Lysophospholipids Control Integrin-dependent Adhesion in Splenic B Cells through Gi and G12/G13 Family G-proteins but Not through Gq/G11

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. 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. 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) 2The 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. 2The 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. 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 (1Stein J.V. Nombela-Arrieta C. Immunology. 2005; 116: 1-12Crossref PubMed Scopus (185) Google Scholar, 2Butcher E.C. Williams M. Youngman K. Rott L. Briskin M. Adv. Immunol. 1999; 72: 209-253Crossref PubMed Google Scholar), 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 (3Martin F. Kearney J.F. Nat. Rev. Immunol. 2002; 2: 323-335Crossref PubMed Scopus (665) Google Scholar, 4Pillai S. Cariappa A. Moran S.T. Annu. Rev. Immunol. 2005; 23: 161-196Crossref PubMed Scopus (388) Google Scholar). Both entry of follicular B cells into the white pulp (5Lo C.G. Lu T.T. Cyster J.G. J. Exp. Med. 2003; 197: 353-361Crossref PubMed Scopus (140) Google Scholar) and MZ B cell localization within the marginal zone (6Lu T.T. Cyster J.G. Science. 2002; 297: 409-412Crossref PubMed Scopus (315) Google Scholar) depend on the interaction between lymphocyte integrins LFA (leukocyte function antigen)-1 and α4β1 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 (5Lo C.G. Lu T.T. Cyster J.G. J. Exp. Med. 2003; 197: 353-361Crossref PubMed Scopus (140) Google Scholar, 7Guinamard R. Okigaki M. Schlessinger J. Ravetch J.V. Nat. Immunol. 2000; 1: 31-36Crossref PubMed Scopus (433) Google Scholar), 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 Gαi2 subunit of Gi (8Dalwadi H. Wei B. Schrage M. Spicher K. Su T.T. Birnbaumer L. Rawlings D.J. Braun J. J. Immunol. 2003; 170: 1707-1715Crossref PubMed Scopus (111) Google Scholar) or different effector molecules of the Gi family like Pyk-2, Rac2, or Dock2 (7Guinamard R. Okigaki M. Schlessinger J. Ravetch J.V. Nat. Immunol. 2000; 1: 31-36Crossref PubMed Scopus (433) Google Scholar, 9Croker B.A. Handman E. Hayball J.D. Baldwin T.M. Voigt V. Cluse L.A. Yang F.C. Williams D.A. Roberts A.W. Immunol. Cell Biol. 2002; 80: 231-240Crossref PubMed Scopus (53) Google Scholar, 10Fukui Y. Hashimoto O. Sanui T. Oono T. Koga H. Abe M. Inayoshi A. Noda M. Oike M. Shirai T. Sasazuki T. Nature. 2001; 412: 826-831Crossref PubMed Scopus (361) Google Scholar). 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 (11Rieken S. Sassmann A. Herroeder S. Wallenwein B. Moers A. Offermanns S. Wettschureck N. J. Immunol. 2006; 177: 2985-2993Crossref PubMed Scopus (32) Google Scholar) and in mice lacking the G12/G13 effector Lsc/p115RhoGEF (12Girkontaite I. Missy K. Sakk V. Harenberg A. Tedford K. Potzel T. Pfeffer K. Fischer K.D. Nat. Immunol. 2001; 2: 855-862Crossref PubMed Scopus (145) Google Scholar). 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. 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 (13Rickert R.C. Roes J. Rajewsky K. Nucleic Acids Res. 1997; 25: 1317-1318Crossref PubMed Scopus (557) Google Scholar) to Gna13fl/fl;Gna12-/- mice (14Moers A. Nieswandt B. Massberg S. Wettschureck N. Gruner S. Konrad I. Schulte V. Aktas B. Gratacap M.P. Simon M.I. Gawaz M. Offermanns S. Nat. Med. 2003; 9: 1418-1422Crossref PubMed Scopus (208) Google Scholar) and Gnaqfl/fl;Gna11-/- mice (15Wettschureck N. Rutten H. Zywietz A. Gehring D. Wilkie T.M. Chen J. Chien K.R. Offermanns S. Nat. Med. 2001; 7: 1236-1240Crossref PubMed Scopus (311) Google Scholar), 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 Gαq/Gα11-deficient mice were kept on a C57BL6/N background (eighth generation backcross), B cell-specific Gα12/Gα13-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 (14Moers A. Nieswandt B. Massberg S. Wettschureck N. Gruner S. Konrad I. Schulte V. Aktas B. Gratacap M.P. Simon M.I. Gawaz M. Offermanns S. Nat. Med. 2003; 9: 1418-1422Crossref PubMed Scopus (208) Google Scholar, 15Wettschureck N. Rutten H. Zywietz A. Gehring D. Wilkie T.M. Chen J. Chien K.R. Offermanns S. Nat. Med. 2001; 7: 1236-1240Crossref PubMed Scopus (311) Google Scholar). 