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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC Mar 1, 2012.
Published in final edited form as:
PMCID: PMC3167479
NIHMSID: NIHMS306772

FTY720 blocks egress of T cells in part by abrogation of their adhesion on the lymph node sinus

Abstract

Egress of lymphocytes from lymphoid tissues is a complex process in which Gαi-mediated signals play a decisive role. We show here that although FTY720, an agonist of the sphingosine 1-phosphate 1 receptor (S1P1R), induces S1P1R internalization sufficiently in the presence or absence of Gαi2 or Gαi3, the drug blocks egress of wild type (WT) and Gαi3-deficent T cells, but not Gαi2-deficient T cells in both WT and Gαi2-deficient hosts. Intravital imaging of lymph nodes revealed that all three groups of T cells approached and engaged cortical sinusoids similarly in the presence or absence of FTY720. The cells also entered and departed the sinus at an almost identical frequency in the absence of the drug. However, after engagement of the sinus, most WT and Gαi3-deficient T cells retracted and migrated back into the parenchyma in FTY720-treated animals, due to a failure of the cells to establish adhesion on the sinus, whereas Gαi2-deficient T cells adhered firmly on the sinus that prevented their retraction, facilitating their transmigration of the lymphatic endothelial barrier. These data confirm egress of Gαi2−/− T cells independent of S1P-mediated chemotaxis and failure of FTY720 to close lymphatic stromal channels, and argue for the first time that FTY720 induces lymphopenia in part by impairing T cell adhesion to the sinus in a manner dependent on Gαi2.

Introduction

FTY720, a sphingosine 1-phosphate (S1P) analogue, was recently approved by the US Food and Drug Administration as first-line treatment for relapsing forms of multiple sclerosis (1). It suppresses immune responses by blocking lymphocyte egress, but the underlying mechanism is not completely understood (2,3). Early studies suggested that binding of FTY720 to the sphingosine 1-phosphate (S1P) 1 receptor (S1P1R) caused internalization of the receptor in lymph node lymphocytes and thus prevented them from responding to a high concentration of S1P in the lymphatic sinus (47). This receptor internalization-dependent mechanism has been challenged by several investigations (8,9). Sinha et al. reported that egress of B lymphocytes from lymph nodes was not solely dependent of S1P-mediated chemotaxis (8). And, pertussis toxin (PTX)-treated, S1P1R-deficient T cells could exit lymph nodes, albeit at a reduced frequency (9). Moreover, in comparison with CCR7-positive T cells, CCR7-deficient T cells displayed a compromised response to FTY720-induced lymphopenia (9). These investigations argue that a receptor internalization-independent mechanism, either alone or in conjunction with receptor internalization, is involved in FTY720-mediated blockade on lymphocyte egress.

The S1P1R is a G protein couple receptor (GPCR) and activates exclusively PTX-sensitive Gαi/o proteins to propagate the entirety of its signal (10). The primary PTX substrates in T lymphocytes are Gαi2 and Gαi3 as shown by in vitro PTX labeling and cDNA cloning (11,12). Our previous study has demonstrated that these two Gαi proteins can be functionally overlapping, distinct, or antagonistic in chemotactic responses in a receptor-specific fashion (13,14). In the present study, we use Gαi2- and Gαi3-deficient T cells, in conjunction with intravital time-lapse imaging, to delineate potentially distinct activities of these two Gαi proteins in the multiple-step process of T cell egress. We find that in marked contrast to WT and Gαi3-deficient T cells, Gαi2-deficient T cells cannot be sufficiently sequestered in the lymphoid tissue by FTY720. The drug appeared to diminish interactions between T cells and the sinus in the presence of Gαi2 and to promote them migrating away from an exit, causing lymphopenia. The study suggests for the first time involvement of T cell adhesion to the sinus for T cell egress.

