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The Journal of Immunology, 2003, 170: 1707-1715.
Copyright © 2003 by The American Association of Immunologists

B Cell Developmental Requirement for the G{alpha}i2 Gene 1

Harnisha Dalwadi2,*, Bo Wei2,*, Matthew Schrage*, Tom T. Su{dagger}, David J. Rawlings{ddagger} and Jonathan Braun3,*,{dagger}

* Department of Pathology and Laboratory Medicine and {dagger} Molecular Biology Institute, University of California, Los Angeles, CA 90095; and {ddagger} Departments of Pediatrics and Immunology, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Null mutation of the G{alpha}i2 trimeric G protein results in a discrete and profound mucosal disorder, including inflammatory bowel disease (IBD), attenuation of IL-10 expression, and immune function polarized to Th1 activity. Genetic and adoptive transfer experiments have established a role for B cells and IL-10 in mucosal immunologic homeostasis and IBD resistance. In this study, we addressed the hypothesis that G{alpha}i2 is required for the development of IL-10-producing B cells. G{alpha}i2-/- mice were reduced in the relative abundance of marginal zone (MZ), transitional type 2 (T2), and B-1a B cells and significantly increased in follicular mature and B-1b B cells. Reconstitution of RAG2-/- mice with G{alpha}i2-/- bone marrow induced an IBD-like colitis and a deficiency in absolute numbers of MZ, T2, and B-1 B cells. Thus, the G{alpha}i2-/- genotype in colitis susceptibility and B cell development involved a cis effect within the hemopoietic compartment. In vitro, the B cell population of G{alpha}i2-/- mice was functionally deficient in LPS-induced proliferation and IL-10 production, consistent with the exclusive capacity of T2 and MZ cell subpopulations for LPS responsiveness. In vivo, G{alpha}i2-/- mice were selectively impaired for the IgM response to T-independent type II, consistent with the relative depletion of MZ and peritoneal B-1 subpopulations. Collectively, these results reveal a selective role for G{alpha}i2 in MZ and B-1 B cell development. Disorders of this G{alpha}i2-dependent process in B cell development may represent a mechanism for IBD susceptibility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mice with targeted disruption of G protein {alpha} inhibitory subunit (G{alpha}i2)4 develop colonic inflammation with features similar to those of human inflammatory bowel disease (IBD) (1). IBD, including Crohn’s disease and ulcerative colitis, is a immune-mediated disease distinguished by aberrant mucosal T cell and cytokine activity, resulting in cellular inflammation and intestinal tissue damage (2, 3, 4). G{alpha}i2 is a potential candidate gene involved in the pathogenesis of human IBD, because linkage studies in humans have implicated an IBD at chromosome 3p21, overlapping with the G{alpha}i2 gene (1, 5). However, how G{alpha}i2 plays a role in IBD pathogenesis is presently unclear.

G proteins are heterotrimeric signaling complexes that couple to seven transmembrane domain receptors (6). There are four families of the G {alpha} subunit: {alpha}s, {alpha}q, {alpha}i, and 12/13. G{alpha}i proteins are involved in the inhibition of adenyl cyclases, activation of phosphoinositide 3-kinase (PI3-kinase) {alpha}, and activation of certain voltage-independent calcium channels, including CD20. There are three known isoforms of G{alpha}i (G{alpha}i1, G{alpha}i2, and G{alpha}i3), and all are pertussis toxin (PT) sensitive (7). Immune cells express both G{alpha}i2 and G{alpha}i3, and (6), and the mucosal and peripheral immune system in G{alpha}i2-/- animals is biased toward proinflammatory cytokines related to colitis (1, 8, 9). Consistent with this genetic phenotype, pharmacologic inhibition of G{alpha}i by PT enhances autoimmunity and Th1 responses (10, 11), and G{alpha}i2 in immune cells is important for the regulation of IL-12 production (12, 13). Overall, these studies indicate that G{alpha}i2 expression is important in immune regulation.

