Src Homology 2-Domain Containing Leukocyte-Specific Phosphoprotein of 76 kDa (SLP-76) Is Required for Optimal CXCR4-Stimulated T Lymphocyte Firm Arrest to ICAM-1 Under Shear Flow

Summary

Rapid arrest of T cells at target sites upon engagement of chemokine receptors is crucial to the proper functioning of the immune system. Although T cell arrest always occurs under hydrodynamic forces in vivo, most studies investigating the molecular mechanisms of arrest have been performed under static conditions. While the requirement of the adaptor protein SLP-76 in TCR-induced integrin activation has been demonstrated, its role in chemokine-triggered T cell adhesion is unknown. Using a flow chamber system, we show that SLP-76 plays an important role in regulating the transition from tethering and rolling to firm adhesion of T cells under physiological shear flow in response to CXCL12α SDF-1α); SLP-76-deficient primary T cells exhibited defective adhesion with a significant decrease in the number of firmly arrested cells. We further demonstrate the N-terminal phosphotyrosines of SLP-76 play a critical role in this T cell adhesion under flow. These findings reveal a novel role for SLP-76 in CXCR4-mediated T lymphocyte trafficking.

Introduction

A successful immune response relies on rapid changes in the adhesive properties of T cells to allow arrest and subsequent migration to secondary lymphoid organs (SLOs) or inflammatory sites. Intravascular T cells are subjected to the shear stresses of blood flow, requiring multiple adhesive and chemotactic events for cells to exit blood vessels. These processes result from integration of mechanical and biochemical signals initiated by engagement of selectins, integrins, and chemokine receptors, on both T cells and host tissues [1–4].

Entry of T cells to inflammatory sites requires a complex multistep process. In response to inflammatory stimuli, cells are first captured by weak binding to P- or E-selectin expressed on the inflamed vascular endothelium, resulting in tethering and rolling adhesion. Exposure to chemokines present on the vessel wall leads to rapid activation of integrins on T cells and to firm adhesion through binding of these integrins to their ligands. Subsequent actin reorganization following the enhancement of integrin activity contributes to shear-resistant firm arrest [5]. Within seconds to minutes of firm arrest, adherent T cells extravasate and eventually transmigrate to the site of inflammation.

During the transition to adhesion, multiple signaling pathways are activated inside the cell. Key among these are signals downstream of chemokine receptors and signaling to and by integrins. Circulating T cells maintain the integrin lymphocyte function-associated protein-1 (LFA-1; also known as α₁Lβ₂) in a closed and non-adhesive state. Chemokines or TCR activation triggers “inside-out” signaling, which induces LFA-1 activation leading to up-regulation of its ligand-binding affinity and avidity. Once the open and activated LFA-1 binds to its ligand, signaling events collectively designated “outside-in” signaling ensue [6].

In TCR-induced integrin activation, SLP-76 (SH2-domain containing leukocyte-specific phosphoprotein of 76 kDa) is a key mediator of inside-out signaling [7]. TCR stimulation induces the recruitment of SLP-76 to the phosphorylated transmembrane adapter protein linker for activation of T cells (LAT) via its binding partner Grb2-related adapter downstream of Shc (Gads) [8]. This multi-component signaling complex containing LAT, Gads, and SLP-76 is recruited to the plasma membrane, resulting in association of activated effector proteins and reorganization of the actin cytoskeleton.

We recently showed that SLP-76 is recruited to the plasma membrane by the LFA-1 ligation with ICAM-1, indicating that SLP-76 also functions in outside-in signaling downstream of integrins [9]. However, it is unclear whether SLP-76 additionally plays a role in chemokine-mediated integrin inside-out activation and subsequent T cell adhesion. Although it has been shown that SLP-76 is required for CXCL12α-induced intracellular Ca²⁺ mobilization and Erk activation [10, 11], LAT and the binding of Gads/SLP-76 to LAT have not been implicated in this process. We and others confirmed that the LAT/Gads/SLP-76 complex is neither assembled nor recruited to the plasma membrane in response to LFA-1 ligation or CXCL12α binding in T cells [9, 12]. These data imply that SLP-76 may have different roles in outside-in integrin signaling or chemokine-induced inside-out integrin activation compared to TCR-stimulated inside-out integrin activation.

