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Originally published In Press as doi:10.1074/jbc.M605287200 on October 4, 2006

J. Biol. Chem., Vol. 281, Issue 48, 36985-36992, December 1, 2006
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Lysophospholipids Control Integrin-dependent Adhesion in Splenic B Cells through Gi and G12/G13 Family G-proteins but Not through Gq/G11*

Stefan Rieken{ddagger}, Susanne Herroeder{ddagger}§, Antonia Sassmann{ddagger}, Barbara Wallenwein{ddagger}, Alexandra Moers{ddagger}, Stefan Offermanns{ddagger}, and Nina Wettschureck{ddagger}1

From the {ddagger}Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, and the §Institute of Anesthesiology, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany

Received for publication, June 2, 2006 , and in revised form, October 4, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin-mediated adhesion is a crucial step in lymphocyte extravasation and homing. We show here that not only the chemokines CXCL12 and CXCL13 but also the lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) enhance adhesion of murine follicular and marginal zone B cells to ICAM-1 in vitro. This process involves clustering of integrin LFA-1 and is blocked by pertussis toxin, suggesting that Gi family G-proteins are involved. In addition, lysophospholipid-induced adhesion on ICAM-1 depends on Rho and Rhokinase, indicative of an involvement of G12/G13, possibly also Gq/G11 family G-proteins. We used G12/G13- or Gq/G11-deficient B cells to study the role of these G-protein families in lysophospholipid-induced adhesion and found that the pro-adhesive effects of LPA and S1P are completely abrogated in G12/G13-deficient marginal zone B cells, reduced in G12/G13-deficient follicular B cells, and normal in Gq/G11-deficient B cells. We also show that loss of lysophospholipid-induced adhesion results in disinhibition of migration in response to the follicular chemokine CXCL13, which might contribute to the abnormal localization of splenic B cell populations observed in B cell-specific G12/G13-deficient mice in vivo. Taken together, this study shows that lysophospholipids regulate integrin-mediated adhesion of splenic B cells to ICAM-1 through Gi and G12/G13 family G-proteins but not through Gq/G11.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are adhesion receptors that connect cells to extracellular matrix or to counter-receptors on other cells. Integrin-mediated adhesion is critically involved in a variety of processes such as tissue morphogenesis, wound healing, blood clotting, and leukocyte trafficking.

In leukocytes, chemokines and their G-protein coupled receptors (GPCRs)2 play an important role in the regulation of integrin activity. While a wealth of studies addressed the role of chemokine receptors in lymphocyte homing to lymph nodes and Peyer's patches (1, 2), not much is known about the regulation of B lymphocyte adhesion in the spleen. The spleen contains two subpopulations of mature B cells, follicular B cells and marginal zone B (MZ B) cells, the latter residing at the interface between red and white pulp, the marginal zone. Due to this localization at a site of early antigen contact and due to their high proliferative capacity, MZ B cells play an important role in the rapid defense against blood-borne bacterial antigens (3, 4). Both entry of follicular B cells into the white pulp (5) and MZ B cell localization within the marginal zone (6) depend on the interaction between lymphocyte integrins LFA (leukocyte function antigen)-1 and {alpha}4beta1 and their respective ligands on stromal cells, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), respectively. A role for GPCRs in the regulation of this interaction was suggested based on pharmacological or genetic inactivation of different G-protein families in mice. For example, treatment with pertussis toxin (PTX), which ADP-ribosylates and thereby inactivates Gi family G-proteins, causes displacement of both follicular B cells and MZ B cells from their physiological niches (5, 7), suggesting that Gi-coupled receptors normally contribute to splenic B cell homing. This hypothesis is strengthened by the finding that numbers of MZ B cells are low in mice lacking either the G{alpha}i2 subunit of Gi (8) or different effector molecules of the Gi family like Pyk-2, Rac2, or Dock2 (7, 9, 10). In addition to Gi, also G12/G13 family G-proteins seem to be involved in splenic B cell localization, since MZ B cell numbers are reduced both in B cell-specific G12/G13-deficient mice (11) and in mice lacking the G12/G13 effector Lsc/p115RhoGEF (12). However, whether abnormal B cell localization in these mice is due to loss of GPCR-mediated integrin activation or secondary to other defects is not clear.