Materials and Methods

Mice

Gαi2−/−, Gαi3−/−, and WT control mice on the mixed 129Sv/C57BL/6 background were generous gifts from Dr. Lutz Birnbaumer (15) and the mice were backcrossed with C57BL/6 (B6) mice for six times as described (16). Both female and male mice were used at 4–6 weeks of age unless otherwise indicated. The mice were housed in specific pathogen-free cages at the animal facilities of the Massachusetts General Hospital in accordance with institutional guidelines. The study was reviewed and approved by the MGH Subcommittee of Research Animal Studies.

In vivo flow cytometry

T cells were isolated from lymph nodes of indicated mice as previously described (17), suspended in RPMI medium supplemented with 2% FBS, and stained with 10 μM DiD (Invitrogen) for 30 minutes in a humidified incubator at 37°C followed by two washes with serum-free RPMI. The resultant cells at 5 × 106 cells/mouse were adoptively transferred into naïve WT, Gαi2−/− or Gαi3−/− mice via the tail vein. The recipients were anesthetized 16 hr later by intraperitoneal injection of a mixture of ketamine (80mg/kg) and xylazine (20mg/kg). Fluorescently labeled donor cells were then counted by in vivo flow cytometry to establish control values before gavage of mice with either 1mg/kg of FTY720 or 10 mg/kg of SEW2871 (Cayman Chemical Company, Michigan). SEW2871 was administrated every 12 hr to maintain a sufficient level of the drug in the plasma (18). Fluorescently labeled cells in circulation were counted by in vivo flow cytometer at indicated time points under anesthesia. The mouse to be examined by in vivo flow cytometry was positioned on a custom-built inverted microscope stage equipped with a warming tube to keep body temperature constant as described (19). A stationary laser beam was focused through cylindrical optics onto the ear blood vessel, forming a ~5 μm wide slit whose long axis was perpendicular to the vessel, and the length was adjusted to match its diameter. As individual fluorescently labeled cells flew through the excitation beam, a burst of fluorescence was generated with a light-emitting diode at 633nm, collected by the microscope objective lens, and detected by a photomultiplier tube through a confocal slit aperture. Matlab software developed in house was used to determine the number of peaks per unit time, as well as peak height and pulse width.

Infection of Gαi2−/− T cells with Gαi2/3-expressing lentivirus

To generate a lentivirus expressing Gαi2 or Gαi3, Gαi2 or Gαi3 cDNA was subcloned into the multiple cloning site of a HIV-based lentiviral vector, pCDH1-copGFP (System Biosciences). The constructed and control GFP lentivirus were produced by transfection of the constructs individually into 293TN cells, along with a ViralPower lentiviral packaging mix (System Biosciences). The culture supernatant was collected 48 hr later and viral titers were determined in 293TN cells based on the number of GFP+ cells at the highest titration of the supernatant. For infection, T cells prepared from Gαi2−/− mice were first stimulated with anti-CD3/anti-CD28 for 24 hrs to enhance their susceptibility to viral infection. Lentiviral culture supernatant was then added to the T cell culture at a ratio of a virus to cell 20~50:1 overnight in the presence of 8 μg/ml of polybrene followed by culturing these cells for additional 48 hr after replenishment of half the medium. Percentage of GFP+ cells was about 35% on average as determined by flow cytometry. The infected cells were i.v. administrated into cognate WT mice at 4 × 106/mouse. GFP+ cells were counted by in vivo flow cytometry before and after FTY720 treatment as above.

