Ligand-engaged chemoattractant receptors trigger Gαi subunit nucleotide exchange, stimulating the activation of downstream effector molecules. Activated chemoattractant receptors also dock G protein–coupled receptor kinases (GRKs) that help mediate receptor desensitization. In this study, we show that the B cell–specific loss of GRK2 severely disrupts B cell trafficking and immune cell homeostasis. The GRK2 deficiency in developing murine B cells leads to a severe immune phenotype, including a major reduction of bone marrow IgD+ cells, splenomegaly with a loss of white pulp and grossly expanded red pulp, a deficit of Peyer patches, and small lymph nodes with marked reductions in B cell numbers. The major phenotypes in these mice arise from excessive S1PR1 signaling combined with inadequate homeostatic chemokine receptor signaling. CXCL13 signaling is the most severely compromised. In B cells, our data also indicate that S1PR1 signals constitutively, as blocking S1PR1 signaling with an S1PR1 antagonist enhanced CXCL13-triggered wild-type B cell migration. Furthermore, blocking S1PR1 signaling in the GRK2-deficient B cells partially corrected their poor response to chemokines. Treating mice lacking GRK2 expression in their B cells with an S1PR1 antagonist partially normalized B cell trafficking into lymph node and splenic follicles. These findings reveal the critical interdependence of Gαi-linked signaling pathways in controlling B lymphocyte trafficking.
This article is featured in In This Issue, p.2353
An array of chemoattractant receptors modulates the positioning and trafficking of B lymphocytes (1–3). Each of these receptors use Gαi proteins to activate downstream effector molecules (4, 5). Mice whose lymphocytes lack Gαi2 and Gαi3 proteins or that possess Gαi proteins unable to interact with regulator of G protein–signaling proteins exhibit severe defects in chemoattractant receptor signaling and in the organization of B cell compartments (5, 6). By adopting an active conformation ligand, engaged receptors recruit heterotrimeric G proteins (7). The activated receptor opens a cleft between the helical and ras domains in the Gαi subunit, which facilitates GDP release, allowing GTP to bind. The GTP-bound Gαi and -freed Gβγ subunits can then stimulate the activity of downstream effector molecules. Precise regulation of Gβγ– and GTP–Gαi signaling coordinate an assortment of signaling pathways that enable B cell integrin activation and directed migration.
Activated G protein–coupled receptors (GPCRs) also recruit GPCR kinases (GRKs). They are proposed to dock the 20-aa N-terminal α helix of GRKs much like they bind the C-terminal α5 helix of the Gα subunit of GDP-bound heterotrimeric G proteins (8). Consequently, GRKs and heterotrimeric G proteins compete for the same site on the activated receptor. Typically, G protein binding precludes GRK binding and vice versa. However, specific receptor conformations may favor the binding of one protein versus the other. Structural studies indicate that activated GPCRs adopt a dynamic conformational landscape rather a single “active” conformation (7). Based on the structural analysis of GRK5/β2–adrenergic receptors interactions, receptor docking causes a GRK conformation change reorienting the catalytic domain increasing catalytic activity (9). This contrasts with most other protein kinases, whose catalytic activity depends upon a posttranslational modification such as phosphorylation. Once recruited and activated, GRKs phosphorylate GPCRs, typically on their C-terminal serine/threonine residues. An activated, phosphorylated receptor becomes a target for β-arrestins, whose recruitment leads to receptor desensitization and receptor endocytosis. Following endocytosis, the receptor is degraded or recycles to the plasma membrane (10).
The GRKs are the serine/threonine protein kinases most related to the AGC kinase subfamily (8). They have a central catalytic domain located within a regulator of G protein–signaling homology (RH) domain, which is flanked by a short N-terminal α-helical domain and a variable C-terminal lipid-binding region. Among the seven mammalian GRKs, Grk2 and Grk6 are most prominently expressed in lymphocytes (http://www.immgen.org/databrowser/index.html). Linking heterotrimeric G protein signaling to GRK2 regulation, the C-terminal lipid-binding domain in GRK2 (PH domain) allows Gβγ subunits to recruit GRK2 to the plasma membrane. In contrast, GRK6 undergoes C-terminal palmitoylation to mediate membrane localization (11, 12). A limited immune phenotyping of Grk6-deficient mice revealed normal B cell chemotaxis to CXCL12 but reduced transendothelial migration (13). Whereas GRK2 deficiency causes embryonic lethality, an analysis of mice with conditional deletion of Grk2 in B or T lymphocytes has been reported (14). Follicular (FO) B cells from these mice resisted sphingosine-1-phospate (S1P) R1 desensitization, migrated better to S1P in standard chemotaxis assays, but entered lymph nodes (LNs) poorly. The S1PR1 receptors on marginal zone (MZ) B cells also resisted desensitization, which impaired MZ B cell shuttling. In contrast, CXCR4 and CXCR5 signaling was reported as not significantly altered. This study implicated GRK2 as a central regulator of S1PR1 desensitization.
Building on these results, we have examined in greater detail the origins of the phenotypes when B cells lack GRK2. We confirmed the finding of impaired S1PR1 desensitization but have also found severe defects in B cell responses to homeostatic chemokines. At least some of these defects result from the dysregulated S1PR1 signaling. Together, they led to defective B cell physiology and, surprisingly, abnormalities in immune homeostasis. These include impaired bone marrow and splenic B cell development, pronounced splenomegaly with a marked disruption of the splenic architecture, a loss of Peyer patches, reduced LN homing because of transmigration defects, small lymphoid organ B cell follicles, and accelerated B cell LN and bone marrow egress.
Materials and Methods
Mice and bone marrow reconstitutions
C57BL/6, C57BL/6 Grk2fl/fl, and B6.SJL-PtprcaPepcb/BoyJ (CD45.1) mice were obtained from The Jackson Laboratory. The C57/BL6 mb1-cre mice were kindly provided by Dr. M. Reth and used to breed with the Grk2fl/fl mice to generate the Grk2fl/flmb1-cre mice. For the bone marrow reconstitutions, 6-wk-old CD45.1 mice were twice irradiated with 550 rad for total of 1100 rad. Mixed chimeric mice were made by reconstituting the irradiated CD45.1 mice with a 1:1 mixture of bone marrow prepared from C57BL/6 CD45.1 wild-type (WT) mice and either CD45.2 Grk2fl/fl mice or CD45.2 Grk2fl/flmb1-cre mice. The engraftment was monitored by sampling the blood 28 d later. The mice were used 6–8 wk after reconstitution. All mice used in this study were 6–14 wk of age. Mice were housed under specific pathogen–free conditions. All the animal experiments and protocols used in the study were approved by the National Institute of Allergy and Infectious Diseases’ Animal Care and Use Committee at the National Institutes of Health.