In this study, we address whether SLP-76 plays a role in CXCR4-mediated T cell adhesion. To mimic physiological environment, we used a flow chamber system in which cells are exposed to continuous shear flow while undergoing the tethering, rolling, and adhesion cascade. Primary T cells deficient in SLP-76 show defective chemokine-induced integrin-mediated firm adhesion under shear flow. Furthermore, we demonstrate that N-terminal phosphotyrosines of SLP-76 are important in regulating T cell arrest under shear flow in response to stimulation by immobilized CXCL12α. Our data thus suggest that SLP-76 plays a critical role in chemokine-induced firm arrest of T cells under physiological shear conditions.

Results and Discussion

SLP-76 is required for optimal CXCL12α-induced T cell firm arrest on ICAM-1 under shear flow

To investigate the role of SLP-76 in CXCL12α-induced firm arrest under shear flow, we first analyzed cell adhesion using Jurkat T cells, a model system that has been a reliable surrogate for primary T cells in many studies investigating T cell signaling and function [13]. Jurkat cells were flowed over immobilized P-selectin/ICAM-1/CXCL12α under a wall shear rate of 100 s⁻¹ (Fig. 1A(i)). Adherent cells were classified as tethered, rolling, or firmly arrested based on their instantaneous velocities and the duration of their stopping (Fig. 1A(ii)). In response to immobilized CXCL12α, SLP-76 deficient J14 cells exhibited a significant reduction in firm arrest and a correspondingly increased tethering and rolling population, compared to WT cells (Fig. 1B and C). Since re-expression of WT SLP-76 in the J14 clone does not rescue the defect in firm arrest or migration in transwell assays (Fig. 1B and C) [11], we analyzed the surface expression levels of integrin α_L (CD11a) of these cells using flow cytometry. As shown in Fig. 1D, the integrin α_L levels of both J14 and SLP-76 retransfected J14 cells were 4-fold lower than that of WT Jurkat cells. We found that the lack of firm arrest of both cell types was well correlated with the inherent defect in the amount of integrin α_L expression. Therefore, we explored the role of SLP-76 for adhesion by “knocking down” SLP-76 expression using RNA interference in WT Jurkat cells. As shown in Fig. 2A, we achieved 80% knock-down using three sequential electroporations of a mix of two siRNAs specific for SLP-76. We found that individual SLP-76 siRNA treatment also significantly lowered the expression levels of SLP-76, indicating that the SLP-76 knock-down using the SLP-76 mix was a specific on-target silencing. When introduced to immobilized CXCL12α under shear flow, SLP-76 knocked-down Jurkat cells showed a substantial decrease in the percentage of firmly arrested cells (36% compared to 52% of the control, Fig. 2B). In addition, for the SLP-76 knocked-down cells that did arrest, the time to stop was significantly decreased and the rolling velocity increased (Fig. 2C). These observations indicate that SLP-76 is necessary for optimal activation of LFA-1 and adhesion stabilization in the gradual transition from rolling to arrest.

Figure 1. SLP-76-deficient Jurkat cells fail to arrest on ICAM-1 under shear flow with the CXCL12α stimulation.

(A) (i) Schematic of the recruitment of T cells on immobilized P-selectin/ICAM-1/CXCL12α substrates under shear flow. T cells were perfused into a parallel-plate flow chamber with having the functionalized substrate at a wall shear rate of 100 s^-1 (flow direction is from left to right). Distance (i.e., rolling distance before stable arrest) and time to stop (i.e., time for rolling prior to stopping) were analyzed from the trajectory of each individual firmly arrested cell. (ii) Each adherent T cells, for instance, was categorized into tethering, rolling or firm arrest based on its terminal velocity and its instantaneous velocity changes within the field of view. (B) WT Jurkat E6-1 cells, SLP-76 deficient J14 cells or SLP-76-retransfected J14 cells (J14 + WT SLP-76) were perfused into a flow chamber with immobilized P-selectin/ICAM-1/CXCL12α at a wall shear rate of 100 s⁻¹ for 5 min. The percent of firmly arrested cells in total adherent cells with each Jurkat SLP-76 variants was shown as the mean ± SEM of three independent experiments. *: P < 0.05. (C) Displacements of adherent E6-1, J14 and J14 + WT SLP-76 cells as a function of time at 100 s⁻¹. Light gray lines represent trajectories of tethering cells, dark gray lines for rolling cells and thick black lines for firmly arrested cells. (D) (i) E6-1, J14 or J14 + WT SLP-76 cells were incubated with CD11a (integrin α_L; thick black lined and gray shaded histograms) or isotype control (thin black lined histograms) mAb and the surface expression levels of CD11a were quantified using flow cytometry. Representative results of three independent experiments are shown. (ii) Relative median fluorescence intensity (MFI) for each Jurkat variants was calculated by dividing the value of (MFI_{CD11a mAb} minus MFI_{control mAb}) by MFI_{control mAb}. Results are presented as the mean of relative MFI ± SEM of three independent experiments. *: P < 0.001; N.S.: not statistically significant.