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 IgG2aκ 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, in and analyzed on a To the of S1P or LPA on LFA-1 the of cell was at by an to and were performed in as described (6Lu T.T. Cyster J.G. Science. 2002; 297: 409-412Crossref PubMed Scopus (315) Google Scholar) with For ICAM-1, were with antibody at washed twice with PBS, incubated with in PBS for h, and blocked with for were with For VCAM-1, were incubated for 1 at 37 with 100 in and blocked with for 30 min In CXCL12 or CXCL13 were with ICAM-1 or at a of were enriched with Lympholyte-M from splenocyte and incubated for 30 min in with and at 37 and to cells were washed and at a of in In cells were for 1 at and with from or E. E. 2003; PubMed Scopus Google Scholar) the adhesion In these the were also against or or isotype (all from BD 1:200) were only For the adhesion of cell suspension were to of the and mixed with of GPCR The following agonists were used nm not S1P 1 LPA After 30 min of adhesion at 37 °C/5% cells were and cells in for min on Cells were then for B cell subpopulations with (all from BD Biosciences) and in of PBS, and were for 1 The of follicular and marginal zone B cells was and as of into adhesion splenocytes were in for 30 min at 37 and and then to through or at a of The of either chemokine or with 100 nm 100 nm LPA was to the After of at 37 and were kept on for min and at for were and cells were for B cell subpopulations as described Cell numbers were as of were in and marginal zone B cells were with a and was prepared with a kit was performed to LPA were with the following and and and and and and and and of were in and splenic B cells and 3, and min with 1 LPA using a activation kit and of and in response to 1 LPA splenic were incubated at 37 in the presence or absence of then fixed for min in and in for To in MZ B cells, cells were incubated for 30 min with a antibody (BD and and the was in and cells (MZ B cells and B To in MZ B cells, cells were incubated for 30 min with a antibody (BD and and the was in and cells (MZ B are as as by the BD CellQuestPro B cells from and mice were by cell (Miltenyi, Bergisch-Gladbach, Germany), and were as described S. A. PubMed Scopus Google Scholar). from cells were on and to were incubated at with against and (all from or for were then incubated with secondary for 1 h. was using the kit Biosciences, are as between two were performed with were performed with of and To whether GPCR activation to B cell adhesion in we incubated murine splenic on or in the presence or absence of different GPCR agonists and the of follicular B or MZ B cells, respectively. We found that the chemokines CXCL12 and CXCL13, as as the phorbol 12-myristate adhesion on ICAM-1-coated in both cell A and adhesion was and a but in adhesion of both MZ B cells and follicular B cell PMA, MZ B PMA, CXCL12 and CXCL13 to in adhesion not also the lysophospholipids S1P and LPA adhesion to ICAM-1 in both cell A and while adhesion to was not not While effects of CXCL12 and CXCL13 on B cell adhesion described T. R. M. Cyster J.G. J. Exp. Med. 2002; PubMed Scopus Google Scholar), with to lysophospholipid-induced ICAM-1 To the pro-adhesive effects of S1P and LPA in we the involvement of the interaction of ICAM-1 on PubMed Google Scholar). We cells with against LFA-1 or and found that both completely abrogated or adhesion of MZ B and follicular B cells, while with isotype not of integrin activation and clustering N. R. B. A. J. Immunol. Rev. 2002; PubMed Scopus Google Scholar, M. N. J. PubMed Scopus Google Scholar), and we found that treatment with 100 nm S1P or LPA the of LFA-1 cell while we to of ICAM-1 