Spleens and LNs and were removed and gently dissociated into single-cell suspensions. Bone marrow cells were collected by flushing isolated femurs with PBS. Peripheral blood samples were collected by retro-orbital eye bleeding. After removing RBCs with Tris–NH4Cl, the cells were resuspended in PBS containing 1% fatty acid–free BSA at 4°C. B cells were isolated by negative depletion using biotinylated Abs to CD4, CD8, Gr-1 (Ly-6C and Ly-6G), and CD11c and Dynabeads M-280 Streptavidin (Thermo Fisher Scientific). The B cell purities were >95%. When needed, splenic, lymph, bone marrow, or B cells were cultured in RPMI 1640 containing 10% charcoal-stripped FCS (Life Technologies), 2 mM l-glutamine, antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin), 1 mM sodium pyruvate, and 50 μM 2-ME.
Standard flow cytometry
Single cells were resuspended in PBS, 2% FCS, and strained with fluorochrome-conjugated or biotinylated Abs against B220 (RA3-6B2), CD19 (1D3), CD23 (B3B4), CD21/35 (4E3), CD93 (AA4.1), CD43 (S7), IgD (11-26c-2a), IgM (R6-60.2), CD24 (M1/69), BP-1 (6C3), CD3 (145-2C11), CD4 (GK1.5 or RM4-5), CD8 (53-6.7), CD11c (HL3), CD11b (M1/70), CD184 (CXCR4, 2B11), CCR7 (4B12), CXCR5 (2G8), CD11a (M17/4), CD49d (9C10, MFR4.B), CD54 (3E2), CD62L (MEL-16), NK1.1 (PK136), TCRγδ (GL3), Ly-6G (1A8), Ly-6C (AL-21), CD45.1 (A20), or CD45.2 (104) (all from eBioscience, BioLegend, or BD Pharmingen). Biotin-labeled Abs were visualized with fluorochrome-conjugated streptavidin (eBioscience). A LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used in all experiments to exclude dead cells. Compensation was performed using CompBeads and ArC Amine Reactive Compensation Beads (Thermo Fisher Scientific) individually stained with each fluorochrome. Compensation matrices were calculated with FACSDiva software. Data acquisition was done on FACSCanto II (BD Biosciences) flow cytometer and analyzed with FlowJo software version 9.9.6 (Treestar).
S1PR1 immunostaining, internalization, and recovery
For the S1PR1 immunostaining, cells were fixed in 2% paraformaldehyde for 10 min, washed, and stained with S1PR1-PE, which is directed against an external S1PR1 epitope (R & D Systems). To assess S1PR1 internalization, the splenocytes were rested in DMEM/10 mM HEPES for 30 min at 37°C/5% CO2 before stimulation with 1 μM S1P or solvent (DMSO) for 10 or 30 min. Cells were stained with S1PR1-PE prior to stimulation and at the indicated time points. To examine S1PR1 recovery, cells were incubated for 10, 30, or 60 min in DMEM/10 mM HEPES at 37°C/5% CO2. Cells were washed and stained with S1PR1-PE prior to incubation and at the indicated times.
Intracellular flow cytometry
Purified B cells were rested in DMEM containing 1% charcoal-stripped FCS, antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin), 1 mM sodium pyruvate, and 50 μM 2-ME for 30 min at 37°C/5% CO2 before stimulation for varying durations with 1 μg/ml CXCL13 (R&D Systems or PeproTech) or 1 μM S1P (Sigma-Aldrich or Cayman Chemical) for 2, 5, 10, 30, or 60 min. The labeling of dead cells, fixation, and permeabilization were performed as described in the manufacturer’s protocol. Following permeabilization, the cells were immunostained with anti-B220, anti-IgD, anti-IgM, CD21, CD23, and CD93 for 30 min at 4°C. To detect phosphorylated signaling molecules Abs against phospho-Akt (Ser473) Alexa Fluor 647 (D9E), phospho-Erk (Thr202/Tyr204) (197G) Alexa Fluor 647, and phospho–ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) (pERM), all from Cell Signaling Technology, were used. Isotype control staining was performed using rabbit IgG isotype mAb Alexa Fluor 647 (DA1E; Cell Signaling Technology). Secondary F(ab′)2 fragment of goat anti-rabbit IgG (H+L) Alexa Fluor 647 (Thermo Fisher Scientific) was used to detect the pERM Abs. After washing, the cells were resuspended in 250 μl of 1% BSA/PBS and filtered prior to acquisition on a FACSCanto II flow cytometer (BD Biosciences).
Chemotaxis assays were performed using a transwell chamber (Costar). Splenic B cells were immunostained for B cell subsets with fluorochrome-conjugated Abs against B220, CD21, CD23, CD93, anti-IgM, and anti-IgD; they were washed twice, resuspended in complete RPMI 1640 media using charcoal-stripped FCS, incubated for 1 h at 37°C, and added to the upper wells of a 24-well transwell plate containing a 5-μm insert. In some experiments, the indicated concentrations of S1P (Sigma-Aldrich or Cayman Chemical) or chemokine (R & D Systems or PeproTech) were added to the upper well. The lower wells contained various concentrations of CCL19, CXCL12, CXCL13, or S1P in 600 μl of media. The numbers of cells that migrated to the lower well after 3-h incubation were counted using a MACSQuant flow cytometer (Miltenyi Biotec). The percentage of cells that migrated in the absence of chemoattractant was designated as nonspecific migration. The specific migration was calculated by subtracting the nonspecific migration from the migration in the presence of chemoattractant. The percentage migration was calculated as the numbers of cells that migrated into the lower part of the chamber divided by the total number of cells in the starting cell suspension and multiplying the result by 100. For those assays using Ex 26 (Tocris Bioscience), the compound (1 μM) was added during the 1-h preincubation phase and present in the both chambers of the chemotaxis chamber. DMSO alone served as the control.
Intracellular calcium measurements
Splenic B220+ cells were seeded at 3 × 105 cells per 100-μl loading medium (RPMI 1640, 10% charcoal-stripped FCS) into poly-d-lysine–coated 96-well black wall, clear-bottom microtiter plates (Nalgene Nunc). An equal volume of assay loading buffer (FLIPR Calcium 4 Assay Kit; Molecular Devices) in HBSS supplemented with 20 mM HEPES and 2 mM probenecid was added. Cells were incubated for 1 h at 37°C before adding chemokine or S1P, and then the calcium flux peak was measured using a FlexStation 3 (Molecular Devices). For those assays using Ex 26, the compound was added during the 1-h preincubation and added to the upper and lower chamber of the chemotaxis chamber. The data were analyzed with SoftMax Pro 5.2 (Molecular Devices). Data are shown as fluorescent counts, and the y-axis is labeled as Lm1.