Figure 2. SLP-76 is critical in firm arrest of T cells under shear flow with the CXCL12α stimulation.

(A) Knockdown of SLP-76 in E6-1 cells by RNA interference. Western blots of total cell lysates from E6-1 cells at 72 h post three sequential electroporations with a negative control scrambled siRNA (siC), a mix of two siRNAs targeting SLP-76 (siSLP-76 (1,2)) or two individual SLP-76 siRNA (siSLP-76 (1) and siSLP-76 (2)) are shown. The SLP-76 protein level in siC was set to 1 and the protein levels in siSLP-76 sets are presented as the mean ± SEM of three independent experiments. *: P < 0.05. (B) Number of adherent cells and adhesion type with E6-1 cells transfected with siC or with siSLP-76 (1,2). *: P < 0.05. (C) Time and distance to stop of all firmly adherent cells and rolling velocities from E6-1 cells transfected with siC or with siSLP-76 (1,2). Data are shown as the mean ± SEM of three independent experiments. *: P < 0.05. (D) WT or SLP-76 cKO T lymphocytes were perfused on the ICAM-1/CXCL12α-substrates at 100 s⁻¹. Cell adhesion was sorted into stable (i.e., stopping time > 40 s) or transient arrest and data are expressed as the mean ± SEM of three independent experiments. *: P < 0.05.

Based on the results obtained with cell lines, we next investigated the role of SLP-76 in T cell arrest under flow using primary T cells. Flow chamber experiments were performed with primary T cells from WT or SLP-76 conditional knockout (cKO) mice. The arrest of primary T cells on surfaces immobilized with ICAM-1/CXCL12α was classified as either “transient arrest” or “stable arrest”, because selectin which induces rolling adhesion was not applied in these experiments. Arrest sustained for more than 40 s in this study is termed stable arrest. SLP-76 cKO T cells exhibited a significant decrease in stable arrest at 100 s⁻¹ compared to WT T cells (Fig. 2D). These data along with our previous work [9] demonstrate that SLP-76 deficiency in murine T cells decreases the number of stably arrested cells on ICAM-1 in response to immobilized CXCL12α under physiological shear flow, indicating that SLP-76 appears to be required for the transition from transient adhesion to firm adhesion.

In contrast with our results, Horn et al. [12] recently reported that deletion of SLP-76 by siRNA transfection in human T cells did not affect CXCL12α-induced T cell chemokinesis and transwell migration, nor did it affect the ability of T cells to bind either soluble or immobilized ICAM-1. We speculate that the discrepancy in results might be due to differences in assay conditions. Continuous shear forces employed in our study might prevent the unstable adherent cells from becoming firmly arrested, while under static conditions it is possible that T cells in contact with ICAM-1 could activate alternative signaling pathways that do not require SLP-76.

Role of the N-terminal tyrosines of SLP-76 in T cell firm arrest on ICAM-1 under shear flow

The SLP-76 N-terminal tyrosines are critical for its function in TCR signaling [14]. The Y112 and Y128 residues are required for binding to the guanine nucleotide exchange factor, Vav1 and the adapter protein, Nck [14, 15]. Y145 is crucial for the interaction of SLP-76 with Tec family tyrosine kinase IL-2-inducible T cell kinase (Itk) [16]. To examine the role of these N-terminal tyrosines in CXCL12α-induced LFA-1-mediated firm arrest of T cells under shear flow, we analyzed the adhesion of T cells from mutant mice where tyrosine residues were mutated to phenylalanine (Y112/118F or Y145F). Both Y112/128F and Y145F T cells exhibited defective arrest on P-selectin/ICAM-1/CXCL12α-surfaces compared to WT cells (Fig. 3A). In addition, T cells from both SLP-76 Y-F knock-in (KI) lines demonstrated a greater proportion that undergo tethering and rolling compared to WT cells in response to immobilized CXCL12α, suggesting that these mutations lead to defects in the transition from transient adhesion to firm arrest in CXCR4-induced activation. In the absence of immobilized chemokine, the majority of cells simply tethered or rolled (Fig. 3A). When firmly arrested cells were further analyzed for time and distance to stop, we found that the Y-F KI T cells were weakly adherent, indicated by long stop times and rolling distance before stopping (Fig. 3B and C), suggesting that LFA-1-ICAM-1-mediated firm arrest is impaired in both Y-F KI T cells. To ensure that the SLP-76 dependent effects on adhesion were due to impaired signaling through engaged receptors and not due to a global defect in the SLP-76 deficient cells, we bypassed the receptor-mediated signaling by stimulating WT and SLP-76 Y-F KI T cells with PMA. As shown in Fig. 3D, this completely rescued the adhesion defect in the mutant T cells.