To elucidate the mechanisms regulating B cell adhesion we studied the effects of chemokine and lysophospholipid GPCR agonists on integrin-mediated adhesion in splenic B cell populations. We show here that not only chemokines, but also the lysophospholipids S1P and LPA, enhance B cell adhesion to ICAM-1, but not to VCAM-1, and that this process involves LFA-1 clustering. We also show that, in contrast to chemokines, lysophospholipids mediate their effects not only through Gi family G-proteins but also through the G12/13/Rho/Rho-kinase signaling pathway, while Gq/11 family G-proteins are not involved.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—B cell-specific G12/G13-deficient mice and B cell-specific Gq/G11-deficient mice were generated by mating the B cell-specific CD19-Cre line (13) to Gna13fl/fl;Gna12-/- mice (14) and Gnaqfl/fl;Gna11-/- mice (15), respectively. Control mice (CD19-Cre-/-;Gna13fl/fl;Gna12+/+ or CD19-Cre-/-; Gnaqfl/fl;Gna11+/+, respectively) and B cell-specific double-deficient (CD19-Cre+/-;Gna13fl/fl;Gna12-/- or CD19-Cre+/-; Gnaqfl/fl;Gna11-/-, respectively) mice were generated by intercrosses of triple heterozygous parents.

B cell-specific G{alpha}q/G{alpha}11-deficient mice were kept on a C57BL6/N background (eighth generation backcross), B cell-specific G{alpha}12/G{alpha}13-deficient mice were kept on a mixed C57BL6/N x Sv129J background, with a predominant contribution of the C57BL6/N strain (fourth generation backcross). Animals were housed under specific pathogen-free conditions. Genotyping was performed as described previously (14, 15).

Flow Cytometry—Lymphocytes from splenocyte suspension were enriched with Lympholyte-M (Cedarlane, Hornby, Ontario, Canada). B lymphocyte subsets were analyzed with a three-color FACSCalibur flow cytometer and CellQuestPro software (BD Biosciences) using the following antibodies: anti-CD21-FITC, anti-CD23-PE, anti-B220/CD45R-Biotin, anti CD11a-Biotin, anti-CD18-Biotin, rat IgG2a{kappa} isotype control, and SA-PerCP (all from BD Biosciences).

Integrin Staining—Untouched B cells were prepared from splenocytes using a B cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany), adhered to ICAM-1-coated coverslips (coated as described for adhesion assays) for 3, 10, or 30 min in the presence or absence of 100 nM S1P or LPA at 37 °C/5% CO2. Cells were then fixed in ice-cold 4% paraformaldehyde, washed twice in PBS, and incubated with biotinylated anti-CD11a antibody (BD Biosciences, 1:200) for 1 h. After washing in PBS, cells were incubated in SA-TRITC 1:200 in PBS with 2% fetal calf serum (FCS) for 1 h, washed, mounted in Mowiol, and analyzed on a Leica DC300F microscope. To quantify the effect of S1P or LPA on LFA-1 clustering, the number of CD11a clusters per cell was counted at x63 magnification by an investigator blinded to genotype and treatment.

Adhesion Assays—Adhesion assays were performed in 48-well plastic dishes as described (6) with minor modifications. For ICAM-1, wells were coated with 50 µg/ml goat-anti-human-IgG-Fc antibody (Jackson ImmunoResearch, West Grove, PA) at 4 °C overnight, washed twice with PBS, incubated with 2.5 µg/ml mouse ICAM-1-Fc (R&D Systems, Minneapolis, MN) in PBS for 2-3 h, and finally blocked with 0.5% BSA/PBS (100 µl per well for each step). Uncoated wells were treated with 0.5% BSA/PBS only. For VCAM-1, wells were incubated for 1 h at 37 °C with 100 µl/well 3 µg/ml hVCAM-1 (R&D Systems) in carbonate buffer and blocked with 0.5% BSA/PBS for 30 min before use. In some cases CXCL12 or CXCL13 were co-immobilized together with ICAM-1 or VCAM-1 at a concentration of 0.5 µg/ml. Lymphocytes were enriched with Lympholyte-M from splenocyte suspensions and incubated for 30 min in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) with 10 mM HEPES and 10% heat-inactivated FCS (Invitrogen) at 37 °C and 5% CO2 to remove contaminating macrophages. Non-adherent cells were washed twice, counted, and resuspended at a density of 2 x 107 cells/ml in RPMI 1640, 10 mM HEPES, 0.5% FCS. In some cases, cells were pretreated for 1 h at 37°C and 5% CO2 with 200 ng/ml PTX (Calbiochem/EMD Biosciences), 10 µM Y27632 (kind gift from Yoshitomi Pharmaceutical Inc., Osaka, Japan), or 0.2 µM TAT-C3 (16) before the adhesion assay. In these cases, the toxins were also present during adhesion. Blocking antibodies directed against CD11a or CD18 or isotype control antibodies (all from BD Biosciences; 1:200) were added only during adhesion.