Confocal microscopy and immunohistochemistry
Immunohistochemistry was performed as described previously (15). Briefly, freshly isolated LNs or spleens were fixed in newly prepared 4% paraformaldehyde (Electron Microscopy Sciences) overnight at 4°C on an agitation stage. Spleens or LNs were embedded in 4% low-melting agarose (Invitrogen) in PBS and sectioned with a vibratome (Leica VT1000 S) at a 30-μm thickness. Thick sections were blocked in PBS containing 10% FCS, 1 mg/ml anti-Fcγ receptor (BD Biosciences), and 0.1% Triton X-100 (Sigma-Aldrich) for 30 min at room temperature. Sections were stained overnight at 4°C on an agitation stage with Abs directed at B220 (RA3-6B2; BD Biosciences), CD4 (RM4-5; BD Biosciences), CD169 (3D6.112), CD21/35 (4E3), CD45.1 (A20), CD45.2 (104) (all from BioLegend), Ki67 (SolA15; Thermo Fisher Scientific), and LYVE-1 (223322; R&D Systems). Stained thick sections were microscopically analyzed using a Leica SP8 confocal microscope equipped with an HC PL APO CS2 40× (numerical aperture, 1.30) oil objective (Leica Microsystems), and images were processed with Leica LAS AF software (Leica Microsystems) and Imaris software v.9.0.1 x64 (Bitplane AG). The intensities of fluorescent signals in regions of interests were measured by LSA AF Lite software (Leica Microsystems).
Homing, egress, and competition assays
Purified splenic B cells from Grk2fl/fl and Grk2fl/flmb1-cre B cells mice were labeled with 1 μM eFluor 450 or 2.5 μM CMTMR (Thermo Fisher Scientific) for 15 min at 37°C, and equal numbers of viable cells (8–10 million) were injected i.v. into recipient mice. After 2 h, spleen, inguinal LNs, and mesenteric LNs were removed and gently dissociated into single-cell suspensions. Data acquisition was done on a FACSCanto II flow cytometer and analyzed with FlowJo software (Tree Star). The egress assay used a similar initial approach, but 2 h after the cell transfer, mice were injected i.v. with CD62L Ab (100 μg/mouse). After 18 h, spleen, inguinal LNs, and popliteal LNs were removed and gently dissociated into single-cell suspensions. Peripheral blood was collected by retro-orbital eye bleeding. After removing RBCs with Tris–NH4Cl, the cells were resuspended in PBS containing 1% BSA at 4°C. The ratios between the numbers of cells in the various compartments 2 h after transfer versus after CD62L Ab injection were calculated. The competition assays were performed by adoptively transferring 20 million CMTMR-labeled Grk2fl/fl or Grk2fl/flmb1-cre B cells to separate WT mice. One hour later each mouse was injected with 10 million eFluor 450–labeled Grk2fl/fl B cells. Two hours later, multiple LNs were collected, and cell suspensions were made from each of them. The number of labeled cells in each LN cell suspension was assessed by flow cytometry.
Intravital two-photon laser-scanning microscopy
Inguinal LNs were prepared for intravital microscopy as described previously (15). Cell populations were labeled for 10 min at 37°C with 2.5–5 μM CellTracker Red CMTMR (Thermo Fisher Scientific) or 2 μM of eFluor 450 (Thermo Fisher Scientific). Five to ten million labeled cells of each population in 200 μl of PBS were adoptively transferred by tail vein injection into recipient mice. After anesthesia, the skin and fatty tissue over inguinal LN were removed. The mouse was placed in a prewarmed coverglass chamber slide (Nalgene Nunc). The chamber slide was then placed into the temperature control chamber on the microscope. The temperature of air was monitored and maintained at 37.0 ± 0.5°C. Inguinal LN was intravitally imaged from the capsule over a range of depths (10–220 μm). All two-photon imaging was performed with a Leica SP8 inverted five-channel confocal microscope (Leica Microsystems) equipped with 25× water-dipping objective, 0.95 numerical aperture (immersion medium used distilled water). Two-photon excitation was provided by a Mai Tai Ti:Sapphire laser (Spectra-Physics) with a 10-W pump, tuned wavelength ranges from 820 to 920 nm. Emitted fluorescence was collected using a four-channel nondescanned detector. Wavelength separation was through a dichroic mirror at 560 nm and then separated again through a dichroic mirror at 495 nm followed by 525/50-nm emission filter for Alexa Fluor 488 (Thermo Fisher Scientific); the eFluor 450 (Thermo Fisher Scientific), or second harmonic signal, was collected by 460/50-nm emission filter, a dichroic mirror at 650 nm, followed by 610/60-nm emission filter for CMTMR or Alexa Fluor 594 (Thermo Fisher Scientific), and the Evans blue (Sigma-Aldrich). For four-dimensional analysis of cell behavior, stacks of various numbers of section (z step = 2, 3, 4, 5, or 6 μm) were acquired every 2.5, 5, or 30 s to provide an imaging volume of 20–120 μm in depth. Sequences of image stacks were transformed into volume-rendered four-dimensional videos using Imaris software v.9.0.1 x64 (Bitplane AG), and the tracks analysis was used for semiautomated tracking of cell motility in three dimensions by using the following parameters: autoregressive motion algorithm, estimated diameter 10 μm, background subtraction true, maximum distance 20 μm, and maximum gap size 3. Tracks acquired that could be tracked for at least 20% of total imaging duration were used for analysis. Some tracks were manually examined and verified. Calculations of the cell motility parameters (speed, track length, displacement, straightness, and speed variability) were performed using the Imaris software v.9.0.1 x64 (Bitplane AG). Statistical analysis was performed using Prism software (GraphPad Software). Annotations on videos and video editing were performed using Adobe Premiere Pro CS3 (Adobe Systems). Video files were converted to MPEG4 format with ImTOO Video Converter Ultimate (v. 7.8.19) (ImTOO Software Studio).
In vivo results represent samples from 2 to 10 mice per experimental group. Results represent mean values of at least triplicate samples. SEM and p values were calculated with a t test or ANOVA using GraphPad Prism (GraphPad Software).
