PKD2-deficient lymphocytes display normal adhesion responses to ICAM-1 and fibronectin
PKD2 is the major isoform expressed in primary naïve and effector murine lymphocytes, with the other family members either expressed at very low levels (PKD3) or not expressed at all (PKD1) ([] and data not shown). We therefore initially focused on the role of PKD2 as a candidate regulator of integrin-mediated lymphocyte adhesion. Initial experiments confirmed normal expression of both the β1 and β2 integrin chains on the surface of PKD2-deficient lymphocytes (data not shown). We went on to analyse cell adhesion responses of both wild-type (WT) and PKD2-deficient lymphocytes to the β1 and β2 integrin ligands ICAM-1 and fibronectin using static adhesion assays. Adhesion of PKD2-deficient lymph node cells to either integrin ligand was comparable to that observed in WT lymphocytes, whether the cells were left untreated or whether they were stimulated with either phorbol esters or TCR ligands, to activate PKC, and therefore PKD, signalling (Fig. 1A and data not shown). Similarly, no defects were observed in integrin-mediated adhesion of untreated, phorbol ester-treated or antigen-receptor activated PKD2-deficient effector T cells (Fig. 1C and D), PKD2-deficient splenocytes or purified PKD2-deficient B-cells (data not shown). To exclude the possibility that maximal stimulatory conditions and/or excess integrin ligand amounts in the adhesion assays could overcome PKD2 deficiency in these cells, we also examined cell adhesion under different assay conditions. However, integrin-mediated adhesion of WT and PKD2-deficient lymphocytes to lower ligand densities or using shorter assay times did not reveal any observable adhesion defects in the PKD2-deficient lymphocytes compared to WT cells (Fig. 1B and data not shown). Thus, PKD2 does not play a significant role in integrin-mediated cell adhesion responses in primary lymphocytes under static conditions.
Figure 1. Adhesion of WT and PKD2-deficient lymphocytes to the integrin ligands ICAM-1 and fibronectin. (A and B) Lymphocytes isolated from the lymph nodes of wild-type (WT) and PKD2-deficient (PKD2 KO) mice were allowed to adhere to plastic surfaces coated (A) with or without 6 μg/mL ICAM-1 or (B) 1 μg/mL ICAM-1 for 20 min. Cells were left untreated or were treated with either 200n M PdBu or with 10 μg/mL of crosslinking TCR antibodies just prior to their addition to integrin-ligand coated plates. Data are shown as mean + standard error of the mean (SEM) of data pooled from four (A) or three (B) independent experiments, each performed in duplicate. (C/D) Adhesion of WT and PKD2 KO effector T cells to ICAM-1 (6 μg/mL, C) or fibronectin (10 μg/mL, D) coated surfaces was performed as in (A). Data are shown as mean + SEM of data pooled from five to eight independent experiments, each performed in duplicate or triplicate. There were no significant differences in the ability of WT versus PKD2-deficient cells to adhere to integrin ligands any of these assays (p > 0.05).
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One of the most important functions of integrins is to mediate cell adhesion under shear flow conditions, a process that is critical to permit the firm adhesion of cells to high endothelial venules (HEV) []. Hence, we also investigated whether PKD2 was required to mediate high-affinity adhesion of primary lymphocytes to integrin ligands under conditions of fluid shear stress. WT lymphocytes isolated from secondary lymphoid tissues (spleen or lymph nodes) were able to activate their β2-integrins and adhere firmly to ICAM-1 under conditions of fluid shear stress in response to T- or B-cell antigen receptor stimulation or PdBu treatment (Fig. 2A to C). In contrast, resting WT lymphocytes were approximately threefold less efficient at binding to ICAM-1 under shear stress than activated WT lymphocytes (data not shown). Importantly, the ability of activated lymphocytes to adhere to the LFA-1 ligand ICAM-1 under conditions of fluid shear stress was not affected by PKD2-deficiency (Fig. 2A to C). Lowering the ligand density in the assay (Fig. 2D) or increasing the shear flow rate (Fig. 2E and F) did not reveal any adhesion defects in PKD2-deficient cells.