For the adhesion assay itself, 50 µl of cell suspension (1 x 107 cells/ml) were added to each well of the coated plate and mixed with 50 µl of RPMI 1640, 10 mM HEPES, 0.5% FCS containing 2x concentrated GPCR agonists. The following agonists were used (100 nM each if not otherwise indicated): SDF-1{alpha}/CXCL12 (Chemicon, Temecula CA), S1P (Biomol, Plymouth Meeting, PA), 1 µM LPA (Biomol), CXCL13/BCA-1 (R&D Systems). After 30 min of adhesion at 37 °C/5% CO2, non-adherent cells were discarded and adherent cells detached in RPMI 1640, 5 mM EDTA for 20 min on ice. Cells were then stained for B cell subpopulations with CD45R/B220-Biotin/SA-PerCP, CD21-FITC, CD23-PE (all from BD Biosciences) and resuspended in 200 µl of PBS, and data were acquired for 1 min. The absolute number of follicular (CD45R/B220pos, CD21int, CD23hi) and marginal zone (CD45R/B220pos, CD21hi, CD23lo) B cells was calculated and expressed as proportion of input into adhesion assay.

Migration Assays—Lympholyte-M-purified splenocytes were preincubated in RPMI 1640, 10 mM HEPES, 0.1% BSA for 30 min at 37 and 5% CO2 and then allowed to migrate through uncoated, ICAM-1-coated, or VCAM-1-coated (coating see above) transwell inserts (5 µm pore size; Corning, Acton, MA) at a density of 2 x 106 splenocytes/100 µl/well. The lower wells contained 600 µl of RPMI 1640, 10 mM HEPES, 0.1% BSA either without chemokine or with 100 nM CXCL13. If indicated, 100 nM LPA was added to the upper well. After 2 h of incubation at 37 and 5% CO2, transwell plates were kept on ice for 20 min and centrifuged at 180 x g for 3 min, inserts were discarded, and cells were stained for B cell subpopulations as described above. Cell numbers were expressed as proportion of input. All experiments were done in triplicates.

Reverse Transcription-PCR—B220pos, CD21hi, and CD23lo marginal zone B cells were sorted with a FACSVantage, and mRNA was prepared with a RNeasy mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed according to standard protocols. LPA receptor subtypes were amplified with the following primers: S1P1,5'-GTGTAGACCCAGAGTCCTGCG-3' and 5'-AGTACATGGGCCGGTGGAACT-3'; S1P3, 5'-GGAGCCCCTAGACGGGAGT-3' and 5'-AGCCAAGTTGCCGATGAAAAAGT-3'; S1P4,5'-CCTGGAACTCACTTTATAGACCAGG-3' and 5'-CCAGCCTGCCGCTGTGATTGTA-3'; LPA1, 5'-GCAGCTGCCTCTACTTCCAC-3' and 5'-ACGCGCCGGTTGCTCATTC-3'; LPA2, 5'-TGGCGCCCCAAAGATGTGG-3' and 5'-TAGCAACCCCAGACCCAGTG-3'; LPA3, 5'-GCAACACAGACACAGCGGAC-3' and 5'-CCCACACCAGCAGAATGAGC-3'; LPA4, 5'-AACTGCATTCCTTACCAACATC-3' and 5'-GAAAACAAAGAGGCTGAAATACC-3'; LPA5, 5'-GCAACACAGACACAGCGGAC-3' and 5'-CCCACACCAGCAGAATGAGC-3'.

RhoA Activation Assay—Levels of GTP-bound RhoA were determined in wild-type and mutant splenic B cells before and 1, 3, and 10 min after stimulation with 1 µM LPA using a G-LISA RhoA activation kit (Cytoskeleton, Denver, CO).

Extracellular Signal-regulated Kinase (ERK) and Akt Phosphorylation—To determine phosphorylation of ERK and Akt in response to 1 µM LPA splenic lymphocytes were incubated at 37 °C in the presence or absence of agonist, then fixed for 10 min in 3% paraformaldehyde and permeabilized in 0.1% Triton X-100/PBS for 15 min. To determine ERK phosphorylation in MZ B cells, cells were incubated for 30 min with a FITC-labeled anti-ERK1/2 (pT202/pY204) antibody (BD Biosciences), PerCP-labeled anti-CD45R/B220 antibody, and PE-labeled anti-CD23 antibody, and the mean FITC intensity was determined in CD45R/B220pos and CD23neg cells (MZ B cells and newly formed B cells). To determine Akt phosphorylation in MZ B cells, cells were incubated for 30 min with a PE-labeled anti-Akt (pT308) antibody (BD Biosciences), PerCP-labeled anti-CD45R/B220 antibody, and FITC-labeled anti-CD21 antibody, and the mean PE-intensity was determined in CD45R/B220pos and CD21hi cells (MZ B cells). Data are displayed as mean fluorescence intensity as determined by the BD CellQuestPro Software.