Loss of Fr. F cells from the bone marrow B cell compartment in Grk2fl/flmb1-cre mice
Based on Grk2 mRNA expression profiling, bone marrow B cells prominently express Grk2 (http://www.immgen.org/databrowser/index.html). To determine the role of GRK2 in the mouse bone marrow B cell compartment, we crossed Grk2fl/fl mice to mb1-cre mice. Based on the known expression pattern of mb1 and the previous functional assessment of genes deleted with mb1-cre, the loss of GRK2 should become evident in Fr. B cells in the Hardy scheme of B cell development, in which Fr. A-C cells correspond to pro-B cell stages, Fr. D cells correspond to pre-B cells, Fr. E cells correspond to immature B cells, and Fr. F cells correspond to mature recirculating cells (5, 16). Assessing the B cell compartment in bone marrow from control and Grk2fl/flmb1-cre mice revealed similar overall numbers of B cells; however, we noted a 50% increase in Fr. D cells and, strikingly, a 4–5-fold reduction in Fr. F cells in bone marrow prepared from the Grk2fl/flmb1-cre mice (Fig. 1A, 1B). A representative flow pattern of IgM versus IgD on bone marrow cells gated on cells expressing B220 and CD19 but not CD43 is shown (Fig. 1C). Mature IgD+ B cells (Fr. F cells) occupy an anatomically distinct bone marrow niche and can functionally generate specific IgM Abs to blood-borne pathogens (17, 18). Their loss from the Grk2fl/flmb1-cre mouse bone marrow likely results from a recruitment failure, a retention defect, or a major loss of recirculating IgD+ B cells. Among the chemoattractant receptors, CXCR4 has the dominant role in the recruitment of blood-borne B cells and in the retention of B cells within specialized bone marrow niches (19). To assess the impact on the loss of GRK2 on CXCR4-mediated migration, we first checked CXCR4 expression and that of several cell-trafficking molecules on bone marrow B cell fractions of control and Grk2fl/flmb1-cre mice (Fig. 1D). Although we noted some variation in CD11a, CD54, and CD62L (l-selectin), none likely explained the loss of Fr. F cells or the gain of Fr. D cells. The Grk2fl/flmb1-cre Fr. D-F cells expressed 2-fold more CXCR4 cells, predicting an enhanced responsiveness to CXCL12. We also assessed S1PR1 expression because of its known role in B cell bone marrow egress (20). Among the bone marrow fractions, Fr. F cells expressed the most S1PR1, but we did not detect much difference between the cells from the two mouse strains (Fig. 1D). Next, we tested the responsiveness of the different B cell bone marrow fractions to CXCL12 and S1P in a standard chemotaxis assay (Fig. 1E). The GRK2-deficient Fr. F cells had an increased percentage of spontaneously migrating cells, an enhanced S1P migratory response to low concentrations of S1P. Based on their elevated CXCR4 expression, they had a suboptimal response to CXCL12. In addition, the Grk2-deficient Fr. B/C cells responded less well to CXCL12. Together, the enhanced motility, augmented S1P migratory response, and suboptimal CXCR4 responses could interfere with the Fr. F cell retention and/or recruitment.
Splenomegaly and a severely disrupted splenic architecture in the Grk2fl/flmb1-cre mice
The reduction in Fr. F cells could also result from a failure of B cell maturation in the spleen. Immature B cells, which lack IgD, leave the bone marrow via the bloodstream. They enter the marginal sinus and red pulp of the spleen, where they are designated T0 cells (B220+CD93+IgM+CD23−IgD−). These cells are believed to acquire surface IgD following their migration into the splenic white pulp, becoming transitional-type (T) 1 cells, which can further differentiate into T2–FO (B220+CD93+IgM+IgD+CD23+) cells and eventually FO B cells (21). To assess whether Grk2fl/flmb1-cre splenic B cells had acquired surface IgD, we first compared the numbers and composition of splenocytes recovered from the Grk2fl/flmb1-cre mice versus those from control mice (Fig. 2A, 2B). The Grk2fl/flmb1-cre mice had markedly enlarged spleens with twice as many hematopoietic cells, although they had an unremarkable distribution of major cellular subsets. Surprisingly, accompanying the splenic B cell expansion in the Grk2fl/flmb1-cre mice were similar increases in CD4+ and CD8+ T lymphocytes. Immunostaining the splenocytes for expression of B220/CD93/IgM/IgD/CD23/CD21 revealed that, on a percentage basis, the Grk2fl/flmb1-cre mice had an increase in T1 cells and T2–FO cells, but a one-third reduction in FO B cells compared with controls (Fig. 2B). However, because of their large spleens, the Grk2fl/flmb1-cre mice had an absolute increase in FO B cells. Examining IgD versus IgM expression on the B220+CD93− cells revealed that the Grk2fl/flmb1-cre mice had, on a percentage basis, fewer IgD+ splenic cells; however; their absolute number also exceeded that of Grk2fl/fl mice (Fig. 2C). These data indicate that a splenic B cell differentiation defect could not account for the reduction in the mature IgD+ bone marrow cells. However, the large spleen and absolute increase in splenic B cells suggested impaired release of recirculating B cells into the blood of the Grk2fl/flmb1-cre mice.
Splenic FO B cells exit the white pulp via the MZ in an S1PR1-dependent fashion (22). Thereafter, they enter the red pulp prior to their release into the splenic sinuses and, eventually, the blood stream, which allows them access to other immune compartments. To assess the localization of B cells within the splenic compartments in the Grk2fl/flmb1-cre mice, we immunostained thick sections cut perpendicular to the long axis of the spleen with B220, CD21, CD4, CD169, and Ki67, collected standard confocal microscopy images, and tiled the images to give a composite picture of a Grk2fl/fl and Grk2fl/flmb1-cre mouse spleen (Fig. 2D, 2E). We noted a loss of the normal demarcation between the white and red pulps. The white pulp contained usually distorted T cell zones and small B cell zones. The reduction in B cell zones undoubtedly contributed to the loss of FO dendritic cells. Ki67 staining revealed constitutive germinal centers present in several Grk2fl/fl B cell follicles; however, none were revealed in the Grk2fl/flmb1-cre splenic follicles (Fig. 2E). Some MZ macrophages were tightly associated with an underlying T cell zone without an obvious B cell zone in the mice lacking Grk2 in their B cells. The splenic MZs were poorly delineated from the surrounding red pulp, and the expanded red pulp contained numerous B cells, scattered knots of CD169+ cells, and many Ki67+ cells. Interestingly, Grk2fl/flmb1-cre B and CD4 T cells appeared to accumulate at various sites along the periphery of the spleen (data not shown). Overall the architecture of the Grk2fl/flmb1-cre spleens suggests that B cells lacking GRK2 cannot properly access the white pulp, nor can they properly leave the red pulp to enter the circulation.