Intravital imaging and analysis of T cell egress

T cells isolated from WT, Gαi2−/−, or Gαi3−/− mice were stained with 20 μM 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR, Invitrogen) or 10 μM 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) for 20 min at 37°C, respectively. The labeled cells were mixed at 1:1 ratio of two indicated cell types and adoptively transferred by tail intravenous injection of 1 × 107 cells per mouse. The recipient mice were gavaged with FTY720 (1 mg/kg) or saline 4 hr later, followed by subcutaneous injection into a hind footpad of 15 μg anti-LYVE-1 antibody (R&D Systems) conjugated with Alexa Fluor-647 (Monoclonal antibody labeling kit, Invitrogen). The mouse was anaesthetized in 16 hr and placed on an electrically heated plate to maintain temperature at 36°C. The popliteal lymph node was exposed by small skin incisions and bathed with a continuous flow of warm saline to maintain a local temperature at 36°C during the imaging. Intravital imaging of the lymph node was performed by a home-built microscope and images were acquired under the control of in-house developed software (19). The in vivo confocal microscope was equipped with three photomultiplier tubes (PMT, Hamamatsu, R9110) that were optimized to provide bright images with a high contrast. Each xy plane spanned 250 × 250 μm at a resolution of 2 pixels per μm. Stacks of images were acquired with a z-axis resolution of 3 μm per section and time-series images were obtained at a 20-second interval. To determine whether a cell was inside, outside, or on the border of a cortical sinus, each cell located relatively to the sinusoid wall was assessed in x-y plane and/or z plane. The moving distances and velocities of the tacking cells were tracked for each video segment and calculated using ImageJ software.

Statistical analysis

Statistical tests were performed using one-way analysis of variance (ANOVA) for multiple group comparisons. A p value <0.05 was considered significant.

Results

T cells from Gαi2−/− mice cannot be sufficiently sequestered by FTY720

To explore whether Gαi2 and Gαi3 played a distinct role in FTY720-induced lymphopenia, we tracked circulation of adoptively transferred Gαi2- and Gαi3-deficient T cells in real time in a living animal following FTY720 treatment by an in vivo flow cytometer (20). The instrumentation allows detection and quantification of adoptively transferred, fluorescently labeled cells flowing through the same ear vessel in live animals over an extended period of time without extraction of blood samples since repeated blood draws can potentially cause stress and reduce the number of circulating lymphocytes (2022). To quantify circulating cells, T cells isolated from wild type (WT), Gαi2−/−, or Gαi3−/− mice were labeled with a vital red fluorescence dye before administrated into cognate WT mice, and counted 16 hr later via the ear vessel to establish control values (figure 1, time 0). The donor cells were monitored in the same ear vessel at indicated times following gavage of the mice with 1 mg/kg of FTY720. As shown in figure 1A, the number of WT T cells declined precipitously over a 4 hr period of time and reached the lowest level at 8 hr after FTY720 treatment. Gαi3−/− T cells also reached the lowest level at the similar time window, although they were refractory to leave the circulation in an initial 4 hr. The number of Gαi2−/− T cells in circulation diminished similarly as that of WT T cells in the first 4 hrs, but it never reached the lowest level as those seen with WT and Gαi3−/− T cells. Rather, Gαi2-deficient T cells circulated back to the blood, reached the initial level by 8 hr, and remained at the level since. In sharp contrast, WT and Gαi3−/− T cells were trapped in the tissues, returning to the blood slowly and gradually over a period of 3 days as previously described (23). To rule out that a faster recovery of circulating Gαi2−/− T cells following FTY720 treatment resulted from a release of the cells from non-lymphoid tissues like the lung or liver, the number of circulating T cells was also monitored directly in the lymph. A similar reduction in the number of circulating T cells following by a faster recovery was again observed in the lymph only in the absence of Gαi2 (supplement, Figure S1A).

Figure 1
FTY720 fails to sequester Gαi2−/− T cells in either Gαi2−/− or WT recipient mice. T cells from indicated mice were stained with DiD, except for panel D, and injected into cognate WT mice (A, B, and D) or ...

Of note, numbers of circulating Gαi2−/−, Gαi3−/− and WT T cells differed at a time of FTY720 administration as well as in saline control mice (Figure 1A and supplement Figure S1B), even though the same number of cells were adoptively transferred into the mice. An increase in circulating Gαi2−/− T cells but a decrease in Gαi3−/− T cells relative to WT cells was constantly observed and hinted a difference of Gαi2 and Gαi3 in regulation of T cell trafficking. For better comparison of FTY720-induced lymphopenia, the numbers of circulating T cells were normalized to 1 at 0 time point in subsequent studies. After normalization, a slight but significant increase in FTY720-induced sequestration of Gαi3−/− T cells was still evidenced when compared with WT T cells (data not shown).