Although the exact causes of IBD are unknown, present thinking focuses on host genetic susceptibility traits that might promote an aberrant immunologic response to enteric bacteria (2, 3, 14, 15, 16, 17). IL-10 expression in the mucosal environment is critical for colitis resistance and mucosal immunologic homeostasis (18, 19, 20), and model systems indicate the protective role of B cells in chronic colitis (21, 22). B cells are an important source of IL-10, predominantly after LPS activation (23, 24, 25), and serve a protective role for colitis through their enteric production of IL-10 (26). Because enteric microflora include a prominent set of Gram-negative bacterial species expressing bioactive LPS, it is appealing to envision a homeostatic circuitry linking enteric LPS, mucosal B cells, elevated mucosal IL-10, and quiescent inflammation in the healthy mucosa. Accordingly, disordered B cell development might disrupt this circuitry, contributing to the colitis phenotype seen in G{alpha}i2-/- mice.

Certain mature B cell populations, follicular mature (FM), marginal zone (MZ), and B-1 B cells, are notable for their divergence in antigenic repertoire, responsiveness to B cell receptor (BCR) and T lymphocyte receptor signaling, and patterns of cytokine production (27, 28, 29, 30). Developmentally, immature B cells emigrate from the bone marrow to the splenic periarteriolar sheath, forming the transitional type 1 B cells (T1) (CD21lowCD24highCD23-) (27). These cells develop into transitional type 2 cells (T2) (CD21highCD24highCD23+), and take residence in the spleen primary follicle. T2 B cells differentiate into FM B cells (CD21highCD24lowCD23+) or MZ B cells (CD21highCD24highCD23-) (31), localized in the primary lymphoid follicle and perifollicular MZs, respectively. The B-1 B cell subset is predominantly found in the body cavities and arises from either the bone marrow or fetal liver (27, 28). B-1 B cells are similar in surface phenotype to T2 and MZ B cells but may also express Mac-1 (CD11b). MZ and B-1 B cells are functionally distinguished from FM cells by the capacity to produce IL-10, and features of innate immune function, including a repertoire skewed toward microbial Ags, and an elevated responsiveness to T lymphocyte receptor signaling. Innate B cell subsets thus express traits pertinent to the apparent homeostatic role of B cells in mucosal immune homeostasis. Because such homeostasis is impaired in G{alpha}i2-/- mice, we considered the hypothesis that G{alpha}i2 may be required for the formation of these B cell subsets.

In this study, we use G{alpha}i2-/- mice to assess the role of G{alpha}i2 on B cell development. Our findings reveal that native G{alpha}i2-/- B cells are deficient in B-1a B cells, splenic MZ B cells, and their immediate precursor, T2 B cells. Similarly, reconstitution of recombination-activating gene 2-deficient (RAG2-/-) mice with G{alpha}i2-/- bone marrow induce symptoms similar to inflammatory bowel disease, and are deficient in these B-1a, MZ, and T2 B cells. Functionally, G{alpha}i2-/- mice have a reduced LPS B cell response, both at the levels of proliferation and IL-10 production. Collectively, these results suggest that G{alpha}i2 plays a role B cell development, and that disorders of this G{alpha}i2 -dependent process in B cell development may interfere with the formation of B cell subpopulations which reduce IBD susceptibility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

129Sv/C57 and G{alpha}i2-/- mice (129Sv/C57 background) were kindly provided by Dr. L. Birnbaumer (University of California, Los Angeles) and were housed in specific pathogen-free facilities at the University of California, Los Angeles, Center for Health Sciences Vivarium. RAG2-/- mice (C57BL/6 background) were provided by W. McBride from the breeding facility of the University of California, Los Angeles, Center for Health Sciences Vivarium and housed in SPF facilities with sterile food and bedding and laminar flow ventilation. Both male and female mice (8–12 wk old) were used.