Enhanced Grk2fl/flmb1-cre B cell–specific migration to S1P but impaired responses to chemokines
The disrupted splenic architecture in the Grk2fl/flmb1-cre mice suggested serious problems in chemoattractant-directed cell migration, as the splenic phenotype resembled that in mice whose B cells lacked Gαi proteins (5). The previous study of Grk2fl/flCD4-cre mice had shown an enhanced S1P-triggered CD4 T cell migratory response; however; a checkerboard analysis showed that the response did not depend upon an S1P gradient (14). The FO and MZ B cells in the Grk2fl/flmb1-cre mice also had enhanced migratory responses to S1P. Although the FO and MZ B cell migration to CXCL12 and CXCL13 were reduced by 20–25%, the differences did not reach statistical significance (14). We examined the responses of Grk2fl/flmb1-cre and control splenic B cell subsets to S1P, CXCL12, CXCL13, and CCL19 (Fig. 3A). We found that all the B cell subsets exhibited excessive spontaneous migration (no chemoattractant). Subtracting nonspecific migration from the chemoattractant-elicited migration gauges the specific migration. The Grk2fl/flmb1-cre B cell subsets had heightened S1P-specific responses, except for the MZ precursors (data not shown) and MZ B cells, which had lower responses to 100 nM S1P. In contrast, most Grk2fl/flmb1-cre B cell subsets had suboptimal specific responses, especially to high chemokine concentrations. The specific migration to CXCL13 was most affected. All the subsets had elevated S1PR1 expression levels, unperturbed CXCR4, CXCR5, and CCR7 expression, and internalized S1PR1 at a slower rate than did WT cells (Fig. 3B, 3C, data not shown). Placing GRK2-deficient splenic B cells into an S1P-depleted environment marginally affected their S1PR1 expression (Fig. 3C). Together, these results indicate that the B cell GRK2 deficiency had enhanced cell motility, increased responses to low concentrations of S1P, but reduced specific responses to chemokines. As expected, the GRK2 loss reduced S1PR1 receptor internalization following S1P exposure.
Leukocytosis but a dearth of B cells in LNs and Peyer patches of Grk2fl/fmb1-cre mice
Although the splenic analysis revealed multiple severe immune defects in the Grk2fl/flmb1-cre mice, their spleens had plenty of surface IgD+ B cells. To assess whether the Grk2fl/flmb1-cre IgD+ B cells exited the spleen into the bloodstream, we checked the cellular composition of the blood. We found that the Grk2fl/flmb1-cre mice had a nearly 2-fold increase in their blood leukocyte counts versus control mice (Fig. 4A). Although their blood had a lower percentage of B cells, the overall B cell numbers in the blood were similar with controls. Analysis of B cell subsets in the blood revealed an increase in immature and transitional B cells and a slight reduction of IgD+ cells (Fig. 4B). Checking the cell surface expression of the blood B cells revealed modest increases in CXCR4, S1PR1, and ICAM-1 expression but small reductions in CD62L and CD11a (Fig. 4C, 4D). The blood B cells from the Grk2fl/flmb1-cre mice exhibited even more severe reductions in chemokine-directed migration than did the splenic B cells. In contrast to the splenic B cells, we did not observe a difference in nonspecific migration (Fig. 4E). Overall, the blood leukocyte analysis verified the presence of recirculating IgD+ B cells in the Grk2fl/flmb1-cre mice and demonstrated the inability of blood B cells to appropriately respond to chemokines.
The previous analysis of the Grk2fl/flmb1-cre mice noted a significant reduction of B cells in LNs (14). We also found a sharp reduction in Grk2fl/flmb1-cre LN B cells (Fig. 4F). The Grk2fl/flmb1-cre mice consistently had small LNs, which contained small LN follicles, and the mice had a near absence of Peyer patches (Fig. 4F, Supplemental Fig. 1). Again, we found a significant increase in the nonspecific migration of Grk2fl/flmb1-cre LN B cells compared with control cells, although the CD4 T cells from the same mice exhibited no such difference (Fig. 4G). Like the Grk2fl/flmb1-cre B cells from other sites, the LN B cells exhibited reduced specific migration to chemokines but enhanced responses to S1P (Fig. 4H). CXCR4, CCR7, and CXCR5 receptor expression levels did not differ, although the Grk2fl/flmb1-cre LN B cells expressed higher amounts of S1PR1 (Fig. 4I). These results are consistent with poor LN homing and/or accelerated egress and the previously observed altered migration in response to S1P and chemokines.
Impaired Grk2fl/flmb1-cre B cell homing, egress, and positioning within lymphoid organs
Preliminary homing experiments, in which we adoptively transferred a 1:1 ratio of WT and knockout (KO) B cells to WT mice, revealed a poor recovery of KO B relative to WT B cells from LNs (data not shown). To better assess the homing defect, we transferred a 2-fold excess of Grk2fl/flmb1-cre B cells to control cells and sampled the blood, spleen, peripheral LNs, and bone marrow 2 h later. As expected, we found more KO B cells in the blood and spleen, yet the LNs and bone marrow had comparable numbers of cells, consistent with a homing defect (Fig. 5A, left panel). To assess LN egress, 2 h after adoptive transfer, we blocked further LN entrance with an Ab directed against CD62L. Eighteen hours later, we enumerated the numbers of control and mutant B cells at the various sites. Although we transferred a 2-fold excess of KO B cells, we recovered fewer Grk2fl/flmb1-cre B cells from the LNs and bone marrow, consistent with accelerated egress (Fig. 5A, middle panel). Despite the 2:1 transfer ratio, we recovered similar numbers of blood B cells, arguing that many Grk2fl/flmb1-cre B cells failed to recirculate. Examining the ratio between the homing and egress results highlights the increased splenic retention, accelerated LN egress, and poor bone marrow retention (Fig. 5A, right panel). These results likely explain the lack of B cells in LNs, Peyer patches, and the 5-fold reduction of Fr. F cells in the bone marrow.