Western Blotting—Splenic B cells from control and mutant mice were isolated by magnetic cell sorting (Miltenyi, Bergisch-Gladbach, Germany), and membrane bound proteins were extracted as described earlier (17). Extracts from 106 cells per lane were electrophoresed on 10% SDS-PAGE gels and blotted onto nitrocellulose membranes according to standard protocols. Membranes were incubated overnight at 4 °C with polyclonal antibodies directed against G{alpha}13 (1:1000), G{alpha}12 (1:500), G{alpha}i3 (1:500), and G{alpha}q/11 (1:500) (all from Santa Cruz Biotechnology, Santa Cruz, CA) or {alpha}-tubulin antibodies for loading control (Clone DM1a, 1:1000; Sigma). Blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000; Sigma) for 1 h. Chemiluminescence was developed using the ECLplus kit (Amersham Biosciences, Freiburg, Germany).

Statistics—Data are displayed as mean ± S.E. Comparisons between two groups were performed with Student's t test. Multiple comparisons were performed with analysis of variance and Bonferroni's post hoc test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To test whether GPCR activation contributes to B cell adhesion in vitro, we incubated murine splenic lymphocytes on ICAM-1- or VCAM-1-coated dishes in the presence or absence of different GPCR agonists and determined the percentage of adherent follicular B or MZ B cells, respectively. We found that the chemokines CXCL12 and CXCL13, as well as the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), strongly enhanced adhesion on ICAM-1-coated plastic in both cell types (Fig. 1, A and B). On VCAM-1-coated dishes, basal adhesion was generally lower, and although PMA induced a mild but significant increase in adhesion of both MZ B cells and follicular B cell (adherent FoB cells: basal, 4 ± 0.7%; PMA, 10.8 ± 2.8%; adherent MZ B cells: 4.1 ± 0.5%; PMA, 8.5 ± 1.5%; p < 0.05), CXCL12 and CXCL13 failed to induce significant increases in adhesion (data not shown).


Figure 1
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  		FIGURE 1.
Basal and agonist-induced adhesion in B cells. A and B, splenic B cells were allowed to adhere to ICAM-1-coated dishes for 1 h in the absence or presence of different GPCR agonists, and the proportion of adherent marginal zone B (MZ B) cells (A) and follicular B (Fo B) cells (B) was determined by flow cytometry. Chemokines were co-immobilized at 0.5 µM; lysophospholipids were applied at 100 nM each. Data are displayed as percent of cell input (n = 4-6 experiments). Figure 1, untreated sample; *, p < 0.05; **, p < 0.005; ***, p < 0.0005 versus untreated control.

 
Interestingly, also the lysophospholipids S1P and LPA strongly enhanced adhesion to ICAM-1 in both cell types (Fig. 1, A and B), while adhesion to VCAM-1 was not affected (data not shown). While effects of CXCL12 and CXCL13 on B cell adhesion have been described before (18), no direct data exist with respect to lysophospholipid-induced ICAM-1 adhesion. To characterize the pro-adhesive effects of S1P and LPA in more detail, we first tested the involvement of LFA-1, the main interaction partner of ICAM-1 on lymphocytes (19). We pretreated cells with blocking antibodies directed against LFA-1 {alpha}-chain CD11a or its beta-chain CD18 and found that both treatments completely abrogated S1P- or LPA-induced adhesion of MZ B (Fig. 2A) and follicular B (Fig. 2B) cells, while pretreatment with isotype control antibodies did not affect adhesion. Two types of integrin activation have been described, enhanced affinity and increased clustering (20-22), and we found that treatment with 100 nM S1P or LPA significantly enhanced the number of LFA-1 clusters per control cell (Fig. 2C), while we failed to detect increased binding of soluble ICAM-1 (data not shown). Taken together, these results indicate that lysophospholipids stimulate B cell adhesion to ICAM-1 and that this process involves enhanced LFA-1 clustering.


Figure 2
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  		FIGURE 2.
Lysophospholipid-induced adhesion depends on LFA-1 activation. A and B, the effect of blocking antibodies directed against LFA-1 {alpha} chain CD11a, or its beta chain, CD18, on adhesion of MZ B cells (A) and follicular B cells (Fo B, B) in the absence of agonist (Figure 2) or in the presence of 100 nM S1P or LPA was determined. Dishes were coated with 0.5% BSA or ICAM-1 as indicated. Data are displayed as percent of cell input (n = 4 experiments). C, left: immunofluorescence staining for CD11a in B cells after 10 min of adhesion to ICAM-1 coated dishes under basal conditions or in the presence of 100 nM S1P or LPA (magnification x100, representative of two independent experiments). Right: statistical evaluation of the number of CD11a clusters per cell in the absence or presence of 100 nM S1P or LPA. *, p < 0.05; **, p < 0.005; ***, p < 0.0005 versus untreated control.