not Taken together, these results that lysophospholipids B cell adhesion to ICAM-1 and that this process involves LFA-1 clustering. LPA and S1P to Gi family G-proteins S. 2002; PubMed Scopus (111) Google Scholar, Y. N. N. J. 2002; PubMed Scopus Google Scholar), we the role of Gi in lysophospholipid-induced B cell of cells with or adhesion 3, A and suggesting that Gi family G-proteins are involved. A of on in B cells be since activation was not in cells signaling in response to LPA in B cells Gα12/Gα13-deficient B cells and cells A and B, flow of the of or in MZ B cells and min with LPA 1 Cells were and with the following antibodies: for on MZ B and B for B on MZ B of from A and B, are as as by the BD CellQuestPro of in or Gα12/Gα13-deficient B cells at different LPA the in GPCR-mediated integrin activation C. E. L. S. E.C. C. PubMed Scopus Google Scholar), and we the effects of the cell of the E. E. 2003; PubMed Scopus Google Scholar) or the S. T. M. 2000; PubMed Google Scholar). these abrogated the pro-adhesive effects of S1P and LPA both in MZ B cells and in follicular B cells 3, A and suggesting that also the critically to adhesion to However, and are not with activation of Gi but are a effector of the G12/G13 family of G-proteins S. T. N. 1997; PubMed Scopus Google Scholar). this G-protein family also to to LPA and S1P receptors S. 2002; PubMed Scopus (111) Google Scholar, Y. N. N. J. 2002; PubMed Scopus Google Scholar), we lysophospholipid-induced adhesion in B cells from B cell-specific G12/G13-deficient mice. the pro-adhesive effects of S1P and LPA were completely abrogated in Gα12/Gα13-deficient MZ B cells, and reduced in follicular B cells. In adhesion was normal A and We performed response for S1P and LPA and found that while mice a with effects at 100 of the S1P and LPA ICAM-1 adhesion in Gα12/Gα13-deficient B cells In line with this we found that S1P and LPA not LFA-1 clustering in cells with an of and LFA-1 clustering 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 for LFA-1 clustering. To whether genetic inactivation of G12/G13 to of or we performed with and Gα12/Gα13-deficient B cells. We not in the of these between the two To that adhesion is due to reduced integrin we analyzed of and by flow We not in the of between the two suggesting that is integrin and not integrin that is in Gα12/Gα13-deficient B cells. To in the of S1P and LPA receptors between and B cells we performed for the different described by M. Y. C. Lu T. Cyster J.G. Nat. Immunol. PubMed Scopus Google Scholar), MZ B cells and follicular B cells the S1P and and was between and B cells. In addition, we found that MZ B and follicular B cells the LPA and also the described family receptors K. S. T. J. Biol. 2003; PubMed Scopus Google Scholar) and R. S. J. J. Biol. 2006; PubMed Scopus Google Scholar) only was in cells. with to LPA we not in between the In addition to the G12/G13 family of G-proteins, the Gq/G11 family suggested to and H. J. H. J. Biol. 2002; PubMed Scopus Google Scholar, S. R. Offermanns S. J. Biol. 2003; PubMed Scopus Google Scholar). We whether adhesion to ICAM-1 was in B cells from B cell-specific Gαq/Gα11-deficient mice but not between the A and In these both and adhesion to ICAM-1 was in cells from B cell-specific Gα12/Gα13-deficient mice and their respective which is due to in the genetic We which are in response to LPA in control, or B cells. We found that LPA of and the in cells. In addition, LPA activation of in cells. Both and were completely abrogated in cells, while in G12/G13-deficient B cells was normal and only reduced In treatment not the in while activation was completely abrogated in G12/G13-deficient B cells B cell-specific Gα12/Gα13-deficient mice show reduced numbers of marginal zone B cells (11Rieken S. Sassmann A. Herroeder S. Wallenwein B. Moers A. Offermanns S. Wettschureck N. J. Immunol. 