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Antibody Response Cell Antigen Receptor Signaling And Published June 2, 2014, doi:10.4049/jimmunol.1300429 The Journal of Immunology Lysophosphatidic Acid Receptor 5 Inhibits B Cell Antigen Receptor Signaling and Antibody Response Jiancheng Hu,*,1,2 Shannon K. Oda,*,2,3 Kristin Shotts,* Erin E. Donovan,* Pamela Strauch,* Lindsey M. Pujanauski,*,4 Francisco Victorino,* Amin Al-Shami,† Yuko Fujiwara,‡ Gabor Tigyi,‡ Tamas Oravecz,† Roberta Pelanda,* and Raul M. Torres* Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, traf- ficking, and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically, and its levels are elevated in certain pathological settings, such as cancer and infections. In this study, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Ga12/13-Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5- triphosphate receptor activity. We further show that LPA5 also limits Ag-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced Ab responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation, and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity. The Journal of Immunology, 2014, 193: 000–000. ignals transmitted by the B cell AgR are not only required bound by IgG signals simultaneously via the BCR and FcgRIIB; for the Ab response but also for the development and signals transmitted by FcgRIIB dampen BCR signaling, thus at- S survival of B lymphocytes (1, 2). BCR signaling by mature tenuating the Ab response (3, 6). B cells can be either positively or negatively regulated by addi- In addition to these established BCR coreceptors, lymphocytes tional surface coreceptors depending on the developmental stage express G protein–coupled receptors (GPCRs) for chemokines of the B cell, the nature of Ag, and the microenvironment where and lysophospholipids, and both of these ligands were reported to the Ag is encountered (3, 4). For example, BCR signaling is more modify lymphocyte AgR signaling (7, 8). GPCRs signal primarily effective when complement-decorated Ag simultaneously engages via associated abg heterotrimeric G proteins, and a pertussis toxin– the BCR and CD21/CD19 coreceptor complex compared with insensitive Ga subunit has long been known to regulate BCR sig- BCR signaling alone (5). In contrast, a B cell encountering Ag naling (9, 10). However, neither the identity of this Ga protein nor by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. the mechanism(s) by which any of these GPCRs regulate AgR *Integrated Department of Immunology, University of Colorado Denver and National signaling in B lymphocytes has been established. Jewish Health, Denver, CO 80206; †Lexicon Pharmaceuticals, Inc., The Woodlands, Sphingosine-1-phosphate (S1P) and lysophosphatidic acid TX 77381; and ‡Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163 (LPA) are biologically active serum lysophospholipids that can signal extracellularly by engaging cognate GPCRs expressed by 1Current address: Department of Pathology and Immunology, Washington University, St Louis, MO. diverse cell types (11). Notably, S1P has emerged as an important 2J.H. and S.K.O. contributed equally to this work. chemoattractant that guides leukocytes during development, ho- 3Current address: Division of Basic Sciences, Fred Hutchinson Cancer Research meostasis, and inflammation (12–16). LPA is another major lyso- Center, Seattle, WA. phospholipid that can signal via six established LPA GPCRs, http://classic.jimmunol.org 4 Current address: Laboratory of Infectious Diseases, National Institute of Allergy and LPA1–6, each capable of associating with members from distinct Infectious Diseases, National Institutes of Health, Bethesda, MD. Ga families (17, 18). These LPA GPCRs are encoded by two gene Received for publication February 12, 2013. Accepted for publication May 5, 2014. clusters that include LPA1-2-3 in the endothelial differentiation This work was supported by the National Institutes of Health (Grants AI052157 to gene family, whereas LPA4-5-6 are closely related to the purinergic R.M.T., AI052310 to R.P., and AI08405 to G.T.), the Cancer League of Colorado (to GPCR subfamily (18). R.M.T.), Cancer Research Institute Special Emphasis Program in Tumor Immunology awards (to J.H., S.K.O., and K.S.), and National Institute for Allergy and Infectious The plasma and tissue concentrations of LPA are in the hun- Downloaded from Diseases Training Grant T32-AI07405 (to E.E.D. and L.M.P.). dred nanomolar range, whereas, in serum, inflammatory exudates Address correspondence and reprint requests to Dr. Raul M. Torres, University of or tumor cell effusates can reach as high as 10 mM (11, 17, 19, 20). Colorado Denver and National Jewish Health, 1400 Jackson Street, Denver, CO The biological function of LPA has received the most attention 80206. E-mail address: [email protected] with respect to cancer; it