To exclude that insufficient sequestration of Gαi2−/− T cells resulted from overlapping stimulation of other S1P receptors by FTY720, the S1P1R-specific agonist SEW2871 was tested. Unlike FTY720 that can potentially activate other G proteins besides Gαi2 or Gαi3 by binding to additional S1P receptors like the S1P4R on T cells, SEW2871 induces lymphopenia via exclusive activation of the S1P1R (24). Like FTY720, SEW2871 caused lymphopenia persistently in Gαi3−/− and WT T cells but only transiently in Gαi2−/− T cells (Figure 1B). The result suggests involvement of the S1P1R only in the action of FTY720 in Gαi2−/− T cells. Similar results were also attained in the reciprocal experiments in which WT, Gαi2−/−, or Gαi3−/− T cells were introduced into Gαi2−/− mice (Figure 1C) or Gαi3−/− mice (data not shown), with only Gαi2−/− T cells remaining in the circulation after FTY720 administration. Clearly, a defect in T cells, rather than endothelial cells, ablated FTY720-induced lymphopenia in the absence of Gαi2.

Moreover, an insufficient response of Gαi2−/− T cells to FTY720 was also unlikely to be caused by altered T cell differentiation in the absence of Gαi2 as previous studies showed normal T cell development in the animal (15,25). To further corroborate this, Gαi2 expression was restored to a WT level in the cells by infection of the cells with a GFP-lentivirus constructed with Gαi2 or Gαi3 or a control GFP-lentivirus as shown in Figure S1C (supplement). The infected cells were administrated into cognate WT mice and counted before and after FTY720 treatment as above. Gαi2−/− T cells responded to FTY720-mediated sequestration normally after infection with Gαi2/GFP-lentivirus but not with Gαi3/GFP or control GFP-lentivirus (Figure 1D). The ability of ectopic Gαi2 expression to restore FTY720 responses in the cells unambiguously confirms that a loss of Gαi2 in S1P1R-mediated signaling is directly responsible for the insufficient response of Gαi2−/− T cells to FTY720.

Similar down regulation of the S1P1R on T cells in the presence or absence of Gαi2

FTY720 has long been shown to induce internalization of the S1P1R on T cells, abolishing S1P-mediated chemotaxis and thus egress of the cells (47). Lack of Gαi2 might abrogate FTY720-induced receptor internalization, rendering the cells refractory to FTY720-induced lymphopenia. To address this, the level of S1P1R expression was analyzed on lymph node T cells isolated from FTY720-treated and non-treated mice by a polyclonal antibody (Ab) specific for the N-terminal 49 amino residues of mouse S1P1R as described (7). It was found that S1P1R expression diminished indiscriminately to background levels in the presence or absence of Gαi2 in 16 hr after gavage of 1 mg/kg FTY720 (Figure 2). Similar receptor modulation in these three groups of T cells was also observed after 44 or 66 hrs of FTY720 treatment (data not shown). The result confirms that Gαi2−/− T cells exit lymph nodes independent on the cell surface S1P1R receptor.

Figure 2
FTY720 induces S1P1R internalization in the presence or absence of Gαi2. Mice with indicated phenotypes were treated with 1 mg/kg FTY720 or saline. Lymph node (LN) cells were isolated from saline- and FTY720-treated mice at 16 hrs post-treatment, ...