2006; 177: 2985-2993Crossref PubMed Scopus (32) Google Scholar), and we whether adhesion to ICAM-1 might contribute to this suggested that integrin-mediated adhesion within the marginal zone to MZ B cell homing by like CXCL13 from (6Lu T.T. Cyster J.G. Science. 2002; 297: 409-412Crossref PubMed Scopus (315) Google Scholar). We whether of adhesion migration CXCL13 in vitro. S1P effects M. Y. C. Lu T. Cyster J.G. Nat. Immunol. PubMed Scopus Google Scholar) and was also to migration in splenic M. L. Y. J. Immunol. 2005; PubMed Scopus Google Scholar), we used LPA in these We found that addition of LPA to the in a of both and migration through ICAM-1-coated in cells. Gα12/Gα13-deficient cells not from cells with to or the of LPA was reduced in cells A and B, This that the lysophospholipid-induced of B cell depends on the signaling of migration was not on and or not suggesting that adhesion is this Taken together, these results show that not only chemokines, but also enhance B cell adhesion to ICAM-1 in vitro. The pro-adhesive effects of lysophospholipids are both through Gi family G-proteins and the pathway, but not Gq/G11 family G-proteins. 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. The mechanisms integrin-mediated adhesion of splenic B cells are not We show here that not only chemokines, but also lysophospholipids enhance adhesion of murine B cells to ICAM-1 in and that this process involves LFA-1 clustering. 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Biol. 2002; PubMed Scopus Google Scholar, Biol. 2002; PubMed Scopus Google Scholar). We not with to lysophospholipid-induced adhesion between and Gαq/Gα11-deficient B cells, while activation is completely abrogated in G12/G13-deficient B cells. Both that Rho activation is not in this which is in line with the finding that activation through Gq/G11 with Rho activation S. R. Offermanns S. J. Biol. 2003; PubMed Scopus Google Scholar). We were to show that LPA not only activation but also of and in MZ B cells and that the latter is by not that and are critically involved in the effects of LPA, that in addition to the signaling also and are for pro-adhesive effects in MZ B cells. B cell-specific Gα12/Gα13-deficient mice show reduced numbers of splenic MZ B cells but not of follicular B cells (11Rieken S. Sassmann A. Herroeder S. Wallenwein B. Moers A. Offermanns S. Wettschureck N. J. Immunol. 2006; 177: 2985-2993Crossref PubMed Scopus (32) Google Scholar). This loss of MZ B cells to a of migration in these cells (11Rieken S. Sassmann A. Herroeder S. Wallenwein B. Moers A. Offermanns S. Wettschureck N. J. Immunol. 2006; 177: 2985-2993Crossref PubMed Scopus (32) Google Scholar), but that also lysophospholipid-induced adhesion to ICAM-1 to the are in the blood T. Res. 2003; PubMed Scopus Google Scholar, S. S. Nat. Rev. Biol. 2003; PubMed Scopus Google Scholar) and seem to mediate localization of to with low blood flow and high ICAM-1 such as the marginal zone (6Lu T.T. Cyster J.G. Science. 2002; 297: 409-412Crossref PubMed Scopus (315) Google Scholar). Taken together, this study shows that lysophospholipids regulate adhesion of B cells to ICAM-1 and that this depends both on Gi and the G12/13/Rho/Rho-kinase signaling of GPCR-mediated pro-adhesive effects might contribute to low MZ B cell numbers observed in with genetic or pharmacological inactivation of Gi (5Lo C.G. Lu T.T. Cyster J.G. J. Exp. Med. 2003; 197: 353-361Crossref PubMed Scopus (140) Google Scholar, 7Guinamard R. Okigaki M. Schlessinger J. Ravetch J.V. Nat. Immunol. 2000; 1: 31-36Crossref PubMed Scopus (433) Google Scholar, H. Wei B. Schrage M. Spicher K. Su T.T. Birnbaumer L. Rawlings D.J. Braun J. J. Immunol. 2003; 170: 1707-1715Crossref PubMed Scopus (111) Google Scholar) or G12/G13 (11Rieken S. Sassmann A. Herroeder S. Wallenwein B. Moers A. Offermanns S. Wettschureck N. J. Immunol. 2006; 177: 2985-2993Crossref PubMed Scopus (32) Google Scholar). We K. Rajewsky for the CD19-Cre M. for with and A. M. and Y. for