 
Since most LPA and S1P receptor subtypes can couple to Gi family G-proteins (23, 24), we investigated the role of Gi in lysophospholipid-induced B cell adhesion. Pretreatment of cells with PTX strongly inhibited S1P- or LPA-induced adhesion (Figs. 3, A and B), suggesting that Gi family G-proteins are involved. A potential toxic effect of PTX-treatment on Gi-independent pathways in primary B cells can be ruled out, since LPA-induced RhoA activation was not impaired in PTX-treated cells (Fig. 7D).

Also the small GTPase RhoA has been implicated in GPCR-mediated integrin activation (25), and we tested the effects of the cell permeable version of the RhoA inhibitor Clostridium botulinum exoenzyme C3 (TAT-C3) (16) or the Rho-kinase inhibitor Y27632 (26). Like PTX these toxins abrogated the pro-adhesive effects of S1P and LPA both in MZ B cells and in follicular B cells (Fig. 3, A and B), suggesting that also the RhoA/Rho-kinase cascade critically contributes to agonist-induced adhesion to ICAM.


Figure 3
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  		FIGURE 3.
The role of RhoA, Rho-kinase, and Gi in the pro-adhesive effect of lysophospholipids. A and B, effect of Gi inhibitor PTX, RhoA inhibitor C3 exoenzyme (TAT-C3), and Rho-kinase inhibitor Y27632 on ICAM-1 adhesion of control MZ B cells (A) and follicular B cells (Fo B, B) in the absence of agonist or in the presence of either 100 nM S1P or 100 nM LPA. Data are displayed as percent of cell input (%o.i.), n = 3 independent experiments. ***, p < 0.0005 versus untreated control.

 
However, RhoA and Rho-kinase are not typically associated with activation of Gi but are rather a main effector pathway of the G12/G13 family of heterotrimeric G-proteins (27). Since this G-protein family has also been shown to couple to LPA and S1P receptors subtypes (23, 24), we tested lysophospholipid-induced adhesion in B cells from B cell-specific G12/G13-deficient mice. Most strikingly, the pro-adhesive effects of S1P and LPA were completely abrogated in G{alpha}12/G{alpha}13-deficient MZ B cells, and reduced in follicular B cells. In contrast, chemokine-induced adhesion was normal (Fig. 4, A and B). We performed dose response experiments for S1P and LPA and found that while control mice showed a bell-shaped dose-response curve with maximal effects at 100 nM, none of the tested S1P and LPA concentrations enhanced ICAM-1 adhesion in G{alpha}12/G{alpha}13-deficient B cells (Fig. 4, C-F). In line with this we found that S1P and LPA did not induce significant LFA-1 clustering in mutant cells (Fig. 4G). Also pretreatment with PTX caused an abrogation of LPA- and S1P-induced LFA-1 clustering (Fig. 4G). This shows that lysophospholipid receptors mediate their pro-adhesive effects on ICAM-1 not only through Gi family G-proteins but also through G12/G13 family G-proteins, and both G-protein families are required for LFA-1 clustering.

To test whether genetic inactivation of G12/G13 leads to altered expression of Gi3 or Gq/G11, we performed Western blotting experiments with wild-type and G{alpha}12/G{alpha}13-deficient B cells. We did not find significant differences in the protein levels of these {alpha}-subunits between the two genotypes (Fig. 5A). To rule out that impaired agonist-induced adhesion is due to reduced integrin expression, we analyzed expression of CD11a and CD18 by flow cytometry. We did not find major differences in the distribution of fluorescence intensity between the two genotypes (Fig. 5B), suggesting that it is indeed integrin activation, and not integrin expression, that is impaired in G{alpha}12/G{alpha}13-deficient B cells. To exclude major differences in the expression levels of S1P and LPA receptors between control and mutant B cells we performed reverse transcriptase polymerase chain reactions for the different receptor subtypes (Fig. 5C). As described by others (28), MZ B cells and follicular B cells expressed the S1P receptor subtypes S1P1, S1P3, and S1P4, and expression was comparable between control and mutant B cells. In addition, we found that MZ B and follicular B cells expressed the LPA receptor subtypes LPA2, LPA3, and weakly also LPA1 (Fig. 5C). Of the recently described non-Edg family receptors LPA4 (29) and LPA5 (30) only LPA4 was expressed in MZB cells. Also with respect to LPA receptor subtypes we did not find significant differences in expression levels between the genotypes (Fig. 5C).