To better understand the positioning of B cells in the spleen and LNs, we adoptively transferred labeled Grk2fl/fl and twice as many Grk2fl/flmb1-cre B cells into WT mice. At 4 h posttransfer, we found nearly one-third fewer GRK2-deficient B cells had entered the splenic white pulp, whereas 2.6-fold more resided in the red pulp (data not shown). At 18 h, the splenic follicles had one-fourth fewer GRK2-deficient B cells, whereas 4.3-fold more resided in the red pulp (Fig. 5B). Examining LNs from the same animals, we found fewer GRK2-deficient B cells in the LN follicles but more Grk2fl/flmb1-cre B cells than control cells at the B–T cell border (Fig. 5C). To confirm these findings, we made 1:1 bone marrow chimeras by infusing CD45.1 bone marrow with either CD45.2 Grk2fl/fl or CD45.2 Grk2fl/flmb1-cre bone marrow into irradiated CD45.1 mice. Eight weeks after bone marrow reconstitution, we checked the localization of the CD45.1 and CD45.2 cells by flow cytometry and immunohistochemistry (Supplemental Fig. 2). As we had noted following the B cell adoptive transfer, the chimeric mice demonstrated that the GRK2-deficient B cells largely resided in the red pulp, whereas the control B cells localized in the B cell follicles. The B cell follicles had 20-fold fewer Grk2-deficient B cells than control B cells. In contrast, the MZs exhibited a strong preference for GRK2-deficient B cells. In LNs, the GRK2-deficient B cells poorly populated LNs, and those cells that entered LNs largely localized at the B–T border rather than in the center of the follicle (Supplemental Fig. 2). These results confirm the previous data illustrating the defective positioning of the GRK2-deficient B cells in lymphoid organs.
Blood-borne GRK2-deficient B cells adhere to high endothelial venules but exhibit defective transmigration
The LN homing defect suggests that either the GRK2-deficient B cells adhere poorly to high endothelial venules (HEVs) or that they have difficulty in crossing the endothelial and pericyte barriers to enter LNs. To address the cause of the homing defect, we examined the behavior of fluorescently labeled, adoptively transferred Grk2fl/fl and Grk2fl/flmb1-cre B cells in HEVs by intravital microscopy. Imaging between 10 and 70 min following i.v. transfer, we found that similar numbers of Grk2fl/fl and Grk2fl/flmb1-cre B cells arrived and adhered to HEVs (Fig. 5D, Supplemental Video 1). Overall, their movements on the HEVs were similar (Fig. 5D, below); however, many of the Grk2fl/flmb1-cre B cells remained adherent to the HEV, either failing to cross the endothelial barrier or only crossing the endothelial barrier without fully exciting the HEV. We tracked 15 Grk2fl/fl- and 27 Grk2fl/flmb1-cre–adherent B cells in HEVs. We found that 84.6% of the Grk2fl/fl B cells that adhered to the HEV surface transmigrated with a 100% success rate. In contrast, only 54.6% Grk2fl/flmb1-cre–adherent B cells attempted to transmigrate, with a success rate of 37.5%, resulting in an overall transmigration frequency of 20.3%. Some GRK2-deficient B cells remained confined to the perivascular space between the endothelium and pericyte layer (Supplemental Video 1). During these experiments, we noted fewer WT B cells had transmigrated than expected, based on our previous studies. To test whether the GRK2-deficient B cells might impair WT B cell entrance into LN, 1 h prior to adoptively transferring Grk2fl/fl B cells fluorescently marked (blue) we transferred either Grk2fl/flmb1-cre (green) or Grk2fl/fl B cells (red). Two hours later, we assessed the number of Grk2fl/fl B cells (blue) in LNs. We found that the prior transfer of Grk2fl/flmb1-cre B cells had severely compromised the homing of Grk2fl/fl B cells (Fig. 5E).
To track B cells within the LN parenchyma we transferred a 2:1 ratio of Grk2fl/flmb1-cre and Grk2fl/fl B cells and imaged the following day (Fig. 5F). The imaging revealed that the GRK2-deficient B cells that had entered LN tended to remain near the HEVs rather than migrating into the LN follicles. Tracking the control and GRK2-deficient B cells did not reveal any statistical difference in their behavior, except that the GRK2-deficient B cells had a reduced displacement, consistent with their tendency to remain close to the HEVs. To analyze the motility of FO B cells, we transferred a 4:1 ratio of Grk2fl/flmb1-cre and Grk2fl/fl B cells. This allowed enough Grk2-deficient B cells to access the follicle for us to track them (Fig. 5G, Supplemental Video 2). We found that the Grk2-deficient B cells moved faster with greater displacement than did the control cells within the follicle. These results show that the loss of GRK2 in B cells impairs transendothelial migration and interferes with the normal trafficking through LN follicles.
Enhanced S1P signaling but reduced CXCL13 signaling, except for the intracellular Ca2+ response in Grk2-deficient B cells
As we had noted the most significant impairment in the migration to CXCL13, we compared the effect of CXCL13 and S1P on the induction of downstream signaling intermediates. Chemoattractant receptor signaling leads to Gi-dependent increases in AKT and ERK phosphorylation; but it leads to decreases in ezrin, radixin, and moesin (ERM) phosphorylation (14, 23). We used flow cytometry to assess the levels of pAKT and pERK in Grk2fl/flmb1-cre and Grk2fl/fl splenic B cell subsets following exposure to CXCL13 or S1P (Fig. 6A, 6B). The Grk2fl/fl and GRK2-deficient splenic B cell subsets had similar basal levels of pAKT and pERK (data not shown). CXCL13 increased pAKT levels in control B cells, but the GRK2-deficient B cells responded less well. S1P exposure induced weak increases in pAKT levels in the control B cells, whereas the loss of GRK2 raised the response to that observed with CXCL13. The pERK responses mirrored the pAKT responses, as the loss of GRK2 reduced CXCL13 responses and strongly increased S1P responses. Similarly, the lack of GRK2 enhanced the S1P-induced increases in pBTK and reduced the CXCL13-induced increases (Supplemental Fig. 3). We found pERM levels were lower in the Grk2fl/flmb1-cre blood B cells but not in blood CD4 T cells. Basal pERM levels were similar in Grk2fl/flmb1-cre MZ B cells to that of controls, although surprisingly, they were elevated in FO and T0/T1 B cells from the same mice (Fig. 6B). Exposing T0/T1 B and FO B cells to S1P reduced pERM proteins in Grk2fl/fl B cells and GRK2-deficient B cells, although the latter exhibited an exaggerated and more pronounced downregulation. The Grk2fl/fl MZ B cells behaved like the other B cell subsets, whereas the GRK2-deficient MZ B cells had a biphasic response to S1P with an initial increase in pERM levels followed by a prolonged downregulation (Fig. 6B).