Egress of Gαi2−/− T cells in FTY720-treated mice was not ascribed to altered cell motility

Cell motility was tracked by real-time imaging of the popliteal lymph node in WT mice receiving equal numbers of fluorescently labeled T cells prepared from Gαi2−/− or Gαi3−/− and WT mice (19). The average velocity of Gαi3−/− and WT T cells was comparable in control mice and was not altered significantly after FTY720 treatment (figure S2A, supplement). Unexpectedly, the velocity of Gαi2−/− T cells decreased, rather than increased, by approximately 20% compared to WT or Gαi3−/− T cells in the distal area of the cortical sinus, irrespective of FTY720 treatment (figure S2A, supplement), similar to that described with Gαi2−/− B cells (8). A decrease in the velocity of Gαi2-deficient T cells was also described previously (26) and might be associated with an impaired response to retention chemokines like SDF-1 as shown in our previous study (13). Superimposed tracking of 20 randomly selected cells of each phenotype over 15 min confirmed similar tracks between Gαi3−/− and WT T cells but less vigorous movement of Gαi2−/− T cells, in particular, in the presence of FTY720 (figure S2B, supplement). The decreased motility superficially contradicted sufficient egress of Gαi2−/− T cells in FTY720-treated mice.

Gαi2−/− T cells enter, but WT T cells depart, the cortical sinus in the presence of FTY720

We then tracked T cell behavior around cortical sinusoids of lymphoid nodes, as these structures were recently identified to be a site for T cell egress (8,9). We found no significant difference in distribution of T cells lacking either Gαi inside or outside cortical sinusoids in control mice (Figure 3 A–C). Enumeration of about 600 cells inside sinusoids in randomly selected 20~30 imaging stacks collected from six lymph nodes revealed that average numbers of WT, Gαi2−/−, Gαi3−/− T cells were similar in the regions (figure 3A–C and G). Likewise, the numbers of Gαi2−/−, Gαi3−/− and WT T cells within 30 μm of each cortical sinus were comparable (Figure 3 A–C and H) (8). However, there were few WT or Gαi3−/− T cells (red) in sinusoid lumens, after FTY720 administration (Figure 3D–F and G). In sharp contrast, the number of Gαi2−/− T cells inside the sinuses remained almost identical as that in control mice (figure 3 D, F, and G). Clearly, Gαi2−/− T cells exit lymph nodes in FTY720-treated animals through the same anatomic structure. Also noticed was a strong bias in the number of Gαi2−/− T cells (green) in the surrounding area (<30 μm) of sinusoids as delineated by a dotted yellow line in mice receiving Gαi2−/− T cells and WT or Gαi3−/− T cells (Figure 3D, F, and H, p<0.001). Ability of Gαi2−/− T cells into enter the sinus sufficiently, concurrent with a drastic decrease in the entry of WT or Gαi3−/− T cells, gave rise to a majority of Gαi2−/− T cells and a scant fraction of WT or Gαi3−/− T cells inside sinusoids in FTY720-treated mice (figure 3 D, F, and G, p<0.001). These imaging studies demonstrate a similar behavior between WT and Gαi3−/− T cells and distinct between Gαi2−/− and Gαi3−/− or WT cells in the vicinity of the sinus in FTY720-treated mice, in agreement with in vivo flow cytometric analysis (figure 1).

Figure 3
Entry of only Gαi2−/− T cells into the cortical sinus in FTY720-treated mice. Equal numbers of CMTMR-labeled WT or Gαi3−/− T cells (red) and CFSE-labeled Gαi2−/− T cells (green) were ...