Figure 4
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  		FIGURE 4.
Effect of chemokines and lysophospholipids on ICAM-1 adhesion and LFA-1 clustering in G12/G13-deficient B cells (B-G{alpha}12/G{alpha}13-DKO). A and B, ICAM-1 adhesion in MZ B cells (A) and follicular B (Fo B) cells (B) from control mice (gray) and B-G{alpha}12/G{alpha}13-DKOs (white). Chemokines were used at 0.5 µM and other agonists at 100 nM each. Data are displayed as percent of input (n = 4-6 experiments). C-F, dose-response curves for lysophospholipid-mediated B cell adhesion to ICAM-1. Adhesion of MZ B cells (A, C) and follicular B cells (B, D) to ICAM-1-coated dishes in response to 0, 10, 100, and 1000 nM of S1P (A, B) or LPA (C, D). Left, control cells; right, B-G{alpha}12/G{alpha}13-DKOs. Data are displayed as percent of cell input (%o.i.) (n = 4 independent experiments). G, statistical evaluation of the number of CD11a clusters per cell in control B cells (black), G{alpha}12/G{alpha}13-deficient B cells (white), or PTX-treated control B cells (gray) after adhesion to ICAM-1 in the absence or presence of 100 nM S1P or LPA (n = 2 experiments). Figure 4, untreated sample; *, p < 0.05; **, p < 0.005; ***, p < 0.0005 versus untreated control; #, p < 0.05; ##, p < 0.005; ###, p < 0.0005 versus untreated B-G{alpha}12/G{alpha}13-DKO.

 
In addition to the G12/G13 family of G-proteins, the Gq/G11 family has been suggested to activate RhoA and Rho-kinase (31-33). We therefore investigated whether lysophopsholipid-induced adhesion to ICAM-1 was impaired in B cells from B cell-specific G{alpha}q/G{alpha}11-deficient mice but did not find significant differences between the genotypes (Fig. 6, A and B). In these experiments both basal and agonist-induced adhesion to ICAM-1 was lower than in cells from B cell-specific G{alpha}12/G{alpha}13-deficient mice and their respective controls, which is most likely due to differences in the genetic background.

We next investigated which downstream effectors are activated in response to LPA in control, G12/G13-deficient, or PTX-treated B cells. We found that LPA induced phosphorylation of ERK1/2 and the serine/threonine protein kinase Akt/PKBAkt (Akt) (Fig. 7, A-C) in wild-type cells. In addition, LPA induced activation of RhoA (Fig. 7D) in wild-type cells. Both ERK and Akt phosphorylation were completely abrogated in PTX-pretreated cells, while in G12/G13-deficient B cells Akt phosphorylation was normal and ERK phosphorylation only slightly reduced (Fig. 7, A-C). In contrast, PTX treatment did not affect the LPA-induced increase in active, GTP-bound RhoA, while LPA-induced RhoA activation was completely abrogated in G12/G13-deficient B cells (Fig. 7D).

B cell-specific G{alpha}12/G{alpha}13-deficient mice show strongly reduced numbers of marginal zone B cells (11), and we asked whether impaired agonist-induced adhesion to ICAM-1 might contribute to this phenotype. It has been suggested that integrin-mediated adhesion within the marginal zone contributes to MZ B cell homing by counteracting promigratory signals like CXCL13 from adjacent follicles (6). We therefore tested whether lysophospholipid-mediated enhancement of adhesion impairs migration toward CXCL13 in vitro. Since S1P exerts strong promigratory effects itself (28) and was also shown to facilitate chemokine-induced migration in splenic lymphocytes (34), we used LPA in these experiments. We found that addition of LPA to the upper transwell chamber indeed resulted in a significant reduction of both basal (Fig. 8A, left) and CXCL13-induced (Fig. 8B, left) migration through ICAM-1-coated inserts in control cells. G{alpha}12/G{alpha}13-deficient cells did not differ from control cells with respect to basal or CXCL13-induced migration; however, the anti-migratory effect of LPA was strongly reduced in mutant cells (Fig. 8, A and B, right). This indicates that the lysophospholipid-induced inhibition of CXCL13-dependent B cell chemotaxis depends on the G12/G13-mediated signaling pathway. Inhibition of migration was not present on uncoated transwell inserts (Fig. 8, C and D) or VCAM-1 coated inserts (data not shown), suggesting that ICAM-1-mediated adhesion is underlying this effect.