Figure 2. Integrin-mediated cell adhesion responses of WT and PKD2-deficient lymphocytes under conditions of fluid shear stress. Adhesion of WT (black bars) versus PKD2-deficient (PKD2 KO, white bars) spleen and lymph node lymphocytes to ICAM-1-coated surfaces under shear flow conditions was examined and quantified as described in the Materials and Methods. Surfaces were coated with either (D) 1 μg/mL or (A–C, E, F) 6 μg/mL ICAM-1. Lymphocytes were treated with either 10 μg/mL of crosslinking (A, D, E) BCR or (B, F) TCR antibodies or with (C) 200 nM PdBu before the start of the assay. Fluid shear flow rates were set at 0.3–0.5 dynes/cm2 (A–D) or 1 dynes/cm2 (E, F). Data are shown as mean + SEM of data pooled from three to four (A, C) or two (B, D, E, F) independent experiments, each performed in duplicate or triplicate. There were no significant differences in the ability of WT versus PKD2-deficient cells to adhere to integrin ligands in these assays (p > 0.05).
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PKD2 is not required for lymphocyte migration in vitro, nor for trafficking of lymphocytes in vivo
Integrins are essential for the ability of lymphocytes to migrate from the bloodstream into secondary lymphoid tissues via the regulation of cell adhesion, spreading and migration. All three mammalian PKD isoforms have been identified as novel regulators of actin-driven cell migration in many cell types [[33-36, 39, 40]]. As PKD1 is not expressed in naïve or effector lymphocytes and PKD3 is expressed only at very low levels ([] and data not shown), we were interested in whether PKD2 was required to regulate migratory responses of primary lymphocytes. In an in vitro assay, WT or PKD2-deficient lymphocytes were added to transwell filters coated with the β1-integrin ligand fibronectin and left to migrate towards an stromal cell-derived factor 1 (SDF-1α) chemokine gradient for 4 h. As shown in Fig. 3A, fibronectin-mediated, SDF-1α-induced migration of PKD2-deficient lymphocytes was equivalent to that of WT lymphocytes. Similarly, there were no significant differences in the ability of WT and PKD2-deficient lymphocytes to migrate towards SDF-1α in vitro when the assay conditions were varied (shorter assay times, lower chemokine concentrations; data not shown). To confirm these results, we assessed the ability of PKD2-deficient lymphocytes to successfully migrate from the bloodstream into secondary lymphoid tissues in vivo by mixing WT and PKD2-deficient lymphocytes at a ratio 1:1 and injecting them into the tail vein of WT hosts. Subsequently, blood, lymph nodes and spleen were analysed for the presence of these transferred cells. As shown in Fig. 3B, PKD2-deficient and WT T and B cells were equally present in the blood, and importantly, PKD2-deficient lymphocytes could exit the blood and enter secondary lymphoid tissues normally.
Figure 3. In vitro migration and in vivo trafficking of WT and PKD2-deficient lymphocytes to secondary lymphoid tissues. (A) Splenocytes from WT (black bars) and PKD2-deficient mice (PKD2 KO, white bars) were left to migrate across fibronectin-coated membranes for 4 h in response to the chemokine SDF-1α (250 ng/mL). Data are shown as mean ± SEM of data pooled from two independent experiments, each performed in duplicate. (B–D) Lymphocytes from WT mice labelled with CFSE and PKD2-deficient mice labelled with CellTrace Violet or CMTMR were mixed at a ratio of 1:1 before being injected into C57BL/6 host mice. Values indicate recovery of WT (black symbols) or PKD2-deficient (white symbols) TCRβ+ T cells (circles) or B220+ B cells (squares) as a percentage of the total recovered transferred cells from the (B) blood, (C) spleen and (D) lymph nodes 3 h after transfer. The data shown are pooled from two independent experiments, with each dot indicating one individual mouse (n = 8); horizontal bars indicate means.