For bone marrow reconstitution, femoral bone marrow was isolated from wild-type and G{alpha}i2-/- mice, and 2 to 4 x 106 cells were injected i.v. into RAG2-/- (129 Sv background) mice previously irradiated with 900 rads (137Cs whole body irradiation; MARK I 68A Irradiator; J. L. Shepard and Associates, San Fernando, CA). Bone marrow recipients were monitored for weight and rectal prolapse and were sacrificed 6 wk after transplantation for intestinal histology and to collect splenic lymphocytes for flow cytometry and functional immunologic assessment.

Flow cytometry

For surface staining, 1 x 106 cells were stained with various combinations of FITC-, PE-, PerCP-, APC-, and biotin-labeled Abs. The Abs used were: anti-CD21; anti-CD23; anti-CD24 (heat-stable Ag); anti-B220; anti-CD19; anti-CD1d; anti-CD5; anti-CD11b; anti-CD9; anti-IgM; and anti-IgD (BD PharMingen, San Diego, CA). The FACSCalibur flow cytometer using Cell Quest software (BD Biosciences, Mountain View, CA) was used to collect and analyze at least 10,000 events. Statistical significance was calculated using an unpaired two-tailed Student t test. p values < 0.05 were considered significant.

Histology and immunohistochemistry

Intestinal tissue samples were taken from G{alpha}i2-/- and G{alpha}i2+/+ bone marrow recipients and examined for the changes in histology. Immunohistochemistry was performed to evaluate the presence of MZ B cells in the spleens of G{alpha}i2-/- and wild-type mice. Briefly, 6-µm sections of OCT-embedded spleen from G{alpha}i2-/- and wild-type mice were stained with 7-amino-4-methylcoumarin-3-acetic acid-anti–1 and Texas Red-anti-IgM, and examined with an epi-ilumination fluorescence microscope (Zeiss).

B cell activation in vitro

RBC-depleted splenocytes from 129Sv/C57 or G{alpha}i2-/- mice were incubated for 20 min at 4°C with Miltenyi Biotech (Auburn, CA) magnetic beads coated with anti-CD43. The unbound resting B cells were >85% B220 by flow cytometry.

The anti-IgM induced calcium response of resting B cells was performed using a flow cytometric assay (32). Briefly, cells were loaded with 5 µM Indo-1 acetoxymethyl ester (Molecular Probes, Plano, TX) and stained with fluorochrome-conjugated Abs for B220, CD21, and CD24 or CD23. The cells were stimulated at room temperature with polyclonal goat F(ab')2 anti-mouse IgM (20 µg/ml; Jackson ImmunoResearch, West Grove, PA), and data were acquired for 500 s at 440 and 510 nM with an LSR flow cytometer (BD Biosciences). The results were analyzed using the ratio of 510:440 nm signals and compiled graphically using FLOWJO software (Tree Star, San Carlos, CA). For measurement of PT effects, resting B cells were preincubated with 1 µg/ml PT (Sigma-Aldrich, St. Louis, MO; or medium control) for 1 h on ice, loaded with Indo-1, and stimulated as above with anti-IgM, and data were collected in the presence of 0.5 mM EGTA with a spectrofluorometer at 510 nm (Beckman, Duarte, CA). Spectrofluorometry data were expressed as the ratio of signal at baseline.

For proliferation and IL-10 production, 1 x 105 splenic resting B cells were incubated in triplicate with various concentrations of Salmonella typhimurium LPS (Sigma-Aldrich) purified as described (33) or rat anti-mouse-CD40 (BD PharMingen) in a 96-well flat-bottom tissue culture plate for 48 h. After 48 h, 0.5 µCi of [3H]thymidine was added to each culture for another 18 h. A Micro 96 harvester (Skatron Instruments, Tranby, Norway) was used to collect the cells, and a Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD) was used to assess proliferation. Supernatants from the cultures were collected after 48 h to assay IL-10 production by capture ELISA (R&D Systems, Minneapolis, MN). The minimal detection for the IL-10 cytokine assay was 50–100 pg/ml.