Figure 5
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  		FIGURE 5.
Characteristics of G{alpha}12/G{alpha}13-deficient MZ B and follicular B (Fo B) cells. A, Western blots with antibodies directed against G{alpha}13, G{alpha}12, G{alpha}i3, and G{alpha}q/11 on extracts of control B cells (from CD19-Cre-/-; Gna13fl/fl;Gna12wt/wt mice, left) and G{alpha}12/G{alpha}13 double-deficient B cells (from CD19-Cre+/-;Gna13fl/fl;Gna12-/- mice, right). Antibodies directed against {alpha}-tubulin were used as loading control. B, flow cytometric analysis of CD11a and CD18 expression on control (blue) and G{alpha}12/G{alpha}13-deficient (B-G{alpha}12/G{alpha}13-DKO, green) MZ B and Fo B cells. C, reverse transcription-PCR for S1P and LPA receptor subtypes performed on cDNA from control (Contr) and G{alpha}12/G{alpha}13 double-deficient (DKO) MZ B cells.

 


Figure 6
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  		FIGURE 6.
Effect of chemokines and lysophospholipids on ICAM-1 adhesion in Gq/G11-deficient B cells (B-G{alpha}12/G{alpha}13-DKO). A and B, ICAM-1 adhesion in MZ B cells (A) and follicular B (Fo B) cells (B) from control mice (gray) and B-G{alpha}q/G{alpha}11-DKOs (white). Chemokines were used at 0.5 µM and other agonists at 100 nM each. Data are displayed as percent of input (n = 4-6 experiments). Figure 6, untreated sample; *, p < 0.05; **, p < 0.005; ***, p < 0.0005 versus untreated control; #, p < 0.05; ##, p < 0.005 versus untreated B-G{alpha}12/G{alpha}13-DKO.

 
Taken together, these results show that not only chemokines, but also lysophospholipids, can enhance B cell adhesion to ICAM-1 in vitro. The pro-adhesive effects of lysophospholipids are mediated both through Gi family G-proteins and the G12/G13/RhoA/Rho-kinase pathway, but do not involve Gq/G11 family G-proteins. Loss of lysophospholipid-induced adhesion causes disinhibition of migration in response to the follicular chemokine CXCL13, which might contribute to the abnormal localization of splenic B cell populations in B cell-specific G12/G13-deficient mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The molecular mechanisms governing integrin-mediated adhesion of splenic B cells are not well understood. We show here that not only chemokines, but also lysophospholipids enhance adhesion of murine B cells to ICAM-1 in vitro, and that this process involves LFA-1 clustering. Integrin activation by non-chemokine GPCRs has been reported in a variety of cellular systems, e.g. for thrombin and thromboxane A2 receptors in platelets (35-37), or for S1P in human and murine cell lines (38, 39). Indirect evidence for a role of S1P in integrin-mediated processes comes from genetic or pharmacological inactivation of the S1P1 receptor, which causes impaired lymphocyte entry into secondary lymphoid organs (40) and dislocation of MZ B cells from the marginal zone (28, 41).

We also show that lysophospholipid-induced adhesion depends on both Gi and G12/G13 family G-proteins, while chemokine-induced adhesion does not require G12/G13. This finding is in line with the general assumption that chemokine GPCRs signal primarily through Gi family G-proteins (42), while lysophospholipid receptors can utilize a variety of different G-protein families (23). It is not completely clear why activation of Gi through chemokine receptors is sufficient to enhance adhesion to ICAM-1, while activation of lysophospholipid receptors requires both Gi and G12/G13 to do so. Possible explanations might be differences in the expression levels of chemokine versus lysophospholipid receptors or differences in the signaling efficiency of the respective receptors.


Figure 7
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  		FIGURE 7.
Intracellular signaling cascades activated in response to LPA in control B cells (WT), G{alpha}12/G{alpha}13-deficient B cells (G12/G13-/-), and PTX-treated control cells (WT+PTX). A and B, flow cytomertric analysis of the phosphorylation state of ERK1/2 (Thr-202/Tyr-204) or Akt (Thr-308) in MZ B cells without agonist (solid gray) and 20 min after stimulation with LPA 1 µM (unshaded area). Cells were stimulated, fixed, permeabilized and stained with the following antibodies: for A, FITC-anti-ERK1/2, PerCP-anti-CD45R/B220, PE-anti-CD23 (gate on CD45R/B220pos;CD23neg MZ B and newly formed B cells); for B PE-anti-Akt, PerCP-anti-CD45R/B220, FITC-anti-CD21 (gate on CD45R/B220pos;CD21hi MZ B cells). C, statistical evaluation of data from A and B, data are displayed as mean fluorescence intensity as determined by the BD CellQuestPro Software. D, levels of active, GTP-bound RhoA in control (black), PTX-treated control (gray), or G{alpha}12/G{alpha}13-deficient (white) B cells at different time points after LPA stimulation. *, p < 0.05; **, p < 0.005 versus corresponding untreated sample.