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Another essential function for LFA-1 and VLA-4 integrins is to regulate lymphocyte entry into the splenic white pulp []. Absolute numbers of T and B lymphocytes in the spleens of PKD2-deficient mice are normal [] and, as shown in Fig. 4A, splenic white pulp architecture in PKD2-deficient mice is also normal, with B220+ B and TCRβ+ T cells located within B-cell follicles and T cell zones, respectively. Integrins also play an important role in maintaining correct lymphocyte compartmentalization within the spleen, specifically by retaining marginal zone (MZ) B cells at the marginal zone []. We therefore asked whether MZ B-cell numbers and localization within the spleens of PKD2-deficient mice was defective or not. Histological analysis revealed normal marginal zone architecture in the spleens of PKD2-deficient versus WT mice (data not shown). Furthermore, the frequency and absolute numbers of IgMHiCD21HiCD23Lo MZ B cells and IgD+CD21LoCD23Hi follicular B cells in the spleens of PKD2-deficient mice were comparable to that of WT mice (Fig. 4B). Collectively, these data argue that PKD2 does not play an important role in mediating adhesion and migratory responses of lymphocytes into, and positioning within, secondary lymphoid tissues in vivo.
Figure 4. Analysis of lymphocyte compartmentalization within the spleens of WT and PKD2-deficient mice. (A) Spleen sections of WT (left) or PKD2-deficient (right) mice were stained with anti-B220 (green) and anti-TCRβ (red) antibodies. (B) B220+ MZ and follicular B-cell subsets in the spleens of 8–12-week-old WT (top) and PKD2-deficient (PKD2 KO, bottom) mice were analysed by flow cytometry using CD23 and CD21 antibodies, as described in the Materials and Methods. Graph represents the absolute cell numbers of the different B-cell subsets and data are shown as mean + SEM of nine mice, analysed in five separate experiments.
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A B-cell line devoid of any PKD isoforms can mediate normal adhesion to integrin ligands and a specific PKD inhibitor that abolishes PKD activity in cells has no effect on integrin-mediated adhesion in murine lymphocytes.
Functional redundancy between different PKD family members has been described previously []. Given that a second PKD isoform, PKD3, is also expressed in murine lymphocytes, albeit at very low levels [], we wanted to address whether PKD isoforms could redundantly regulate integrin activity/function in lymphocytes. Therefore, we made use of a previously described avian PKD-null DT40 B-cell line that expresses no PKD isoforms at all. As shown in Fig. 5A, the ability of PKD-null DT40 B cells to adhere to the β1-integrin ligand fibronectin was not significantly different to that of WT DT40 B cells, either under resting conditions or after stimulation with either phorbol ester or a B-cell receptor (BCR) ligand. As avian ICAM-1 is not available and DT40 cells do not adhere to human or mouse ICAM-1, we turned to an aggregation assay to assess LFA-1 function in these cells. The LFA-1 ligand ICAM-1 is expressed on the surface of lymphocytes and LFA-1 can mediate cell–cell adhesion by binding to this ligand in-trans in response to specific extracellular stimuli [[42-44]]. We observed that PKD-null DT40 B cells could aggregate normally in response to both a BCR ligand or phorbol ester treatment (Fig. 5B), suggesting that LFA-1-mediated cell adhesion responses are normal in PKD-null DT40 B cells.
Figure 5. Analysis of integrin-mediated cell adhesion responses of WT and PKD-null DT40 B-cells. (A) Adhesion of WT versus PKD-null DT40 B cells to fibronectin-coated surfaces was examined as described in the Materials and Methods. Cells were left unstimulated or were treated with either 200 nM PdBu or were stimulated with a BCR ligand (10 μg/mL M4 mAb) just prior to their addition to fibronectin-coated plates. Data are shown as mean ± SEM of data pooled from four (control, PdBu) and two (BCR) independent experiments, each performed in duplicate. (B) LFA-1 mediated cell:cell adhesion of WT (black bars) and PKD-null (white bars) DT40 B cells was assessed as described in the Materials and Methods. Data are shown as mean + SEM of data pooled from three independent experiments, each performed in duplicate.