Immunizations and ELISA

Wild-type and G{alpha}i2-/- 6- to 8-wk-old mice were immunized i.p., injected with 10 µg of thymus-independent trinitrophenyl (TNP)-Ficoll (Biosearch Technologies, Novato, CA) in PBS or 20 µg of thymus-dependent (TD) TNP-keyhole limpet hemocyanin (KLH) (Biosearch Technologies), and precipitated with alum in PBS. Anti-TNP-specific titers were collected at 0, 1, and 2 wk after immunization and analyzed by ELISA on TNP-BSA with isotype-specific Abs (anti-mouse IgG3, -IgG1, and -IgM) (BD PharMingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Disordered splenic and peritoneal cavity B cell subpopulations in native G{alpha}i2-/- mice

G{alpha}i2-/- mice are susceptible to colitis, and B cells play a protective role in this disease process. We therefore set out to assess the possible connection between these observations, that G{alpha}i2 may affect the formation of protective B cell subpopulations. We first evaluated this idea by examining splenic B cell populations in native G{alpha}i2-/- mice, using surface phenotyping to define FM cells (CD23highCD21intermediateCD24lowCD1intermediateIgMintermediateIgDhigh), MZ B cells (CD23-/low CD21high CD24highCD1highIgMhighIgDlow), and T2 cells (CD23lowCD21low/intermediateCD24highIgMhigh/intermediateIgDlow) (31, 34). Spleen cells were gated on B cells (B220+ or CD19+) and analyzed for expression of CD21, CD24, and CD23 (Fig. 1, A–C). Compared with wild-type mice, G{alpha}i2-/- mice were deficient in the T2 (CD21highCD24highCD23+) and MZ (CD21highCD24highCD23-/low) (Fig. 1, B and C). The tabulated results of several experiments confirmed ~4-fold and ~3-fold deficiencies in G{alpha}i2-/- T2 and MZ B cells, respectively (Fig. 2A). In contrast, FM B cells (CD21highCD24low) were increased in G{alpha}i2-/- mice, and T1 (CD21lowCD24high) were unaffected (Fig. 1A). Absolute number analysis showed that G{alpha}i2-/-, compared with wild-type, were 2-fold decreased in T2 cells (p = 0.020) and MZ (not reaching statistical significance), increased FM B cells (statistically significant), and unchanged T1 (Fig. 2B).



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  		FIGURE 1. Defective splenic B cell formation in G{alpha}i2-/- mice. A–G, CD43- splenocytes from wild-type (WT) and G{alpha}i2-/- mice were analyzed by flow cytometry using B220 or CD19 (total B cell) events. A, CD21/CD24; B, CD21/CD23, gated on the T2 plus MZ population (CD21highCD24high cells), and recalculated to determine the percent T2 and percent MZ cells among total B cell events. C, CD21/CD23; D, IgD/IgM; E, CD21/CD9; F, CD21/CD1d; G, CD21/IgM; H, CD21/IgD. J and K, Frozen sections of spleen were stained with 7-amino-4-methylcoumarin-3-acetic acid-metallophilic macrophage-Ag-1 (blue) and Texas Red-IgM (red) and photomicrographed at x 200. I, Wild-type; J, G{alpha}i2-/-.

 


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  		FIGURE 2. Relative and absolute levels of B cell subsets in wild-type (WT) and G{alpha}i2-/- mice. The relative numbers of B cell subsets as a percentage of gated B cells (Figs. 1 and 3) were tabulated, and absolute numbers of B cell subsets were calculated from these relative values (total cell number x % B220 x % B cell subset). Data for individual experiments are indicated by symbols, and mean values are shown by horizontal dashes. A, Percentage of splenic B cell subsets; B, absolute numbers of splenic B cell subsets; C, percentage of peritoneal B cell subsets; D, absolute numbers of peritoneal B cell subsets.