Chemoattractant receptor signaling predominately raises intracellular Ca2+ levels via Gβγ subunit–mediated increases in phospholipase C activity (24). In contrast, to the other CXCL13-induced signaling pathways we had assessed, the loss of Grk2 enhanced the intracellular Ca2+ responses. As expected, S1P elicited much more potent responses in the KO B cells (Fig. 6C). The lack of GRK2 in B cells raised the basal intracellular Ca2+ (KO 363 ± 3 versus control 283 ± 4) and slightly enhanced the increase intracellular Ca2+ following cross-linking the B cell Ag receptor (data not shown). The Grk2fl/flmb1-cre B cells intracellular Ca2+ responses to chemoattractants remained dependent upon Gαi proteins, as pertussis toxin blocked both the S1P- and CXCL13-elicited increases (Fig. 6C). To assess the contribution of S1PR1 to the S1P-induced increases in intracellular Ca2+, we pretreated the Grk2fl/flmb1-cre and Grk2fl/fl splenic B cells with Ex 26, a specific S1PR1 antagonist (25), prior to the S1P challenge (Fig. 6D). The Ex 26 pretreatment blocked the S1P-induced increases in intracellular Ca2+ in both the control and GRK2-deficient B cells. The Ex 26 pretreatment slightly reduced the CXCL13-induced intracellular Ca2+ response in the control B cells and consistently enhanced the CXCL13 response in the GRK2-deficient B cells.
Ex 26 reverses the CXCL13 chemotaxis defect and partially restores normal in vivo B cell positioning
To determine whether blocking S1PR1 signaling would affect B cell migratory responses, we again used Ex 26 (25), (Fig. 7). Using B cells from the chimeric mice allowed for testing B cells from the same mice in the same assays, distinguishing their genotype based on CD45.1 (WT) versus CD45.2 (GRK2-deficient). Pretreatment of WT B cells with Ex 26 increased the percentage of cells migrating to CXCL13, whereas it marginally affected the responses to CXCL12 and CCL19. The drug treatment reduced the WT T1 B and FO B cell responses to S1P but minimally affected the MZ B cells, as S1PR3 predominately mediates MZ B cell chemotaxis. Treating the GRK2-deficient B cells with Ex 26 reduced the nonspecific migration and raised the percentage cells specifically migrating in response to all the chemokines, although the FO B cell responses to CXCL12 and CCL19 did not reach statistical significance. The Ex 26 pretreatment blocked the exaggerated T1 and FO B cells responses to S1P.
To test whether Ex 26 would affect the positioning of GRK2-deficient B cells in lymphoid organs we treated the CD45.1 (WT):CD45.2 (Grk2fl/flmb1-cre) chimeric mice overnight with Ex 26. The following day we examined the location of the WT and GRK2-deficient B cells in tissue sections from the spleen and LNs. The Ex 26 treatment resulted in a clear shift of the GRK2-deficient B cells from the red pulp into the white pulp, although the ratio between WT and GRK2-deficient B cells varied among the different B cells follicles, with some being well populated with GRK2-deficient B cells and others less so (Supplemental Fig. 4). Similarly, in the LNs, more GRK2-deficient B cells populated the LN follicle, although WT cells predominated in the LN follicle center (Supplemental Fig. 4). Because we only treated with a single injection of Ex 26, a more prolonged treatment regimen may have further normalized the distribution of the GRK2-deficient B cells.
Outputs from chemoattractant-triggered signaling pathways control cellular migration and behavior. Not surprisingly, numerous interacting mechanisms exist to either enhance or dampen these outputs (26). Consequently, disruption of one of these mechanisms can have far-reaching and, sometimes, unanticipated consequences. This is the case with the loss of GRK2 in B cells. A severe phenotype arose, apparently largely because of a failure of GRK2 to phosphorylate S1PR1 receptors. Inappropriate S1PR1 signaling likely usurps signal transduction intermediates needed by other chemoattractant receptor signaling pathways. It also led to compensations that may have exacerbated the phenotypes. This report revealed a major impact of the loss of GRK2 in B cells on CXCR5 signaling. In the end, the misbalance between chemokine and S1PR1 signaling in the GRK2-deficient B cells explains many of the abnormalities in the B cell compartment of these mice.
Because B cell lymphopoiesis in adult mice begins in the bone marrow, we first assessed the impact of the loss of GRK2 on bone marrow B cell development. The noted increase in Fr. D cells may be secondary to the loss of Fr. F cells and the premature release of immature Fr. E cells. We noted a difference in nonspecific migration and a slight reduction in migration to CXCL12. The enhanced motility of the GRK2-deficient immature B cells may have facilitated their premature release into the bloodstream. Consistent with this idea, blood from the Grk2fl/flmb1-cre mice had an excess of immature B cells. The most intriguing bone marrow phenotype was the major of loss Fr. F cells, mature B cells that have recirculated to and lodged in the bone marrow. This loss did not arise from a lack of mature IgD+ B cells in blood but rather results from mature B cells that have failed to access and/or be retained in their designated microenvironment. Future bone marrow imaging experiments may help explain why these cells fail to lodge and be retained in the bone marrow.
The splenic architecture of the Grk2fl/flmb1-cre mice was severely compromised. Whereas dysregulated chemokine and S1P signaling provides an explanation for some of the splenic abnormalities, other abnormalities will require additional study to be fully understood. The Grk2fl/flmb1-cre animals had spleens 2-fold larger than control animals. Furthermore, the spleens had an excess of both B and T cells, many of which accumulated in the red pulp. This suggests that the GRK2-deficient B cells had interfered with the normal lymphocyte egress pathways in the spleen, thereby causing a problem for T cell egress. This may reflect a difficulty in cell migration from the splenic cords into the sinusoids; however, the precise reason for this is unclear. The levels of signaling available S1P in the splenic red pulp is low (27), arguing that a retention mechanism is unlikely to be the sole explanation. S1PR1 signaling supports plasma cell egress from the spleen into the blood (28) and FO B cell egress from the white pulp to the red pulp, but its involvement in the later stages of lymphocyte egress from the spleen are unknown. The reduced white pulp in the spleens of the Grk2fl/flmb1-cre mice reflects the loss of B cells from the white pulp, which in-turn led to the FO dendritic cell loss. The failure of B cells to populate the white pulp is secondary to excessive S1PR1 signaling retaining the cells near the MZ and reduced CXCL13-directed migration, which normally draws cells into the B cell follicle (22). The poor population of the splenic B cell follicles could be partially reversed by treating the mice with an S1PR1 antagonist, Ex 26. Based on our analysis, the drug would be not only expected to reduce the S1PR1 signal, but also to improve signaling through CXCR5.