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To ensure that the normal adhesion properties of PKD-null cells were not an artefact due to changes in signalling pathways in the immortalized DT40 cell line, we also performed experiments with a recently described PKD inhibitor that inhibits all three mammalian PKD isoforms with a high degree of selectivity [[45, 46]]. Pre-treatment of WT murine splenocytes with 5–10 μM of this inhibitor abolished PKD2 activity, as assessed by western blotting with a phosphospecific antibody that detects active, autophosphorylated PKD1/2 (Fig. 6A) []. In contrast, adhesion of lymph node cells and splenocytes to ICAM-1 and fibro-nectin under basal and stimulated conditions were similar for both non-treated and PKD inhibitor-treated cells (Fig. 6B and C and data not shown). Varying ligand concentrations, amounts of stimulatory agents or incubation time for the adhesion assays also did not reveal any adhesion defects in the adhesion properties of the PKD-inhibitor treated cells (Fig. 6D and data not shown). Similarly, the adhesion properties of PKD inhibitor-treated lymphocytes to low or high ICAM-1 ligand densities under conditions of increasing shear flow (0.3–1 dynes/cm2) was comparable to that of WT cells (Fig. 6E to G and data not shown). We also performed detachment assays to assess whether there were any significant differences in the strength of adhesion of PKD inhibitor-treated lymphocytes for ICAM-1. Here, non-treated and PKD inhibitor treated lymphocytes were stimulated with a BCR ligand and then allowed to adhere to ICAM-1 ligands at low shear flow rate (0.3–0.5 dynes/cm2) before the flow rate was increased to 1 dynes/cm2. Under these conditions we observed no significant differences in the rate of detachment of PKD2 inhibitor treated cells versus control cells from ICAM-1 coated surfaces (Fig. 6H). Based upon these experiments, we conclude that PKD isoforms do not play an important role in regulating integrin-mediated cell adhesion responses in lymphocytes.
Figure 6. PKD isoforms do not redundantly regulate adhesion of murine lymphocytes to integrin ligands. (A) Primary murine splenocytes were pre-treated with dimethylsulfoxide (DMSO) solvent or with increasing concentrations of a PKD specific inhibitor (0.625–10 μM) for 1 h before stimulation with either a BCR ligand (10 μg/mL, left) or with 200 nM PdBu (right) for 30 min. The cells were then lysed and protein extracts analysed by SDS-PAGE and western blotting for auto-phosphorylated PKD2 and total PKD2 expression. Data are representative of two independent experiments. (B–D) Static adhesion assays were performed using lymphocytes isolated from the (B) spleens or (C, D) lymph nodes of WT mice. The cells were pre-treated with DMSO solvent or with 5 μM of a PKD specific inhibitor for 1 h before they were left untreated or were activated with either 200 nM PdBu or with 10 μg/mL of crosslinking BCR (splenocytes) or TCR (lymph node cells) antibodies just prior to their addition to plates coated with 6 μg/mL ICAM-1 (B,C) or 1 μg/ml ICAM-1 (D). Data are shown as mean + SEM of data pooled from four (splenocytes) or three (lymph node cells) independent experiments, each performed in duplicate. (E–G) Adhesion of antigen receptor activated control (DMSO, black bars) or PKD-inhibitor treated (5 μM, white bars) WT spleen and lymph node cells to surfaces coated with 6 μg/mL ICAM-1 under low (0.3–0.5 dynes/cm2; E, F) or high (1 dynes/cm2; G) shear flow conditions was analysed as described above. (H) Antigen receptor activated control (DMSO, black bars) or PKD-inhibitor treated (5 μM, white bars) splenocytes were allowed to adhere to ICAM1 (6 μg/mL) coated surfaces under low (0.3–0.5 dynes/cm2) shear flow conditions for 5 min before switching to high shear flow (1 dyne/cm2; arrow). Data (E–H) are shown as mean + SEM of data pooled from two independent experiments, each performed in duplicate. There was no significant effect of the PKD inhibitor on the adhesion of WT lymphocytes to adhere to integrin ligands in any of these assays (p > 0.05).
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