was shown to promote cell migration, The online version of this article contains supplemental material. proliferation, and survival of a number of diverse cancer cell types Abbreviations used in this article: A20-shG12/13, A20 cell line constitutively dox- (18, 21, 22). Moreover, LPA levels were found to be significantly ycyclineinducible expressing Ga12 and Ga13 shRNA; A20/shGaq, A20 cell line constitutively expressing Gaq sh RNA; ER, endoplasmic reticulum; ES, embryonic elevated with infection (23), inflammation (24), and particular stem; faf, fatty acid free; FO, follicular; GPCR, G protein–coupled receptor; IP3, cancers (19, 22). These data led to the notion that LPA contributes inositol 1,4,5-triphosphate; IP3R, inositol 1,4,5-triphosphate receptor; LPA, lysophos- phatidic acid; MZ, marginal zone; NP, 4-hydroxy-3-nitrophenylacetic acid; OTP, to the promotion and metastasis of cancer (22) and have focused octadecenyl thiophosphate; PLCg2, phospholipase C-g2; shRNA, short hairpin attention on modulating LPA in vivo as a possible therapeutic 2+ RNA; SOC, store-operated Ca channel; S1P, sphingosine-1-phosphatase; TI-2, T approach (18). independent, type 2. In this study, we show that the LPA5 GPCR expressed by mature Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 B lymphocytes negatively regulates BCR signaling by inhibiting www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300429 2LPA5 NEGATIVELY CONTROLS BCR SIGNALING AND Ab RESPONSE calcium release from intracellular stores via an LPA5–Ga12/13– MA) for the indicated times and quenched in ice water. Cells were lysed in Arhgef1 pathway. We further demonstrate that LPA also dimin- RIPA buffer and used to prepare whole-cell lysates, as described previously 3 ishes the activation and Ab response of Ag-specific B cells upon (29). Briefly, 30 ml whole-cell lysates was mixed with 6 ml2 SDS loading buffer and run on SDS-PAGE after boiling at 90–95˚C for 5 min. After engaging cognate Ag. Cumulatively, these data show that LPA SDS-PAGE, proteins were transferred to nitrocellulose membranes (Bio-Rad, directly regulates B lymphocyte activation and function via the Hercules, CA) and blocked with 5% nonfat milk–TBS solution at room LPA5 GPCR that serves as a negative coreceptor for the BCR. temperature for 30 min. Membranes were incubated with primary Ab in 5% nonfat milk–TBS solution for 1 h at room temperature or overnight at 4˚C, washed three times with Tween 20 and Tris-buffered saline, incubated with Materials and Methods HRP-labeled secondary Ab in 5% nonfat milk–TBS solution for 45 min, and Mice washed four times with Tween 20 and Tris-buffered saline. Target proteins C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME), C57BL/6-IghB1-8/B1-8 were detected by standard chemiluminescence and quantified by densitometric mice (25) (gift of Dr. Klaus Rajewsky, Harvard University, Boston, MA), analysis using ImageJ software (National Institutes of Health, Bethesda, MD). Arhgef12/2 mice (26), Lpar22/2 mice (27) (gift of Dr. Jerold Chun, Scripps Research Institute, La Jolla, CA), B6.C20 mice (C57BL/6 mice Intracellular calcium measurements congenic for Igha; gift of Dr. Leonore Herzenberg, Stanford University, 3 7 2/2 Approximately 0.5–1 10 cells/ml were resuspended in IMDM (Invi- Stanford,CA),andLpar5 mice were bred and maintained within the trogen, Grand Island, NY) with 2.5% fatty acid–free (faf) BSA plus 5 mM Biological Resource Center at National Jewish Health and used in accor- Indo-1-AM (Molecular Probes, Grand Island, NY) and incubated at 37˚C dance with the regulations of the Institutional Animal Care and Use 2/2 for 30 min. Subsequently, cells were washed twice with IMDM with faf Committee. Lpar5 mice were backcrossed to C57BL/6 mice for at least 2.5% BSA and resuspended at 2 3 106 cells/ml in IMDM with faf 2.5% three or four generations before these analyses. BSA. Cell aliquots were stored at room temperature for analysis. The Indo- Generation of Lpar52/2 mice 1-AM 400/475 fluorescence ratio was acquired as a function of time, using an LSR (BD Biosciences, Sparks, MD) or MoFlo (Beckman Coulter, Brea, The Lpar5-targeting vector was derived using the L KOS system (28). CA) cytometer. After a baseline reading of 30 s, A20 or splenic B cells The L KOS phage library was screened by PCR using Lpar5-specific were stimulated with anti-IgG F(ab9)2 (2 mg/ml) or anti-IgM F(ab9)2 primers: forward 59-GTTCTGCCTGGGCGTGTG-39 and reverse 59- (20 mg/ml), respectively, with or without 1-20 mM LPA (16:0, 1-palmitoyl- GCCAGCAGGAGGCGCAC-39. Five pKOS genomic clones, (pKOS-28, 2-hydroxy-sn-glycero-3-phosphate; Avanti Polar Lipids; critical micelle pKOS-58, pKOS-68, pKOS-81, and pKOS-92) were isolated and confirmed concentration = 0.54 mM), and readings were continued for an additional by sequence and restriction analysis.
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