Figure 3. SLP-76 Y112/128F and Y145F T cells show impaired CXCL12α-triggered and LFA-1-mediated arrest under shear flow.

T cells from WT, SLP-76 Y112/128F, or Y145F mice were perfused into a flow chamber with immobilized P-selectin/ICAM-1/CXCL12α at 100 s⁻¹. (A) Adhesion types are classified as tethering, rolling or firm arrest. Data obtained from three independent experiments were accumulated to show the overall change in the adhesion type. n (number of adherent cells) = 113 in WT without CXCL12α; n = 199 in WT with CXCL12α; n = 132 in Y112/128F without CXCL12α; n = 152 in Y112/128F with CXCL12α; n = 145 in Y145F without CXCL12α; n = 202 in Y145F with CXCL12α. Time (B) and distance (C) to stop of all firmly arrested cells in WT, Y112/128F or Y145F. (D) Adhesion of WT, Y112/128F and Y145F T cells was measured among cells subjected to PMA stimulation. Data are expressed as the mean ± SEM of three independent experiments. *: P < 0.05.

Collectively, our data show that the adapter protein, SLP-76, plays a critical role in the gradual transition of CXCR4-engaged T cell arrest on ICAM-1 under shear flow using an in vitro flow chamber. It is likely that SLP-76 is important in local signaling events in which chemokine receptors engaged with surface-bound chemokines transmit the instantaneous signal to proximal β₂ integrins undergoing rapid increase in adhesiveness to ICAM-1 under flow, as demonstrated by Alon and co-workers [17, 18]. Interestingly, Zarbock and colleagues recently showed that SLP-76 is also involved in global signaling events in which neutrophil binding to E-selectin induces progressive activation of LFA-1 without chemokine stimulation [19]. Further studies are necessary to understand how SLP-76 discerns or integrates two signaling networks to enhance integrin avidity and leukocyte arrest. In addition, we find that the SLP- 76 N-terminal tyrosines are important in CXCL12α-triggered and ICAM-1-mediated T lymphocyte adhesion under shear flow by enhancing integrin affinity and stabilizing T cell arrest. The work by Teixidó’s group has established that T cell exposure to CXCL12 activates Vav1-Rac signaling [20]. This group also demonstrated that Vav1 dissociation from talin under CXCL12 stimulation leads to integrin α₄β₁ activation and T cell adhesion [21]. Our work adds further potential mechanism to the function of Vav1 as a molecular linker and implies that Vav1 must associate with SLP-76 for efficient integrin α_Lβ₂- mediated T cell adhesion.

Modulating integrin-mediated cell adhesion is an attractive strategy for the treatment of inflammatory and autoimmune diseases. While antibodies blocking integrin-ligand binding have shown therapeutic success not only in animal models but in humans for treatment of psoriasis, multiple sclerosis and Crohn’s disease [22–24], this complete and non-specific interference of integrin function has led to a number of serious side effects [25, 26]. An alternative approach is to modulate integrin signaling by targeting molecules in the integrin activation pathway. Our study further reveals the critical role of SLP-76 in integrin signaling, not only in both TCR-mediated inside-out and outside-in pathways, as previously shown, but also in chemokine-induced integrin-mediated cell adhesion. Future studies investigating the functional domains and cellular localization of SLP-76, through which it functions in the distinct integrin signaling pathways, will help better understand the mechanisms of integrin activation and the design of therapeutic strategies to modulate integrin function in a more precise manner.

Materials and methods

Western blot analysis, flow cytometry, data acquisition, and cell tracking and analysis are described in the Supporting Information.

