IFN{alpha} enhances human B-cell chemotaxis by modulating ligand-induced chemokine receptor signaling and internalization

Gamal Badr, Gwenoline Borhis, Dominique Treton and Yolande Richard

Institut National de la Santé et de la Recherche Medicale U 131, Institut Paris-Sud sur les Cytokines, 32 rue des Carnets, 92 140, Clamart, France

Correspondence to: Y. Richard; E-mail: yolande.richard{at}ipsc.u-psud.fr


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
In this study, we show that IFN{alpha} increases the chemotaxis of human B cells to CCL20, CCL21 and CXCL12 in a dose- and time-dependent manner. The effect was maximal with 2000 IU ml–1 IFN{alpha}. It peaked at 24 h and decreased thereafter. At 24 h, IFN{alpha} had increased B-cell chemotaxis to CCL20 by 20 ± 6.2% (n = 9, P < 0.002), to CCL21 by 20 ± 8.5% (n = 14, P < 0.0001) and to CXCL12 by 16.3 ± 4.2% (n = 12, P < 0.003) without changing CCR6, CCR7 or CXCR4 expression. IFN{alpha} enhanced the migration of memory B cells to CCL20, CCL21 and CXCL12 2.6-fold more strongly than that of naive B cells. The triggering of chemokine receptors by their ligands resulted in the activation of phosphatidylinositide-3 kinase (PI3K)/protein kinase B (PKB), inhibitory NF-{kappa}B (I{kappa}B{alpha}) RhoA and extracellular signal-regulated protein kinase 1/2 (ERK1/2). All these effectors except ERK1/2 are crucial for B-cell chemotaxis. IFN{alpha} modulated the requirements for B-cell chemotaxis, which became dependent on ERK1/2, more dependent on PI3K, RhoA and nuclear factor-{kappa}B but less dependent on Gß{gamma} and phospholipase C activation. IFN{alpha} also decreased ligand-induced chemokine receptor internalization in a manner dependent on PI3K/AKT and RhoA but not on I{kappa}B{alpha} and ERK1/2. Our data characterize chemokine receptor signaling in human B cells and clarify the relevance of downstream pathways in B-cell chemotaxis and chemokine receptor internalization. They also suggest that non-class I PI3K are involved in B-cell chemotaxis.

Keywords: CCR6, CCR7, cell migration chemokines, CXCR4


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Type I IFNs (e.g. IFN{alpha}/ß) modulate innate and specific anti-viral immunity. They are also key cytokines, inducing effective adaptive immunity due to their pleiotropic effects on various types of immune cell (1). In particular, they increase the proliferation of anti-IgM-stimulated normal and some leukemic B cells (2) and the survival of circulating B cells in the absence of a mitogenic stimulus (3). They also prevent the antigen receptor-mediated programed death of germinal center (GC)-like cells (4) and act as pro-apoptotic cytokines in hairy cell leukemia (57). Type I IFNs strongly enhance humoral immunity in vivo. They increase the primary response to soluble proteins, promote isotype switching and induce long-term antibody production (8). Type I IFNs also affect the re-circulation of normal and malignant B lymphocytes, both in vitro and in vivo (9, 10). The mechanisms regulating IFN{alpha}-induced cell proliferation and apoptosis/survival have been extensively studied, whereas those mediating the effects of type I IFNs on chemotaxis are largely unknown.

Lymphocyte re-circulation, which is critical for effective immunity, is tightly regulated by the expression of adhesion molecules and chemoattractant receptors on lymphocytes, combined with the spatial and temporal expression of ligands for these receptors by a variety of tissue cells (11,12). Human B cells express several chemokine receptors including CCR6, CXCR4, CCR7 and CXCR5 and respond to their cognate ligands CCL20, CXCL12, CCL21 or CCL19 and CXCL13, respectively. Triggering of the B-cell antigen receptor, CD40 and IL4 receptor, modulates chemokine receptor expression and chemotaxis in B cells (1317). Chemokine receptors are coupled to heterotrimeric G{alpha}ß{gamma} proteins. Agonistic binding to the receptor induces dissociation of the G{alpha} and Gß{gamma} subunits, which then independently activate downstream effectors. Chemokine receptors can couple to various pertussis toxin (PTX)-senstitive (G{alpha}i) and -insensitive (G{alpha}q and G{alpha}15/16) G{alpha} proteins, but chemotaxis is only observed upon activation of G{alpha}i-coupled receptors (18). Several reports have provided strong evidence that Gß{gamma} rather than G{alpha} are the essential intermediates in the initiation of chemotactic response (19). Chemokine receptor–ligand interactions trigger many intracellular signals including phosphatidylinositide-3 kinase {gamma} or {delta} (PI3K{gamma}/{delta}), phospholipase Cß (PLCß) and nuclear factor-{kappa}B (NF-{kappa}B) (2022). PLCß triggers a rapid Ca++ flux and activates protein kinase C, whereas PI3K{gamma}/{delta} activates AKT and extracellular signal-regulated protein kinase 1/2 (ERK1/2) (23). In leukocytes, Gß{gamma} subunits can also activate Ras proteins, which in turn activate all type I PI3Ks (24). Gß{gamma} stimulation also rapidly activates G protein-coupled receptor kinases, which phosphorylate chemokine receptors, induce their association with ß-arrestin (25) and their rapid internalization (26).