Increased adhesion of Gαi2−/− T cells to the sinusoid surface

To identify a potential difference between WT and Gαi2−/− T cells in their ability to approach, engage, crawl along, and retract from the cortical sinus in control and FTY720-treated mice, time-series images of individual cells were zoomed and traced. Representative images are shown in figure 4 in which one WT cell (red, white arrow), followed by a Gαi2−/− cell (green, white arrow), approached and entered a sinusoid by the same exit site in control mice (figure 4A). Another WT cell (yellow arrow) approached, stuck on, and then protruded its leading edge into the sinus before entry. Similarly, two WT cells approached and engaged different sinusoids for about 7 or 3 min, respectively, and established adhesion on the sinus before they successfully crossed the sinusoid wall in control mice (figure 4C, also see Supplement movie #1). In sharp contrast, WT cells approached, engaged, and sometimes even extended a process into the sinus, but retracted and went away in FTY720-treated mice (figure 4B and 4D, arrow, also see Supplement movies #2 and #3). While the drug drastically altered the behavior of WT cells, it appeared to have little impact on Gαi2−/− T cells. As can be seen in Figure 1B, a Gαi2−/− T cell stuck on the sinusoid, polarized, and penetrated into it in the presence of FTY720 (also see Supplemental movies #2 and #4). We observed only occasionally Gαi2−/− T cells migrating away from the sinus during the imaging study. In many cases, Gαi2−/− T cells adhered to a specific site on the sinusoid surface so strongly that they could not be pulled away despite vigorous motion of their pseudopodia for several minus (figure 4D, arrows, also see Supplemental movies #3 and #4).

Figure 4
Distinct behaviors of T cells after T cells approach the sinus in the presence or absence of Gαi2. Time-lapse images of individual WT and Gαi2−/− cells prepared in figure 3 were zoomed and tracked for their behavior on ...

Enhanced adhesion of Gαi2−/− T cells to the sinusoid surface was further illustrated by analyzing 20 randomly selected WT cells and an equal number of Gαi2−/− cells that were either already stuck on the sinus when the recording started or migrating toward and subsequently engaging the sinus in the same region during a 25 min recording period of time. Among the 20 WT cells traced, 9 of them entered and 8 moved away from the sinus in control mice, indicating a balance between sinus-away and -entry signals under a physiological condition (Figure 5A). This probably also held truth for Gαi2−/− T cells in which 8 cells entered and 5 cells departed, albeit at a reduced rate in either a direction (Figure 5B). The diminished rate of both sinus-away and -entry movement brought about a greater number of Gαi2−/− cells remaining on the sinus than WT cells (figure 5B vs. 5A), with seven Gαi2−/− cells firmly adherent on the sinus for more than 2 min without displacement, in contrast to only 3 WT cells sticking on the sinus (figure 5A and 5B). The number of Gαi2−/− cells sticking on the sinus further increased to 14 in the presence of FTY720 (figure 5D). Yet, an opposite trend was seen in the presence of Gαi2, with only one WT cell (#4) sticking on the sinus and this was the cell that was able to enter the sinus (figure 5C). Remarkably, none of the cells that formed sufficient adhesion on the sinus, as marked in black arrows, migrated away, regardless of Gαi2 expression, and all of them were able to enter the sinus in the presence or absence of FTY720. The observation argues that formation of an anchor between T cells and the sinus may be required to “stop” the sinus-away signal and to acquire a polarized cell shape for them squeezing into the sinus. Thus, a failure of establishing sufficient interactions between T cells and sinuses robustly increased the number (17) of WT T cells departing the sinus, preventing their egress (figure 5C). On the other hand, lack of Gαi2 strengthened T cell adhesion to the sinusoid surface, which prevented their migration away and facilitated their transmigration of the sinusoids (figure 5D). A few Gαi2−/− T cells did not establish adherent on the sinusoid surface and departed in the presence of FTY720 (figure 5D, also see Supplemental movie #5). A blockade of T cell-sinus interactions by FTY720 was consistent with a vigorous movement of the cells over the sinusoid surface, reflected by superimposed tracking of 20 randomly selected WT T cells (figure 5E). The unrestricted migratory paths were in sharp contrast to the locally confined migration seen in most of Gαi2−/− T cells.

Figure 5
Lack of Gαi2 facilitates adhesion of T cells on the sinusoid surface. The time spent by which individual donor T cells remained on the outer border of cortical sinuses and their subsequent movements were compared between control (A and B) and ...