Impaired chemokine receptor signaling along with excessive S1PR1 signaling also explains many of the LN phenotypes in the Grk2fl/flmb1-cre mice. The lack of B cells in the LNs results from a homing defect along with their accelerated egress. Imaging in the HEVs revealed no obvious adherence defect but revealed a problem in B cell transendothelial migration. This result suggests that the loss of Grk2 in B cells had not impaired chemokine-induced integrin activation. Some adherent GRK2-deficient B cells migrated on the endothelium for extended periods, adopting a normal polarized morphology, but often failed to transmigrate. Other GRK2-deficient B cells migrated through the endothelial barrier only to get stuck in the perivascular space. This explains the reduced homing of WT B cells in the presence of GRK2-deficient B cells. The B cell transmigration defect may reflect the inability of GRK2-deficient B cells to properly sense the chemokines that direct B cells into the LN or, conversely, to properly limit integrin activation. Alternatively, the inappropriate S1PR1 signaling may have altered the physical properties of the cells, interfering with the normal membrane deformation required for transmigration.
Of those B cells that did enter the LN parenchyma, many did not migrate away from the HEVs into the LN follicle. Rather, they remained localized near the HEVs, which presumably improved their likelihood of leaving the LN by entering the nearby cortical lymphatics, thereby explaining the accelerated egress noted in these mice. The B cells that managed to migrate from the HEVs to the LN follicle either failed to enter the follicle or tended to remain along the edge. These cellular behaviors are best explained by an inability of the GRK2-deficient B cells to properly sense CXCL13 emanating from the LN follicle. The decreased tendency to migrate to the follicle center could also be explained by an enhanced sensitivity to 7α,25-dihydroxyxcholesterol, the GPR183 (Ebi2) ligand (29); however, we could find no in vitro evidence to support that hypothesis, as LPS-activated B cells migrated less well than did control cells to 7α,25-dihydroxyxcholesterol (I.-Y. Hwang, unpublished observations).
Not only did the Grk2fl/flmb1-cre mice have small underpopulated LNs, they had only two to four visible Peyer patches versus the usual 8–10 found in control mice, and we recovered 20-fold fewer cells. Deleting GRK2 from bone marrow B cell progenitors should not impair early Peyer patch development, suggesting that loss of Peyer patch cells results from a major defect in B cell recruitment or retention. CXCR4, CXCR5, and CCR7 all participate in B cell homing to Peyer patches (30). The same issues as outlined with the entrance of B cells into LNs, nondirectional S1PR1 signaling along with inadequate chemokine receptor signaling, likely limit the recruitment of GRK2-deficient B cells from the blood into Peyer patches. Because the Peyer patch cell population normally contains 70–80% B cells, a recruitment/retention defect would lead to a major loss of the cells populating Peyer patches.
Our migration assay results differ from those previously reported, which also analyzed Grk2fl/flmb1-cre mice on a B6 background (14). Like their report, we found that FO B cells had a heighted response to S1P; however, we found that MZ B cells responded less well to 50 and 100 nM S1P. The reason for this discrepancy is unclear. Compared with the usual 2–5% nonspecific migration, as many as 10–15% of the GRK2-deficient B cells migrated in the absence of any chemoattractant. Interestingly, treating the B cells with Ex 26 substantially reduced this nonspecific migration, arguing that in GRK2-deficient cells S1PR1 signals, in part constitutively, and that Ex 26 may function not only as a neutral antagonist, but also as an inverse agonist. Others have noted constitutive S1PR1 signaling, and a previous study showed that the constitutive activity by S1PR1 receptors enhanced platelet-derived growth factor–induced cell migration. A partial S1PR1 agonist, SB649146, behaved as an inverse agonist, inhibiting the migration (31). Treating the GRK2-deficient T1 and FO B cells with Ex 26 largely blocked their migration response to S1P and reversed their decreased specific migration to chemokines. Intriguingly, Ex 26 also enhanced the specific migratory response of control B cells to CXCL13, suggesting that constitutive S1PR1 signaling may limit CXCL13-directed migration in normal B cells.
In general, the pERK and pAKT assays correlated well with the migration assay, as the Grk2fl/flmb1-cre B cells had reduced responses to CXCL13 and heightened responses to S1P. Like the pERK and pAKT responses, S1P elicited a strong increase in phosphorylated Bruton tyrosine kinase in the Grk2fl/flmb1-cre B cells, whereas CXCL13 triggered a poor response. However, the intracellular Ca2+ responses behaved differently, as the GRK2-deficient B cells had heightened responses to CXCL13 and S1P. Typically, S1P elicits a rather anemic intracellular Ca2+ response; however, the loss of GRK2 boosted it to the level typically observed following CXCL13 stimulation of control B cells. Not only did the GRK2 deficiency not impair the CXCL13-induced intracellular Ca2+ response, we consistently found a 1.5–2-fold increase in the peak response, compared with controls. We do not have a good explanation for this disparity. We did note that the Grk2fl/flmb1-Cre splenic B cells had a higher basal level of intracellular Ca2+ and a 20% increase in CD38 expression (I.-Y. Hwang, unpublished observations). Excessive S1PR1 signaling may have increased CD38 expression, which could elevate intracellular Ca2+ responses.
In conclusion, the loss of GRK2 in B cells disrupted the normal S1P-induced S1PR1 desensitization/resensitization cycle. Despite exposure to saturating concentrations of S1P, GRK2-deficient blood B cells maintain S1PR1 expression, whereas control B cells do not. Strikingly, the loss of GRK2 led to a nearly 10-fold increase in the magnitude of S1PR1 signaling as assessed by changes in pAKT, pERK, and intracellular Ca2+ levels. The enhanced signaling likely contributes to the hypermigratory responses of the GRK2-deficient B cells to S1P. Although the heightened receptor expression may explain the increased S1PR1 signaling, a role for GRK2 in limiting signal transduction by some form of negative feedback remains plausible. Although GRK2 predominately targeted S1PR1, the loss of GRK2 in B cells also affected signaling through chemokine receptors. The reversal of the GRK2-deficient B cell CXCL13 migratory defect by an S1P antagonist in vitro and the partial reversal of the B cell FO homing defect in vivo argues that GRK2-mediated S1PR1 desensitization allows normal chemokine receptor signaling. Future studies will focus on understanding the mechanism by which signaling through S1PR1 impairs chemokine responses and vice versa. Overall, the misbalanced S1PR1 and homeostatic chemokine receptor signaling in the Grk2fl/flmb1-cre mice causes a surprisingly severe B cell–trafficking defect that markedly disrupts normal immune organ architecture and function.
We thank Dr. Anthony Fauci for continued support.
This work was supported by an intramural program of the National Institute of Allergy and Infectious Disease.
The online version of this article contains supplemental material.
Abbreviations used in this article:
G protein–coupled receptor
high endothelial venule
The authors have no financial conflicts of interest.