In this study, we analyzed the effect of IFN{alpha} on the expression of CCR6, CCR7 and CXCR4 on the surface of human B cells and on the chemotactic response of these cells to the corresponding ligands: CCL20, CCL21 and CXCL12. IFN{alpha} had no chemotactic effect alone, but instead increased B-cell chemotaxis by modulating ligand-induced cell signaling and chemokine receptor internalization. Both effects depended on IFN{alpha}-induced PI3K and RhoA activation. This work shows that ERK1/2 is not required for the chemotaxis of medium-treated B cells but seems to play some role in that of IFN{alpha}-treated B cells. In contrast to the widely accepted view that chemotaxis is regulated by PI3K{gamma} or {delta}, our data indicate that non-class I PI3K are also probably involved in B-cell chemotaxis.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Flow cytometry
Chemokine receptor expression was analyzed by flow cytometry using PE-conjugated anti-CCR6, anti-CCR7 and anti-CXCR4 mAbs purchased from R&D Systems (Abingdon, UK). Mouse isotype-matched PE-conjugated control IgG1 and IgG2a were purchased from BD Bioscences (Le Pont de Claix, France). A FACScanTM flow cytometer was used for data acquisition and CellQuest® software (BD Biosciences) was used for data analysis. After gating on viable cells, 10 000 cells per sample were analyzed. For each marker, the threshold of positivity was defined by the non-specific binding observed in the presence of the relevant control IgG.

B-cell preparation and culture
B cells were obtained from palatine tonsils as previously described (27). Briefly, after one cycle of rosette formation, residual T cells and monocytes were depleted with CD2- and CD14-coated magnetic beads (Dynabeads M-450, Dynal AS, Oslo, Norway). The total B-cell population was depleted of CD38+ GC B cells by Percoll gradient separation as previously described (28). The resulting B-cell population, referred to hereafter as B cells, was 98 ± 6% CD19+, 93 ± 3% CD44+, 6 ± 3% CD38+, ≤2 ± 3% CD3+ (n = 25). The viability of these cells was consistently >98%.

For in vitro culture assays, B cells (1.5 x 106 cells ml–1) were cultured in RPMI 1640 medium (Invitrogen SARL, Cergy-Pontoise, France) containing 10 mM HEPES, 2 mM L-glutamine, 100 U ml–1 penicillin, 100 µg ml–1 streptomycin, 1 mM sodium pyruvate and 10% heat-inactivated FCS for various periods of time, with or without 500–5000 IU ml–1 IFN{alpha}2 (R&D Systems). In some experiments, B cells were cultured with 2000 IU ml–1 IFN{alpha}2 and 50 µg ml–1 of blocking anti-IFN{alpha}R1 mAb (clone 64G12, IgG1) (29) or mouse IgG1 for 24 h before assaying for chemotaxis.

In vitro chemotaxis assay
The chemotaxis assay was performed in 24-well plates (Costar, Cambridge, MA, USA) carrying Transwell permeable supports, with a 5-µm pore size polycarbonate membrane. Assays were performed in pre-warmed migration buffer (RPMI 1640 containing 10 mM HEPES and 1% FCS). Migration buffer (600 µl) containing no chemokine, 250 ng ml–1 chemokine (CXCL12, CCL21) or 500 ng ml–1 chemokine (CCL20) (all from R&D Systems) was added to the lower chamber and B cells were loaded onto the inserts at a density of 0.3 x 106 cells per 100 µl for each individual assay. After 3 h at 37°C, the number of cells migrating into the lower chamber was determined by flow cytometry. Briefly, cells from the lower chamber were centrifuged and fixed in 300 µl of 1 x PBS and 1% formaldehyde before counting by FACscanTM for 60 s, gating on forward and side light scatter to exclude cell debris. The number of live cells was compared with a 100% migration control in which 100 µl of input cell suspension (0.3 x 106 cells) was treated in the same manner. The percentage of cells migrating to medium without chemokine was subtracted from the percentage of cells migrating to the medium with chemokines to calculate the percentage specific migration. To determine the phenotype of the migrating cells, input cells and migrating cells (lower chamber) were stained with FITC-labeled anti-CD44 and PE-labeled anti-IgD mAbs for flow cytometry. Events were analyzed separately within gated sIgDhigh and sIgD populations of B cells. The number of naive or memory cells migrating to the lower chamber is expressed as a percentage of the naive or memory cells added at the start of the assay (input cells).

In some experiments, medium- and IFN{alpha}-treated B cells were incubated with 100 nM or 1 µM wortmannin (wortmannin (WN), PI3K/PI4K inhibitor), 10 µM PD98059 (PD, mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor), 100 nM U73122 [GenBank] (PLC inhibitor) or its inactive control (U73343 [GenBank] ), 1 µM SN50 (inhibitor of NF-{kappa}B nuclear translocation), 100 ng ml–1 PTX (all from Calbiochem, San Diego, CA, USA), 10 µM SH5 (phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor, Alexis, Coger, France) or dimethylsulfoxide (DMSO) as a control, for 1 h before being subjected to the chemotaxis assay. We blocked RhoA functions with 50 µg ml–1 Tat-C3, a cell-permeable form of Clostridium botulinum C3 exoenzyme (30). Tat-C3 was added in the presence of 10 µg ml–1 polymyxin B to prevent endotoxin effects. The addition of 10 µg ml–1 polymyxin B alone had no effect on B-cell chemotaxis (data not shown).

Ligand-induced chemokine receptor internalization
IFN{alpha}-treated and medium-treated B cells were incubated for 60 min with medium or 100 ng ml–1 chemokine at 37°C. Cells were washed in ice-cold medium and stained with PE-conjugated anti-CD19, anti-CXCR4, anti-CCR7 and anti-CCR6 mAbs and control mouse PE-conjugated IgG for 30 min at 4°C. Single-color immunofluorescence analysis was performed on 5000 viable cells. In some experiments, IFN{alpha}- and medium-treated B cells were incubated with inhibitors or DMSO, as a control, for 1 h at 37°C before the addition of chemokine.

Western blots
Medium- and IFN{alpha}-treated B cells were re-suspended at a density of 1 x 107 cells ml–1 in pre-warmed RPMI 1640 without FCS and stimulated for 2 min at 37°C with medium or 100 ng ml–1 chemokine. Lysates were prepared as previously described (28). Equal amounts of total cellular protein were subjected to SDSP and analyzed by western blotting. Antibodies recognizing phospho-PKB/AKT (S473), PKB/AKT, phospho-ERK1/2 (T202/Y204), phospho-I{kappa}B{alpha} (S32/36), I{kappa}B{alpha} (all from New England Biolabs, Beverly, MA, USA) or ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used with HRP-conjugated secondary antibodies. Protein bands were detected by enhanced chemiluminescence (ECL, Supersignal Westpico chemiluminescent substrate, Perbio, Bezons, France) reagents. The ECL signal was recorded on ECL hyperfilm. To quantify band intensities, films were scanned, saved as TIFF files and analyzed with NIH Image software.

Statistical analysis
Data are expressed as means ± SD. Differences between groups were assessed using the unpaired Student's t-test and P values <0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
IFN{alpha} increases human B-cell chemotaxis in a dose- and time-dependent manner
We assessed the chemotactic response of B cells to CCL20 and CCL21 after incubation for 24 h with medium or increasing amounts of IFN{alpha}. IFN{alpha} gradually increased B-cell chemotaxis: no significant effect was detected up to 500 IU ml–1 and the effect was maximal at 2000 IU ml–1. The effect was specific because it was prevented by the addition of blocking anti-IFN{alpha}R1 chain mAb (Fig. 1A). Similar results were obtained for CXCL12 (data not shown). Kinetic studies showed that B-cell chemotaxis to CCL20 and CCL21 increased with time up to 24 h, but more strongly for IFN{alpha}-treated B cells than for medium-treated B cells (Fig. 1B). At 24 h, specific migration to CCL20 was 11 and 31% for medium- and IFN{alpha}-treated cells, respectively. Similarly, specific migration to CCL21 peaked at 24 h, at 56 and 71% in medium- and IFN{alpha}-treated cells, respectively. Similar results were obtained for CXCL12 and CXCL13 (data not shown). Given the well-known variability of chemokine responsiveness among individuals, B cells from 9 (CCL20), 14 (CCL21) and 12 (CXCL12) donors were tested. IFN{alpha} increased chemotaxis to CCL20 by 20 ± 6.2% (n = 9, P < 0.002), to CCL21 by 20 ± 8.5% (n = 14, P < 0.0001) and to CXCL12 by 16.3 ± 4.2% (n = 12, P < 0.003) (Fig. 1C).



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Fig. 1. Dose- and time-dependent effect of IFN{alpha} on B-cell chemotaxis. A. B cells were incubated for 24 h with medium alone or with increasing amounts of IFN{alpha} and analyzed for migration to 500 ng ml–1 CCL20 (white bars) and 250 ng ml–1 CCL21 (hatched bars). The chemotactic response to CCL20 (gray bar) or CCL21 (horizontal-line bar) was analyzed after the addition of 50 µg ml–1 anti-IFN{alpha}R1 mAb to 5000 IU ml–1 IFN{alpha}. The experiment was performed on three different donors and results are expressed as the mean ± SD percentage of cells specifically migrating in response to each chemokine. B. B cells were incubated for 2, 12, 24 and 48 h with medium or 2000 IU ml–1 IFN{alpha}. Medium-treated (open symbols) and IFN{alpha}-treated (filled symbols) B cells were analyzed for migration to 500 ng ml–1 CCL20 (circles) and 250 ng ml–1 CCL21 (triangles). Each condition was tested in duplicate. Results are expressed as the percentage ± SD specific chemotaxis. One experiment representative of three is shown. C. Medium-treated (white bars) and IFN{alpha}-treated (black bars) B cells were analyzed for migration to 500 ng ml–1 CCL20, 250 ng ml–1 CCL21 and 250 ng ml–1 CXCL12. Results are expressed as the mean ± SD percentage of specifically migrating cells obtained for each donor. *P < 0.002, n = 9; **P < 0.0001, n = 14; ***P < 0.003, n = 12.

 
IFN{alpha} increases the chemotaxis of memory B cells more strongly than that of naive B cells
We compared the ability of naive (CD44+, IgD+) and memory (CD44+, IgD) B cells to migrate in response to chemokines after 24 h with medium or 2000 IU ml–1 IFN{alpha} (Fig. 2). The percentage of memory B cells in untreated (31 ± 9.3%, n = 6), medium-treated (33.2 ± 10.1%, n = 6) and IFN{alpha}-treated (30.8 ± 8.8%, n = 6) input populations was similar. The percentages of naive B cells in untreated (66.4 ± 8.7%, n = 6), medium-treated (65.8 ± 10.4%, n = 6) and IFN{alpha}-treated (68.4 ± 8.8%, n = 6) input populations were also similar. Results from one representative experiment are shown in Fig. 2, 32% of medium-treated B cells specifically migrated in response to CCL21 (panel E) compared with 54% of IFN{alpha}-treated B cells (panel F). This IFN{alpha}-induced increase was due to a 14% increase in the amount of specifically migrating naive B cells (28% in medium-treated B cells versus 42% in IFN{alpha}-treated B cells) and a 45% increase in the amount of migrating memory B cells (43% in medium-treated B cells versus 88% in IFN{alpha}-treated B cells) (panels E–F).



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Fig. 2. IFN{alpha} enhances the chemotaxis of memory B cells more strongly than that of naive B cells. B cells were incubated for 24 h with medium or 2000 IU ml–1 IFN{alpha}. Medium- and IFN{alpha}-treated B cells were analyzed for migration to 500 ng ml–1 CCL20, CCL21 and CXCL12. Input populations and migrated cell populations were stained with CD44–FITC and IgD–PE. Representative dot plots of input cells (A, B) and transmigrated cells to medium versus CCL21 (C, D) are shown. The numbers of cells in the input and transmigrated populations from each B-cell subset are given in each quadrant (A–D). Percentage of naive (dotted bars) and memory B cells (hatched bars) specifically migrating to chemokines are shown in panels E and F for medium- and IFN{alpha}-treated cells, respectively. The percentages of total B cells migrating to each chemokine are indicated below the histograms (E–F). Data from one representative experiment are shown in panels A–F. The experiment was repeated on six different donors and results are expressed as the mean ± SD IFN{alpha}-induced percentage increase of total (black bars), naive (dotted bars) and memory B cells (hatched bars) specifically migrating to chemokines (G).

 
Similarly, the percentage of B cells specifically migrating in response to CCL20 was 19% in medium-treated cells compared with 34% in IFN{alpha}-treated cells, with 9 and 34% more naive and memory B cells, respectively, in the migrating population. The percentage of total B cells migrating in response to CXCL12 was 32% in medium-treated cells versus 54% in IFN{alpha}-treated cells, with 14 and 47% more naive and memory B cells, respectively, in the migrating population (panels E–F). Similar results were obtained in six independent experiments, showing that IFN{alpha} increased the chemotaxis of memory B cells by 2.6-fold compared with naive B cells on average (panel G)

IFN{alpha} decreases ligand-induced chemokine receptor internalization
We investigated whether IFN{alpha} up-regulated chemokine receptor expression by culturing B cells for 6 and 24 h with medium or 2000 IU ml–1 IFN{alpha} before staining. Incubation with medium induced a time-dependent increase in surface CCR7 expression until 24 h but did not significantly change the expression of CCR6 and only slightly increased that of CXCR4. A similar pattern of chemokine receptor expression was observed on medium- and IFN{alpha}-treated cells (Fig. 3A).



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Fig. 3. IFN{alpha} does not change chemokine receptor expression but decreases ligand-induced internalization of chemokine receptors. A. B cells were incubated for 6 and 24 h with medium or 2000 IU ml–1 IFN{alpha}. CCR7, CCR6 and CXCR4 expression in medium-treated (gray traces) and IFN{alpha}-treated (unfilled traces, bold lines) B cells was analyzed by flow cytometry before (0 h) and after incubation. One experiment representative of four is shown. B. Medium-treated (open symbols) and IFN{alpha}-treated (filled symbols) B cells from five different donors were incubated for 30 or 60 min at 37°C with medium (triangles), 100 ng ml–1 CCL21 (circles, left panel) or 100 ng ml–1 CXCL12 (squares, right panel). Cells were washed in ice-cold medium and stained with PE-conjugated anti-CCR7 (left panel) or anti-CXCR4 (right panel) and control IgG antibodies for 30 min at 4°C. Data are expressed as the mean ± SD percentage of mean channel fluorescence intensity (MFI) values for remaining surface expression. C. CCR7 and CXCR4 expression on medium- (gray traces) and IFN{alpha}-treated (unfilled traces, bold line) cells after 60 min in the presence of CCL21 (left) and CXCL12 (right). Chemokine receptor expression before incubation with chemokine is shown (unfilled traces, dotted line). One experiment representative of three is shown.

 
We investigated whether IFN{alpha} affected the internalization of the chemokine receptors by incubating medium- and IFN{alpha}-treated B cells with medium, 100 ng ml–1 CXCL12 or 100 ng ml–1 CCL21 for 30 or 60 min at 37°C. The cells were then stained with anti-CXCR4-PE mAb (CXCL12-incubated cells), anti-CCR7 mAb (CCL21-incubated cells) or mouse IgG-PE for 30 min at 4°C. In the absence of ligand, IFN{alpha} had no effect on the level of CXCR4 and CCR7 expression, whereas in the presence of ligand, IFN{alpha} significantly decreased the internalization of CXCR4 and CCR7 (Fig. 3B). After 60 min with CCL21, the amount of CCR7 expressed had decreased by 65.4 ± 10.4% in medium-treated B cells and by 28.6 ± 19.2% (n = 5, P < 0.003) in IFN{alpha}-treated B cells. Similarly, after 60 min with CXCL12, surface expression levels of CXCR4 had decreased by 86 ± 4.8% and by 65.8 ± 11% (n = 5, P < 0.003) in medium- and IFN{alpha}-treated B cells, respectively. After 60 min, levels of CXCR4 and CCR7 were clearly higher on IFN{alpha}-treated cells than on medium-treated cells, suggesting that IFN{alpha} decreases ligand-induced chemokine receptor internalization (Fig. 3C). Similar results were obtained for CCL20-induced CCR6 and CXCL13-induced CXCR5 internalization (data not shown).

The IFN{alpha}-mediated increase in B-cell chemotaxis requires PI3K, RhoA and NF-{kappa}B
The signaling pathways involved in the regulation of B-cell chemotaxis remain poorly defined. We therefore analyzed the effects of various inhibitors on the chemotaxis of medium- and IFN{alpha}-treated B cells from five different donors. Migration towards CCL21 was strongly inhibited by 100 ng ml–1 PTX and 100 nM U73122 [GenBank] (87 ± 4% and 79 ± 4%, respectively) in medium-treated B cells, but less strongly inhibited (68 ± 9% and 64 ± 7%, respectively) in IFN{alpha}-treated cells. The addition of 100 nM U73343 [GenBank] , the inactive form of the PLC inhibitor U73122 [GenBank] , did not inhibit chemotaxis of medium- or IFN{alpha}-treated B cells. The addition of 10 µM PD98059 had no effect on the chemotaxis of medium-treated B cells but decreased that of IFN{alpha}-treated B cells by 36 ± 6% (Fig. 4).



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Fig. 4. PI3K/AKT, ERK1/2, RhoA and NF-{kappa}B participate differently in the chemotaxis of medium- and IFN{alpha}-treated B cells. Medium-treated (white bars) and IFN{alpha}-treated (hatched bars) B cells from six different donors were incubated for 1 h at 37°C with various inhibitors or DMSO before being subjected to the chemotaxis assay in the presence of medium or 250 ng ml–1 CCL21. Results are expressed as the mean ± SD percentage of specific migration. *P < 0.02, **P < 0.0003 and ***P < 0.0009.

 
CCL21-mediated migration was not inhibited by 100 nM wortmannin but was decreased by 34 ± 2% and 47 ± 6% in the presence of 1 µM wortmannin in medium- and IFN{alpha}-treated cells, respectively. In contrast, 10 µM SH5 decreased the CCL21-mediated migration of medium- and IFN{alpha}-treated cells to a similar extent (45 ± 12% and 43 ± 11%, respectively), suggesting that downstream effectors of PI3K other than AKT are involved in chemotaxis. RhoA is involved in B-cell chemotaxis as CCL21-mediated migration was inhibited by 27 ± 4% (medium-treated cells) and by 47 ± 8% (IFN{alpha}-treated cells) in the presence of Tat-C3. The inhibition of NF-{kappa}B activation by SN50 led to the inhibition of CCL21-mediated migration by 35 ± 8% and 49 ± 7% in medium- and IFN{alpha}-treated cells, respectively. A similar pattern of inhibition was observed for CXCL12- and CCL20-induced B-cell chemotaxis (data not shown).

Our data suggest that, in the absence of IFN{alpha}, B-cell chemotaxis is dependent on the activation of PI3K/AKT, RhoA and NF-{kappa}B but not of ERK1/2, downstream from Gß{gamma}. Treatment with IFN{alpha} increased the involvement of NF-{kappa}B, RhoA and PI3K, but not that of AKT in B-cell chemotaxis. After IFN{alpha} treatment, ERK1/2 was also involved in the control of B-cell chemotaxis.

IFN{alpha} enhances chemokine-induced ERK1/2, PI3K/AKT and I{kappa}B{alpha} phosphorylation
Preliminary experiments showed that the chemokine-induced phosphorylation of ERK1/2, I{kappa}B{alpha} and AKT was maximal between 1 and 5 min in B cells (data not shown). We thus compared the phosphorylation of ERK1/2, AKT and I{kappa}B{alpha} in medium- and IFN{alpha}-treated cells after 2 min of stimulation by chemokine, in the absence or presence of various inhibitors. CCL21 strongly stimulated the phosphorylation of ERK1/2 in medium-treated cells (Fig. 5A). This effect was twice as strong in IFN{alpha}-treated cells. PTX, U73122 [GenBank] , wortmannin and PD98059 strongly decreased the CCL21-induced phosphorylation of ERK1/2 in medium- and IFN{alpha}-treated cells.



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Fig. 5. Chemokine-induced phosphorylation of (A) ERK1/2, (B) AKT and (C) I{kappa}B{alpha} in medium- and IFN{alpha}-treated B cells. Medium- and IFN{alpha}-treated B cells were incubated for 1 h at 37°C with medium or various inhibitors and were then incubated for 2 min with medium or 100 ng ml–1 CCL21. Phosphorylation of ERK1/2, AKT and IKB{alpha} was corrected for total relevant protein on stripped blots. Data from one representative experiment are shown (left panel). Values of specific phosphorylation are mean ± SD for three separate experiments (right panel).

 
AKT was already phosphorylated in B cells before chemokine stimulation, the amount of phosphorylated AKT being three times higher in IFN{alpha}-treated cells than in medium-treated cells (Fig. 5B). CCL21 increased the phosphorylation of AKT by factors of 1.7 and 2.6 in medium- and IFN{alpha}-treated cells, respectively. This phosphorylation, which was inhibited by SH5 by 72 and 39% in medium- and IFN{alpha}-treated cells, respectively, was totally abolished by 1 µM wortmannin in both populations. Strikingly, U73122 [GenBank] also abolished AKT phosphorylation in medium-treated cells but had no effect in IFN{alpha}-treated cells. The effect of PTX on AKT phosphorylation was also less marked in IFN{alpha}-treated cells than in medium-treated cells.

The phosphorylation of I{kappa}B{alpha}, already detectable in B cells prior to chemokine stimulation, was tripled by IFN{alpha} (Fig. 5C). CCL21 increased the level of I{kappa}B{alpha} phosphorylation by factors of 6.7 and 2.8 in medium- and IFN{alpha}-treated cells, respectively. In medium-treated cells, the CCL21-induced phosphorylation of I{kappa}B{alpha}, which was completely abolished by PTX and U73122 [GenBank] , was decreased by 59 and 51% by wortmannin and SH5, respectively. In IFN{alpha}-treated cells, neither PTX nor SH5 inhibited the CCL21-induced phosphorylation of I{kappa}B{alpha}, which was inhibited by wortmannin (25% inhibition) and U73122 [GenBank] (33% inhibition), respectively.

IFN{alpha}-induced PI3K/AKT and RhoA activation decreases the ligand-induced internalization of chemokine receptor
The ligand-induced internalization of CCR7 was 66.3 ± 14.2% in medium-treated cells and 36 ± 14.8% (n = 3) in IFN{alpha}-treated cells (Fig. 6). The decrease in internalization due to IFN{alpha} treatment was not affected by 100 ng ml–1 PTX, 100 nM U73122 [GenBank] , 10 µM PD98059 or 1 µM SN50 but was totally abolished by 1 µM wortmannin, 50 µg ml–1 Tat-C3 and 10 µM SH5. None of these inhibitors impaired the CCL21-induced internalization of CCR7 in medium-treated B cells. Similar results were obtained for ligand-induced CXCR4, CCR6 and CXCR5 receptor internalization (data not shown).



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Fig. 6. PI3K/AKT and RhoA mediate the IFN{alpha}-induced decrease in ligand-induced chemokine receptor internalization. Medium-treated (white bars) and IFN{alpha}-treated (hatched bars) B cells from three different donors were incubated for 1 h at 37°C with various inhibitors and were then subjected to the chemokine-induced receptor internalization assay. Data are expressed as mean ± SD mean channel fluorescence intensity (MFI) values after staining with anti-CCR7-PE mAb.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
The expression or activity of chemokine receptors on various lymphoid cells, including CD4+ T cells and monocytes, is up- or down-regulated by cytokines (3133). As IFN{alpha} has been shown to regulate B-cell phenotype and functions, we tested its effects on B-cell chemotaxis. IFN{alpha} increased the chemotaxis of B cells by interfering with chemokine-induced signaling cascades and by decreasing chemokine-induced receptor internalization.