Tracking of 400 cells from 20 samples, except for Gαi3−/− T cells in which 12 samples were analyzed, further corroborated an inverse relationship between sinus-away migration and adhesive responses both in the presence and absence of FTY720 (figure 5F vs. 5G). The frequency of WT or Gαi3−/− T cells departing the sinus increased from 39% or 43% in the absence of FTY720, to 85 or 86% in the presence of the drug (figure 5F), with only a few cells remaining on the sinus (figure 5G), conferring a very few chances for the cells to exit (figure 5H). Opposingly, the drug greatly enhanced adhesion of Gαi2−/− T cells on the sinus (figure 5G), concomitant with a sharp decrease in the number of cells migrating away (Figure 5F). The enhanced adhesion was in a good agreement with their reduced motility on or around the sinus (Figure 5E). In spite of their ability to enter the sinus, Gαi2−/− T cells transmigrated the sinusoids at a significantly low level in the presence compared to the absence of FTY720 (Figure 5H), presumably owing to S1P1R receptor internalization. The diminished transmigration might be responsible for an initial decline in the number of circulating Gαi2−/− T cells (<4 hr) after FTY720 treatment (Figure.1). In support of a role of S1P-induced chemotaxis in T cell egress, Gαi2- and Gαi3-deficient T cells entered the sinus at an efficiency slightly lower than WT cells in the control mice (p<0.01 or 0.05, respectively, figure 5H), consistent with a lightly reduced chemotaxis induced by S1P in these cells described previously (14). The reduced entry of Gαi2−/− T cells into the sinus might be eventually compensated by an increasing number of cells adherent on the sinus, resulting in normalization of circulating Gαi2−/− T cells in 8 hr after FTY720 treatment (Fig. 1). The results suggest that both receptor internalization and enhanced sinus-away migration contribute to FTY720-induced sequestration of T cells in the lymphoid tissue.

Discussion

The investigation reveals, for the first time, possible involvement of T cell adhesion on the sinus for regulating egress of lymphocytes, in a good agreement with a multi-step model of T cell egress that requires a S1P1R-independent probing of the sinus before S1P-guided entry (27). The role of T cell adhesion to the sinus in intravasation may be similar to that in extravasation. During the process of extravasation, leukocytes adhere to endothelial cells or extracellular matrix by which they stop a shear force and acquire a polarized cell shape enabling them to squeeze into the tissues through a vessel wall. Sticking of T cells on the sinusoid surface appeared to “stop” the tissue-retention or sinus-away signal in favor of T cells transmigrating across the sinus during T cell egress. An importance of halting the sinus-away signal for T cell egress is underscored by the observation that none of the cells that had formed firm adhesion on the sinus moved away in the presence or absence of FTY720. Based on these observations and published data, we propose a model for T cell egress as depicted in figure S3 in the supplement. T cells crawl along chemokine-decorated fiber paths generated by the fibroblastic reticular cells (FRC) in the T cell zone, approach and engage the sinus (28), following which T cells form adhesion on the sinus, prohibited the cells from migrating away. The adhesion is presumably followed by de-adhesion and entry of the cells into the sinus following a cue of S1P. This process is likely to take place quickly in most of cells under a physiological condition and thus may not be readily captured by imaging studies. FTY720 impairs the adhesion or accelerates deadhesion, permitting relatively unrestricted migration of the cells along or away from the sinus, as reflected by vigorous movements of WT cells on the sinusoid surface and frequent retraction to the parenchyma even after they protrude their leading edges into the sinus. In the absence of Gαi2, FTY720 treatment strengthens the adhesion that stops the sinus-away signal and promotes T cells to enter the sinus with or without an S1P cue (low panel), although the underlying mechanism remains unknown. Ligation of the S1P1R has been shown to activate integrin-mediated firm arrest in high endothelial venules(29). The adhesion molecules involved in this intravasation are not known at present, which are presumably different from those adhesion molecules and selectins such as αLβ2 (LFA-1 or CD11a/CD18), α4β1 (VLA-4), and L-selectin for diapedesis or S1P1R-induced tissue retention in the skin (30), because sticking of Gαi2−/− T cells occurs only on the surface of the sinus in the presence or absence of FTY720. Moreover, injection of neutralizing antibodies against integrins such as αL, α4 or β2 did not reveal any role for these integrins in lymphocyte egress from second lymph organs (7,31). Gαi2-deficient T cells may provide us with a unique opportunity to unravel the nature of the adhesion molecules involved in T cell egress because of their extended adhesion on the sinus.