 
To further confirm the deficiency in MZ cells, we assessed the levels of CD1d and CD9, the expression of which is elevated in MZ B cells (35, 36, 37, 38). As expected, CD1d- and CD9-expressing B cells were profoundly deficient in the CD21high B cell population of G{alpha}i2-/- mice (Fig. 1, E and F). Similarly, the percentage of IgMhighIgDlowCD21high B cells was also decreased in G{alpha}i2-/- mice (Fig. 1, G and H), consistent with the deficiency of T2 and MZ B cells that bear this membrane Ig phenotype (27, 34, 39, 40). The T2 and MZ deficiency was also reflected in the diminution of IgMhighIgDlow B cells in G{alpha}i2-/- mice (Fig. 1D).

Tissue immunofluorescence was used to assess the microanatomy of splenic B cells in wild-type and G{alpha}i2-/- mice. In wild-type mice, a prominent population of IgMhigh cells was present in the splenic MZ (demarcated by layer of metallophilic macrophage Ag-1-positive MZ macrophages) (30) (Fig. 1J). In contrast, G{alpha}i2-/- mice lacked the MZ B cell population, and instead displayed a population of follicular B cells with aberrantly bright IgM+ expression (Fig. 1K). This aberrant IgM expression was confirmed in flow cytometry by the presence of an increased B population displaying a CD21lowIgMhigh (Fig. 1G) and IgMhighIgDhigh (Fig. 1D) phenotype.

We next examined whether G{alpha}i2 affected the formation of peritoneal B-1 B cells (CD19+CD11b+), and their CD5+ and CD5- subpopulations (B-1a and B-1b, respectively). Compared with wild-type mice, G{alpha}i2-/- mice displayed a 9-fold reduction in the percentage of B-1a B cells and an increase in the percentage of B-1b B cells (Fig. 3A). A tabulation of several experiments confirmed that this was a consistent change in both the relative and absolute numbers of these B cell subpopulations (Fig. 2, C and D). B-1a and B-1b B cells in G{alpha}i2-/- mice were also distinguished from wild-type mice (37) by a prominent subpopulation of CD9- (Fig. 3, B and C) and IgMintermediateIgDhigh B cells (Fig. 3, C and F). There was also a marked expansion of IgMintermediateCD23+ B cells in G{alpha}i2-/- mice (Fig. 3D). Backgating confirmed that the anomalous peritoneal B cell population in G{alpha}i2-/- mice (~35% of CD19+ cells) was CD5-CD11bintermediateCD23+IgMintermediateIgDhigh (data not shown). This population may correspond to the peritoneal B-2 cells identified by Erickson et al. (41).



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  		FIGURE 3. Phenotypic analysis of peritoneal B-1 B cells in G{alpha}i2-/- and wild-type (WT) mice. Peritoneal cavity cells were analyzed by flow cytometry, gating on CD19+ events. A, CD11b/CD5; B, IgM/CD9; C, IgD/CD9; D, IgM/CD23; E, IgD/CD23; F, IgD/IgM. The percentage of each gated population is indicated.

 
Depletion of B cell subpopulations and colitis after G{alpha}i2-/- bone marrow reconstitution

To assess whether the G{alpha}i2 null mutation in hemopoietic cells alone could confer this phenotype, we compared the formation of B cell subpopulations 6 wk after wild-type or G{alpha}i2-/- bone marrow transfer into irradiated, MHC-matched RAG2-/- mice. Reconstitution with bone marrow from wild-type mice resulted in a normal distribution (Fig. 4, A and B), percentage (Fig. 4C) and absolute number (Fig. 4D) of T1, T2, FM, and MZ B subsets in splenic B cell populations. In contrast, reconstitution with G{alpha}i2-/- bone marrow resulted in a disordered B cell compartment. Splenic T2 and MZ B cells were very low, FM B cells were expanded, and T1 populations were normal (Fig. 4, A, C, and D). In the peritoneum, the percent of B cells was significantly reduced (4-fold), apparently due to a reduction in absolute numbers of total peritoneal B cells (8-fold, although with substantial data scatter and not statistically significant) (Fig. 4E). These results indicate that G{alpha}i2 expression in hemopoietic cells affects transitional T2, MZ, and B-1 B cell development.