 
MZ B cells and follicular B cells express the S1P receptor subtypes S1P1, S1P3, and S1P4 (28) and according to our results also LPA1, LPA2, and LPA3. While S1P1 and LPA3 are believed to activate predominantly Gi family G-proteins, LPA1, LPA2, S1P3, and S1P4 were shown to couple to both Gi and G12/G13 (24, 43, 44). Our results show that the pro-adhesive effects of lysophospholipids are mediated both through G12/G13 and Gi-type G-proteins. Such co-operativity between different G-protein families has been described in other systems, e.g. in platelets, where activation of Gi-mediated signaling alone is unable to trigger full activation of integrin {alpha}IIbbeta3 but requires co-activation of Gi and G12/G13 family G-proteins (45-47).


Figure 8
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  		FIGURE 8.
CXCL13-induced migration in control and G{alpha}12/G{alpha}13-deficient marginal zone B cells. A-D, migration of control (control, left) and G{alpha}12/G{alpha}13 deficient (G{alpha}12/G{alpha}13-/-, right) MZ B cells toward medium alone (Figure 8) or CXCL13-containing medium in the lower chamber in the absence or presence of 100 nM LPA in the upper chamber. Transwell inserts were either ICAM-1-coated (A, B) or uncoated (C, D) (n = 3 independent experiments). Figure 8, untreated sample; *, p < 0.05; **, p < 0.005 versus untreated control.

 
In addition to the role of G-proteins, we were able to show that lysophospholipid-induced integrin activation involves the small GTPase RhoA and its effector Rho-kinase. This is consistent with a variety of studies showing an involvement of RhoA in integrin-mediated adhesion (25, 48-50). Although G12/G13 are believed to be the major mediators of RhoA activation in response to GPCR stimulation, it has been shown that also Gq/G11 family G-proteins can activate RhoA (31, 32). We did not find differences with respect to lysophospholipid-induced adhesion between control and G{alpha}q/G{alpha}11-deficient B cells, while LPA-induced RhoA activation is completely abrogated in G12/G13-deficient B cells. Both findings strongly suggest that Gq/G11-mediated Rho activation is not critical in this setting, which is in line with the finding that ligand-induced RhoA activation through Gq/G11 occurs with lower potency than G12/G13-mediated Rho activation (33). We were able to show that LPA induces not only RhoA activation but also phosphorylation of ERK and Akt in MZ B cells and that the latter effect is predominantly mediated by Gi. Although our data do not prove that ERK- and Akt-phosphorylation are critically involved in the proadhesive effects of LPA, our findings suggest that in addition to the G12/13/RhoA/ROCK signaling cascade also Gi-mediated ERK and Akt phosphorylation are required for LPA-induced pro-adhesive effects in MZ B cells.

B cell-specific G{alpha}12/G{alpha}13-deficient mice show strongly reduced numbers of splenic MZ B cells but not of follicular B cells (11). This selective loss of MZ B cells has been attributed to a strong enhancement of migration in these cells (11), but it seems likely that also impaired lysophospholipid-induced adhesion to ICAM-1 contributes to the phenotype. Lysophospholipids are highly abundant in the blood (51, 52) and therefore seem well suited to mediate localization of lymphocytes to specialized anatomical sites with low blood flow and high ICAM-1 expression such as the marginal zone (6).

Taken together, this study shows that lysophospholipids regulate LFA-1-dependent adhesion of B cells to ICAM-1 and that this effect depends both on Gi and the G12/13/Rho/Rho-kinase signaling pathway. Loss of GPCR-mediated pro-adhesive effects might contribute to low MZ B cell numbers observed in mouse models with genetic or pharmacological inactivation of Gi (5, 7, 8) or G12/G13 (11).


   FOOTNOTES
 
* This work was supported by the Collaborative Research Center 405 of the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 49-6221-548255; Fax: 49-6221-548549; E-mail: Nina.Wettschureck{at}urz.uni-heidelberg.de.

2 The abbreviations used are: GPCR, G-protein coupled receptor; MZ B, marginal zone B; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; PTX, pertussis toxin; S1P, sphingosine 1-phosphate; LPA, lysophosphatidic acid; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; BSA, bovine serum albumin; FCS, fetal calf serum; ERK, extracellular signal-regulated kinase; PMA, phorbol 12-myristate 13-acetate. Back


   ACKNOWLEDGMENTS
 
We thank K. Rajewsky for providing the CD19-Cre mouse line, M. Scheuermann for help with FACS sorting, and A. Rogatzki, M. Hillesheim, and Y. Teschner for expert technical assistance.



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 DISCUSSION
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