The S1P1R has been shown to exclusively couple with PTX-sensitive heterotrimeric Gαi/o proteins (10). T cells express only Gαi2 and Gαi3 and no Gαi1 or Gαo is expressed to compensate for a loss of either Gαi protein in Gαi2 and Gαi3-deficient mice as analyzed by RT-PCR (data not shown) (32). These two Gαi proteins were thought redundant in coupling to the S1P1R, because Gαi2- and Gαi3-deficeint T cells both displayed similar chemotactic responses to S1P, albeit at a slightly lesser degree compared to WT cells (14). T cell egress and emigration of thymocytes were normal in these two strains of mice (16). Binding of FTY720 to the S1P1R also induced S1P1R internalization on these three groups of cells, but sequestered WT and Gαi3−/− T cells not Gαi2−/− T cells. Importance of Gαi2 in FTY720-stimulated sequestration was strongly implicated by restoring the response in Gαi2-deficient T cells after ectopic Gαi2 expression. Mullershausen et al. recently showed long persistent signaling induced by pFTY720 after S1P1R internalization in CHO-S1P1R cells or primary HUVECs (33). Persistent activation of Gαi2 proteins by internalized S1P1R may play a critical role in abrogation of T cell and sinusoid interaction, probably in part through inhibition of cAMP production, in light of an importance of cAMP in cell-cell adhesion and the well-documented cAMP inhibitory activity of Gαi protein (34), which is currently under investigation.

Our study also provides compelling evidence demonstrating that FTY720 modulation of T cell egress is directly attributed to its action on T cells rather than endothelial cells and FTY720 doesn't close the lymphatic endothelial “stromal portal”, if it exists (35). Hence, FTY720 could not block egress of Gαi2−/− T cells, irrespective of whether the cells were adoptively transferred into WT or Gαi2-KO mice. Its action is also unlikely via other S1P receptors, other than the S1P1R, since SEW2871 that is specific for the S1P1R cannot inhibit egress of Gαi2−/− T cells either. Moreover, restoration of FTY720-mediated responses in Gαi2−/− T cells after transfection of Gαi2, but not Gαi3, into the cells corroborated that failure of FTY720 to sequester Gαi2−/− T cells in the secondary lymphoid tissues did not result from a developmental defect in the cells (figure 1D). Consistent with this was our early study showing that thymic emigration of T cells was normal in Gαi2−/− mice despite an increase in the number of CD4+ and CD8+ cells in the thymus (25). The latter was attributed to an accelerated transition from double positive to single positive thymocytes (25). There was also no significant alteration in T cell distribution in Gαi2 and Gαi3-deficient mice at adulthood, in spite of a defect in new born mice in the absence of either Gαi2 or Gαi3 (16).

The current investigation demonstrates that FTY720 blocks egress of T cells by abrogation of T cell and sinusoid interactions, in addition to induction of S1P1R internalization. These novel mechanistic insights into T cell egress can potentially serve as a basis for identifying new therapeutic targets.

Supplementary Material

Acknowledgments

The authors would like to thank members in Dr. Wu's group for stimulating discussion, Dr. Volker Brinkmann at Novartis Pharma AG for pFTY720 and FTY720, and Dr. Lutz Birnbaumer for Gαi2−/− and Gαi3−/− mice.

Funding Source: This work is supported by the National Institutes of Health grants AI050822 and AI070785, and a Senior Research Award from the Crohn's & Colitis Foundation of America (to M.X.W.).

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