Protein kinase D isoforms are dispensable for integrin‐mediated lymphocyte adhesion and homing to lymphoid tissues

Abstract

Leukocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins are essential for lymphocyte adhesion, trafficking and effector functions. Protein kinase D (PKD) has previously been implicated in lymphocyte integrin regulation through regulation of Rap1 activity. However, the true role of PKD in integrin regulation in primary lymphocytes has not previously been investigated. The major PKD isoform in lymphocytes is PKD2. Here we employed PKD2-deficient mice, a specific PKD kinase inhibitor, as well as PKD-null DT40 B cells to investigate the role of PKD in integrin regulation in lymphocytes. We report that PKD2-deficient lymphocytes bound normally to integrin ligands in static and shear flow adhesion assays. They also homed normally to lymphoid organs after adoptive transfer into wild-type mice. DT40 B cells devoid of any PKD isoforms and primary lymphocytes pretreated with a specific PKD inhibitor bound normally to integrin ligands, indicating that multiple PKD isoforms do not redundantly regulate lymphocyte integrins. In addition, PKD2-deficient lymphocytes, as well as DT40 cells devoid of any PKD isoforms, could activate Rap1 in response to B-cell receptor ligation or phorbol ester treatment. Together, these results show that the PKD family does not play a critical role in lymphocyte integrin-mediated cell adhesion or lymphocyte trafficking in vivo.

The Spontaneously Adhesive Leukocyte Function-associated Antigen-1 (LFA-1) Integrin in Effector T Cells Mediates Rapid Actin- and Calmodulin-dependent Adhesion Strengthening to Ligand under Shear Flow

“…PKC/protein kinase D (PKD) signaling are downstream of phospholipase C in cells, and PKC isoforms have recently been implicated in neutrophil adhesion strengthening under conditions of shear flow (17). However, effector T cell adhesion to ICAM-1 under shear flow conditions is independent of PKC (this report), and we have previously reported that it is also independent of PKD (9). In contrast, we now show the process to be dependent on calcium/calmodulin signaling downstream of the active integrin.…”

“…Adhesion Assay under Shear Flow-The assay was performed essentially as described (8,9). Ibidi -slides VI 0.4 were coated with 6 g/ml (unless otherwise stated) ICAM-1 overnight at 4°C.…”

“…We therefore further focused on LFA-1-ICAM-1 interactions under shear flow conditions and the requirements for inside-out and outside-in signals for this process. To do this, we used a shear flow assay we have previously described and characterized, where lymphocytes are allowed to adhere to ICAM-1 under relatively low shear flow in the presence or absence of integrin inside-out stimulation (8,9). This allows for the analysis of the integrin component of cell adhesion under shear flow conditions, without interference from other adhesion molecules such as selectins (which are required for cell rolling/capture at higher shear forces).…”

Optimal T Cell Activation and B Cell Antibody Responses In Vivo Require the Interaction between Leukocyte Function–Associated Antigen-1 and Kindlin-3

“…Where appropriate, cells were stimulated with 200 nM PdBu (Sigma-Aldrich), 10 mg/ml anti-BCR (R&D Systems), 10 mg/ml anti-CD3 (R&D Systems), 0.2 mg/ml LPS (Sigma-Aldrich), or 5 mM MgCl 2 + 1 mM EGTA immediately before being added to the plate. Cells were allowed to adhere for 30 min at 37˚C before gentle washing and detection, as described (12).…”

The β2 integrin–kindlin-3 interaction is essential for T-cell homing but dispensable for T-cell activation in vivo

“…Where indicated, cells were stimulated with 200 nM PdBu (Sigma-Aldrich) or 10 mg/mL anti-CD3 (clone 2C11; R&D Systems) immediately before addition to the plate for 30 minutes at 37°C, before gentle washing and detection as previously described. 19 Adhesion under shear flow Ibidi VI 0.4 m-slides were coated with 6 mg/mL ICAM-1 overnight at 4°C. Alternatively, mouse endothelial cells (bEnd.3 cells) were grown on plates and stimulated for 24 hours with 10 ng/mL TNFa.…”