Cell lines and mice

Jurkat T cells (E6-1 clone), SLP-76-deficient stable Jurkat cells, J14, and J14 retransfected with WT SLP-76 have been described previously [13]. SLP-76 cKO and SLP-76 Y-F KI mice (Y112/128F and Y145F) have been described previously [9, 27, 28]. Mouse handling and procedures were in strict accordance with University of Pennsylvania and IACUC protocols. Primary T cells from spleens of 6-8-week-old mice were purified by negative selection and magnetic separation (Miltenyi Biotec, Auburn, CA). Cells were activated by plate-bound anti-CD3 (1 μg/ml; eBioscience, San Diego, CA) and anti-CD28 (5 μg/ml; eBioscience) in IMDM medium (supplemented with 10% FBS) for 2–3 days.

siRNA transfection

Jurkat cells (ATCC, Manassas, VA) were maintained in RPMI1640 medium supplemented with 10% FBS at 37°C with 5% CO₂. For electroporation of siRNA, E6-1 cells were resuspended in 100 μl resuspension buffer R (Invitrogen, Carlsbad, CA). a mix of two siRNAs (SLP-76 (1,2))(SLP-76 (1): SASI_Hs01_00219492 and SLP-76 (2): SASI_Hs01_00219493 from Sigma-Aldrich, St. Louis, MO) against SLP-76 (official symbol: LCP2; gene ID: 3937) or Cy3-labeled negative control #1 siRNA (Ambion, Cat #: AM4621, Austin, TX) was added to 2×10⁶ cells/transfection (with a 100 μl electroporation pipet tip). Within 10 min, cells were transfected by electroporation (pulse voltage: 1,350 V; pulse width: 10 ms; pulse number: 3 with MicroPorator MP-100 from Digital-Bio, Hopkinton, MA). The cells were then added to prewarmed cell culture medium, as described above. The transfection was repeated at 24 and 48 h, and cells were used at 72 h for Western blotting and flow chamber experiments.

Flow chamber assays

Cell adhesion assays were performed in a parallel-plate flow chamber (GlycoTech, Gaithersburg, MD). Polystyrene Petri dishes (Corning 430588, Corning, NY) enclosed using a single well flexiPERM (Sigma-Aldrich) were coated with a mixed solution of 2 μg/ml Protein A/G (Thermo Scientific, Rockford, IL) and 1 μg/ml CXCL12α (R&D Systems, Minneapolis, MN) at 4°C overnight. The surfaces were then washed with 1% BSA in PBS. A mixture of 0.2 μg/ml P-selectin/Fc and 0.4 μg/ml ICAM-1/Fc (for primary T cells; 2 μg/ml P-selectin/Fc and 4 μg/ml ICAM-1/Fc for Jurkat cells transfected with siRNA; 2 μg/ml P-selectin/Fc and 8 μg/ml ICAM-1/Fc for E6-1, J14 and J14 + WT SLP-76) was applied to the surfaces and allowed to bind the Protein A/G-coated surfaces at room temperature for 3 h. The surfaces were then blocked for non-specific adhesion with 2% BSA in PBS at room temperature for 1 h. An assembled flow chamber was mounted on an inverted microscope, Axiovert 200 (Carl Zeiss, Gottingen, Germany) enclosed by a microscope incubator, XL-3 (Carl Zeiss) to perform the flow chamber experiments at 37°C. The T cells (5×10⁵/ml) were perfused into the chamber at a flow rate corresponding to a calculated wall shear rate of 100 s⁻¹. Adhesion of T cells on immobilized P-selectin/ICAM-1 in response to immobilized CXCL12α was observed using phase contrast microscopy under a 10× objective (NA = 0.20; Type: A-Plan). Adherent T lymphocytes were classified as tethering, rolling, or firmly arrested cells based on their instantaneous velocities and the duration of their stopping.

Statistical Analyses

Data were analyzed by unpaired and one-tailed Student’s t-test for comparisons between two sets or one-way analysis of variance (ANOVA), followed by Tukey-Kramer test for multiple comparisons. In both analyses, the minimum acceptable level of significance was P < 0.05.

Abbreviations

cKO

conditional knockout

Gads

Grb2-related adapter downstream of Shc

Itk

IL-2-inducible T cell kinase

KI

knock-in

LAT

linker for activation of T cells

LFA-1

lymphocyte function-associated protein-1

N.S

not statistically significant

SDF-1α

stromal cell-derived factor-1α

SLO

secondary lymphoid organ

SLP-76

Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa