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
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Abstract |
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Keywords: CCR6, CCR7, cell migration chemokines, CXCR4
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Introduction |
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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 Gß
proteins. Agonistic binding to the receptor induces dissociation of the G
and Gß
subunits, which then independently activate downstream effectors. Chemokine receptors can couple to various pertussis toxin (PTX)-senstitive (G
i) and -insensitive (G
q and G
15/16) G
proteins, but chemotaxis is only observed upon activation of G
i-coupled receptors (18). Several reports have provided strong evidence that Gß
rather than G
are the essential intermediates in the initiation of chemotactic response (19). Chemokine receptorligand interactions trigger many intracellular signals including phosphatidylinositide-3 kinase
or
(PI3K
/
), phospholipase Cß (PLCß) and nuclear factor-
B (NF-
B) (2022). PLCß triggers a rapid Ca++ flux and activates protein kinase C, whereas PI3K
/
activates AKT and extracellular signal-regulated protein kinase 1/2 (ERK1/2) (23). In leukocytes, Gß
subunits can also activate Ras proteins, which in turn activate all type I PI3Ks (24). Gß
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 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
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
-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
-treated B cells. In contrast to the widely accepted view that chemotaxis is regulated by PI3K
or
, our data indicate that non-class I PI3K are also probably involved in B-cell chemotaxis.
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Methods |
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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 ml1) were cultured in RPMI 1640 medium (Invitrogen SARL, Cergy-Pontoise, France) containing 10 mM HEPES, 2 mM L-glutamine, 100 U ml1 penicillin, 100 µg ml1 streptomycin, 1 mM sodium pyruvate and 10% heat-inactivated FCS for various periods of time, with or without 5005000 IU ml1 IFN2 (R&D Systems). In some experiments, B cells were cultured with 2000 IU ml1 IFN
2 and 50 µg ml1 of blocking anti-IFN
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 ml1 chemokine (CXCL12, CCL21) or 500 ng ml1 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-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-
B nuclear translocation), 100 ng ml1 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 ml1 Tat-C3, a cell-permeable form of Clostridium botulinum C3 exoenzyme (30). Tat-C3 was added in the presence of 10 µg ml1 polymyxin B to prevent endotoxin effects. The addition of 10 µg ml1 polymyxin B alone had no effect on B-cell chemotaxis (data not shown).
Ligand-induced chemokine receptor internalization
IFN-treated and medium-treated B cells were incubated for 60 min with medium or 100 ng ml1 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
- 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-treated B cells were re-suspended at a density of 1 x 107 cells ml1 in pre-warmed RPMI 1640 without FCS and stimulated for 2 min at 37°C with medium or 100 ng ml1 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
B
(S32/36), I
B
(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.
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Results |
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IFN decreases ligand-induced chemokine receptor internalization
We investigated whether IFN up-regulated chemokine receptor expression by culturing B cells for 6 and 24 h with medium or 2000 IU ml1 IFN
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
-treated cells (Fig. 3A).
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The IFN-mediated increase in B-cell chemotaxis requires PI3K, RhoA and NF-
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-treated B cells from five different donors. Migration towards CCL21 was strongly inhibited by 100 ng ml1 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
-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
-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
-treated B cells by 36 ± 6% (Fig. 4).
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Our data suggest that, in the absence of IFN, B-cell chemotaxis is dependent on the activation of PI3K/AKT, RhoA and NF-
B but not of ERK1/2, downstream from Gß
. Treatment with IFN
increased the involvement of NF-
B, RhoA and PI3K, but not that of AKT in B-cell chemotaxis. After IFN
treatment, ERK1/2 was also involved in the control of B-cell chemotaxis.
IFN enhances chemokine-induced ERK1/2, PI3K/AKT and I
B
phosphorylation
Preliminary experiments showed that the chemokine-induced phosphorylation of ERK1/2, IB
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
B
in medium- and IFN
-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
-treated cells. PTX, U73122
[GenBank]
, wortmannin and PD98059 strongly decreased the CCL21-induced phosphorylation of ERK1/2 in medium- and IFN
-treated cells.
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The phosphorylation of IB
, already detectable in B cells prior to chemokine stimulation, was tripled by IFN
(Fig. 5C). CCL21 increased the level of I
B
phosphorylation by factors of 6.7 and 2.8 in medium- and IFN
-treated cells, respectively. In medium-treated cells, the CCL21-induced phosphorylation of I
B
, which was completely abolished by PTX and U73122
[GenBank]
, was decreased by 59 and 51% by wortmannin and SH5, respectively. In IFN
-treated cells, neither PTX nor SH5 inhibited the CCL21-induced phosphorylation of I
B
, which was inhibited by wortmannin (25% inhibition) and U73122
[GenBank]
(33% inhibition), respectively.
IFN-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-treated cells (Fig. 6). The decrease in internalization due to IFN
treatment was not affected by 100 ng ml1 PTX, 100 nM U73122
[GenBank]
, 10 µM PD98059 or 1 µM SN50 but was totally abolished by 1 µM wortmannin, 50 µg ml1 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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Discussion |
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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 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 ml1. It was also time-dependent, peaking at 24 h and decreasing thereafter. A short-term treatment with IFN
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
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
-mediated effects on B-cell chemotaxis do not require de novo transcription. Consistent with the observation that IFN
and IFNß share identical receptors, IFNß also increased B-cell chemotaxis (data not shown) and both effects were totally prevented by blocking anti-IFN
R1 chain mAb. Although the percentage of naive and memory B cells was not modified by incubation with medium or IFN
, IFN
increased the chemokine-induced migration of memory B cells more strongly than that of naive B cells.
IFN 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-cellPDC interactions and PDC-derived soluble factors, including IFN
, contribute to B-cell activation (38).
Few human B-cell lines with a mature phenotype are responsive to IFN 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
, 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
-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
and PI3K. The phosphorylated ERK1/2 produced after stimulation with IFN
(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
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
/
, 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-
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
/PI3K activation. In IFN
-treated B cells, AKT phosphorylation was strongly dependent on PI3K, but only partially dependent on Gß
. This suggests that AKT is phosphorylated independent of G
ß in IFN
-treated B cells, probably by IFN
-induced class IA PI3K.
The phosphorylation of IB
was totally dependent on G
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
activation in IFN
-treated B cells. In contrast, B-cell chemotaxis was more heavily dependent on NF-
B activation in IFN
-treated B cells, suggesting that IFN
-induced NF-
B activation is sufficient to promote chemotaxis. This is consistent with the increase in the amounts of phosphorylated I
B
observed in IFN
-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-
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
release or occurs after PI3K activation, as suggested by the work of Huang et al. (46). Our work suggests that IFN
, 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
, abolished the IFN
-mediated increase in B-cell chemotaxis (data not shown).
In addition to affecting receptor signaling, IFN 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
-treated B cells. PI3K, by increasing the amounts of phosphatidyl inositol-3,4,5-triphosphate (PIP3), may delay the feedback control of G
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, PLC, PI3K/AKT, RhoA and NF-
B, but not on ERK1/2 activation. IFN
increases B-cell chemotaxis by interacting with ligand-induced chemokine receptor signaling: It increases the involvement of PI3K, RhoA and NF-
B and triggers the involvement of ERK1/2, without changing the level of involvement of AKT. In contrast, IFN
decreases ligand-induced chemokine receptor internalization in a manner dependent on PI3K/AKT and RhoA, but not on NF-
B or ERK1/2.
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Supplementary data |
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Acknowledgements |
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Abbreviations |
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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-![]() | nuclear factor-![]() |
PDC | plasmacytoid dendritic cells |
PDK1 | phosphoinositide-dependent kinase 1 |
PI3K | phosphatidylinositide-3 kinase |
PLC | phospholipase C |
PTX | pertussis toxin |
WM | wortmannin |
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Notes |
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Received 11 May 2004, accepted 24 January 2005.
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References |
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