1971–1980. 15. Paulus, A., K. Chitta, S. Akhtar, D. Personett, K. C. Miller, K. J. Thompson, J. Carr, S. Kumar, V. Roy, S. M. Ansell, et al. 2014. AT-101 downregulates BCL2 and MCL1 and potentiates the cytotoxic effects of lenalidomide and dexamethasone in preclinical models of multiple myeloma and Waldenstro¨m macroglobulinaemia. Br. J. Haematol. 164: 352–365. 16. Rechavi, O., I. Goldstein, and Y. Kloog. 2009. Intercellular exchange of proteins: the immune cell habit of sharing. FEBS Lett. 583: 1792–1799. 17. Li, X., Y. Zhang, S. C. Yeung, Y. Liang, X. Liang, Y. Ding, M. S. Ip, H. F. Tse, J. C. Mak, and Q. Lian. 2014. Mitochondrial transfer of induced pluripotent stem cells-derived MSCs to airway epithelial cells attenuates cigarette smoke-induced damage. Am. J. Respir. Cell Mol. Biol. 51: 455–465. 18. Lehmann, T. P., K. Filipiak, W. Juzwa, P. Sujka-Kordowska, P. P. Jagodzinski, M. Zabel, J. Głowacki, E. Misterska, M. Walczak, and M. Głowacki. 2014. Co‑culture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes. Mol. Med. Rep. 9: 574–582. 19. Tavi, P., T. Korhonen, S. L. Ha¨nninen, J. D. Bruton, S. Lo¨o¨f, A. Simon, and H. Westerblad. 2010. Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes. J. Cell. Physiol. 223: 376–383. 20. Watkins, S. C., and R. D. Salter. 2005. Functional connectivity between immune cells mediated by tunneling nanotubules. Immunity 23: 309–318. 21. Plotnikov, E. Y., T. G. Khryapenkova, A. K. Vasileva, M. V. Marey, S. I. Galkina, N. K. Isaev, E. V. Sheval, V. Y. Polyakov, G. T. Sukhikh, and D. B. Zorov. 2008. Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture. J. Cell. Mol. Med. 12: 1622–1631. 22. Halayko, A. J., H. Salari, X. Ma, and N. L. Stephens. 1996. Markers of airway smooth muscle cell phenotype. Am. J. Physiol. 270: L1040–L1051. 23. Hakonarson, H., C. Kim, R. Whelan, D. Campbell, and M. M. Grunstein. 2001. Bi-directional activation between human airway smooth muscle cells and
9