Freshly isolated human and murine B cells migrate poorly in response to chemokines and a short period of incubation strongly enhances chemotaxis (17, 34, 35). Incubation in medium for 24 h increased B-cell chemotaxis to CCL20 and CXCL12 without significantly modifying the expression of CCR6 and CXCR4 (this article and [35]). In contrast, the increase in B-cell chemotaxis to CCL21 was correlated with an increase in CCR7 cell surface expression induced by incubation in medium (this article and [17]). In addition to this medium-induced increase in chemotaxis, IFN{alpha} further increased B-cell chemotaxis to CCL20 by 20 ± 6.2% (n = 9, P < 0.002), to CCL21 by 20 ± 8.5% (n = 14, P < 0.0001) and to CXCL12 by 16.3 ± 4.2% (n = 12, P < 0.003). The effect was dose dependent, reaching a plateau at 2000 IU ml–1. It was also time-dependent, peaking at 24 h and decreasing thereafter. A short-term treatment with IFN{alpha} after a 24-h pre-incubation with medium was sufficient to induce a similar or even stronger increase in B-cell chemotaxis (Supplementary Fig. S1, available at International Immunology Online). Indeed, a 2-h treatment with IFN{alpha} increased chemotaxis to CCL20 by 25.3 ± 1.5% (n = 3, P < 0.0003), to CCL21 by 32 ± 2% (n = 3, P < 0.0013) and to CXCL12 by 34 ± 2% (n = 3, P < 0.0004). This indicates that IFN{alpha}-mediated effects on B-cell chemotaxis do not require de novo transcription. Consistent with the observation that IFN{alpha} and IFNß share identical receptors, IFNß also increased B-cell chemotaxis (data not shown) and both effects were totally prevented by blocking anti-IFN{alpha}R1 chain mAb. Although the percentage of naive and memory B cells was not modified by incubation with medium or IFN{alpha}, IFN{alpha} increased the chemokine-induced migration of memory B cells more strongly than that of naive B cells.

IFN{alpha} produced during inflammatory responses may therefore increase the chemotaxis of B cells, possibly accelerating the initiation of an antigen-specific B-cell response within secondary lymphoid organs. In the tonsil, CCL20 expression is restricted to crypt epithelium, a major site of antigen entry (17), whereas CXCL12 is present both in crypt epithelium and in GC where it contributes to GC organization (36). Recent data showed that plasmacytoid dendritic cells (PDC) strongly enhance plasma cell differentiation and increase Ig production of blood B cells, in a T-cell-independent manner (37, 38). Direct B-cell–PDC interactions and PDC-derived soluble factors, including IFN{alpha}, contribute to B-cell activation (38).

Few human B-cell lines with a mature phenotype are responsive to IFN{alpha} and most of them fail to migrate in response to chemokines, even if they express chemokine receptors. In our hands, <5% of Daudi cells, a Burkitt cell line sensitive to IFN{alpha}, specifically migrate in response to CXCL12, CXCL13 or CCL21, whereas CXCR4, CXCR5 and CCR7 were strongly expressed (data not shown). We thus tried to identify the pathways critical for B-cell chemotaxis by treating B cells with specific inhibitors of various signaling cascades before adding chemokines. As expected, PTX and U73122 [GenBank] strongly blocked B-cell chemotaxis (39). In contrast to previous data showing that the blockade of MEK1/2 activity prevents the CCL11-induced migration of neutrophils and the CXCL12-mediated chemotaxis of T cells (40, 41), our results showed that CXCL12-, CCL20- and CCL21-mediated B-cell chemotaxis was independent of MEK1/2 activity. Similarly, Smit et al. recently reported that CXCR3-mediated chemotaxis in primary T cells is also independent of MEK1/2 activity (39). The involvement of MEK1/2 activity in IFN{alpha}-treated cells but not in medium-treated cells is puzzling because chemokines increase the phosphorylation of ERK1/2 in both populations by a mechanism dependent on Gß{gamma} and PI3K. The phosphorylated ERK1/2 produced after stimulation with IFN{alpha} (probably via Ras/Grb2 activation) and chemokine stimulation may display different cellular distributions, leading to the phosphorylation of a different set of effectors, as suggested for ERK activation downstream from Gß{gamma} and ß-arrestin complexes (42).

Low concentrations of wortmannin (≤100 nM), selective for class I PI3Ks, did not block B-cell chemotaxis. However, high concentrations of wortmaninn (≥1 µM) or LY294002 (≥20 µM), two structurally different PI3/PI4K inhibitors, exerted a significant inhibition. This suggests that class II PI3K or PI4K, rather than PI3K{gamma}/{delta}, are involved in the regulation of B-cell chemotaxis. A similar conclusion was drawn by Smit et al. in their study on primary T cells (39). Our data also showed that AKT, NF-{kappa}B and RhoA play a role in B-cell chemotaxis. PTX and wortmannin totally abolished the phosphorylation of AKT in medium-treated B cells, suggesting that this process is essentially dependent on Gß{gamma}/PI3K activation. In IFN{alpha}-treated B cells, AKT phosphorylation was strongly dependent on PI3K, but only partially dependent on {gamma}. This suggests that AKT is phosphorylated independent of G{gamma}ß in IFN{alpha}-treated B cells, probably by IFN{alpha}-induced class IA PI3K.

The phosphorylation of I{kappa}B{alpha} was totally dependent on G{gamma}ß activation and partially dependent on chemokine-induced PI3K/AKT activation in medium-treated B cells but was largely independent of chemokine-induced PI3K/AKT and Gß{gamma} activation in IFN{alpha}-treated B cells. In contrast, B-cell chemotaxis was more heavily dependent on NF-{kappa}B activation in IFN{alpha}-treated B cells, suggesting that IFN{alpha}-induced NF-{kappa}B activation is sufficient to promote chemotaxis. This is consistent with the increase in the amounts of phosphorylated I{kappa}B{alpha} observed in IFN{alpha}-treated cells before chemokine receptor stimulation. Our results for Tat-C3 protein indicate that RhoA is involved in B-cell chemotaxis. Low-molecular weight G proteins of the Rho family (RhoA, Cdc42 and Rac) have been shown to regulate the actin cytoskeleton and cell motility (43). RhoA can activate various downstream effectors, including NF-{kappa}B, PLC, Rho kinase and MEK kinase (44, 45). Experiments are under way to identify targets of RhoA involved in B-cell chemotaxis. It is currently unknown whether chemokine-induced RhoA activation results from direct Gß{gamma} release or occurs after PI3K activation, as suggested by the work of Huang et al. (46). Our work suggests that IFN{alpha}, by activating class IA PI3K, increases the amount of phosphorylated AKT and activated RhoA, thereby promoting the chemotaxis of B cells. High doses of wortmannin were required to block B-cell chemotaxis, but the addition of 100 nM wortmannin, together with IFN{alpha}, abolished the IFN{alpha}-mediated increase in B-cell chemotaxis (data not shown).

In addition to affecting receptor signaling, IFN{alpha} decreases ligand-induced chemokine receptor internalization but does not interfere with spontaneous chemokine receptor internalization. None of the inhibitors used prevented the ligand-induced internalization of chemokine receptors in medium-treated B cells, whereas wortmannin, SH5 and Tat-C3 totally abolished this increase in IFN{alpha}-treated B cells. PI3K, by increasing the amounts of phosphatidyl inositol-3,4,5-triphosphate (PIP3), may delay the feedback control of G{alpha} signaling by regulators of G protein signaling (47). The mechanisms by which RhoA and AKT decrease chemokine receptor internalization remain to be studied.

Our results show that B-cell chemotaxis is dependent on the activation of Gß{gamma}, PLC, PI3K/AKT, RhoA and NF-{kappa}B, but not on ERK1/2 activation. IFN{alpha} increases B-cell chemotaxis by interacting with ligand-induced chemokine receptor signaling: It increases the involvement of PI3K, RhoA and NF-{kappa}B and triggers the involvement of ERK1/2, without changing the level of involvement of AKT. In contrast, IFN{alpha} decreases ligand-induced chemokine receptor internalization in a manner dependent on PI3K/AKT and RhoA, but not on NF-{kappa}B or ERK1/2.


    Supplementary data
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Supplementary data are available at International Immunology Online.


    Acknowledgements
 
This work was supported by grants from Institut National de la Santé et de la recherche Medicale (INSERM) and Université Paris-Sud (Paris XI). The authors would like to thank J. Bertoglio (INSERM U461, France) and P. Eid (CNRS-UPR 9045, France) for kindly providing the purified Tat-C3 protein and the blocking anti-IFN{alpha}R1 mAb (clone 64G12), respectively.


    Abbreviations
 
AKT/PKB   protein kinase B
DMSO   dimethylsulfoxide
ECL   enhanced chemiluminescence
GC   germinal center
ERK   extracellular signal-regulated protein kinase
MEK   mitogen-activated kinase kinase
NF-{kappa}B   nuclear factor-{kappa}B
PDC   plasmacytoid dendritic cells
PDK1   phosphoinositide-dependent kinase 1
PI3K   phosphatidylinositide-3 kinase
PLC   phospholipase C
PTX   pertussis toxin
WM   wortmannin

    Notes
 
Transmitting editor: T. Kurosaki

Received 11 May 2004, accepted 24 January 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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