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  		FIGURE 4. Disordered B cell development in G{alpha}i2-/- bone marrow chimeras. Six weeks after wild-type (WT) or G{alpha}i2-/- bone marrow transfer into MHC-matched irradiated RAG2-/- mice, resting splenic B cells were isolated from wild-type and G{alpha}i2-/- bone marrow chimeras, and analyzed for B cell subsets by flow cytometry. B220+ events were used to calculate percentages of each subpopulation. A, CD21/CD24; B, CD21/CD23. Results represent one of three similar experiments. C, Percentage of splenic B cell subsets; D, absolute numbers of splenic B cell subsets; E, percentage of peritoneal B cell subsets; F, absolute numbers of peritoneal B cells.

 
RAG2-/- reconstituted with G{alpha}i2-/- vs wild-type bone marrow was also distinguished by the development of colitis. Among 2 groups of 10 mice, all G{alpha}i2-/- recipients began losing weight 5–6 wk after bone marrow transfer, most displayed rectal prolapse, and all showed histologic features of colitis (mucosal lymphocyte infiltration and epithelial damage in the colon and ileum). No wild-type recipients displayed any of these features of colitis.

Augmented BCR calcium response in G{alpha}i2-/- mice

Increased strength of BCR signaling is a factor favoring FM vs MZ differentiation. We therefore tested the prediction that pharmacologic or genetic G{alpha}i2 inactivation would augment BCR signaling. We first tested PT, a selective inhibitor of G{alpha}i, for its effect on BCR-induced intracellular calcium mobilization. PT treatment of splenic B cells resulted in an increased amplitude and persistence of calcium mobilization (Fig. 5A). We then determined the effect of the G{alpha}i2 genotype on this calcium response among B cell subsets. As previously reported, MZ and T2 B cells displayed elevated calcium response compared with FM B cells (42, 43, 44). Among FM B cells, the calcium response was elevated in G{alpha}i2-/- vs wild-type cells (Fig. 5B), providing concordant genetic evidence for an inhibitory effect of G{alpha}i2. However, for the MZ and T2 subsets, no difference in the calcium response was observed with respect to G{alpha}i2 genotype (Fig. 5, C and D). T1 cells minimally responded to anti-IgM (Ref.42 and data not shown). Thus, pharmacologic inactivation of G{alpha}i2 enhances the BCR-induced calcium response, but genetic absence of G{alpha}i2 does not uniformly increase the response among B cell subsets. This is particularly notable for T2, which is the transitional stage at which the MZ vs FM fate choice takes place.



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  		FIGURE 5. B cell activation in G{alpha}i2-/- mice. A—D, Resting B cells, loaded with Indo-1, were stimulated with anti-IgM and assayed for calcium-dependent fluorescence. A, Cells pretreated with PT or medium control; B—D, wild-type (WT) or G{alpha}i2-/- B cells prestained with subset-specific mAbs and gated for FM (B220+CD21intCD24-) (B), MZ (B220+CD21highCD23-) (C), and T2 (B220+CD21highCD24+) (D) B cells. E—H, Resting B cells from wild-type and G{alpha}i2-/- mice were cultured with different concentrations of anti-CD40 (E) or LPS (F–H). Proliferation was assessed at 48 h by [3H]thymidine incorporation (E and F). LPS-induced IL-10 production in spleen cells (G) and peritoneal B cells (PEC) (H). Rel. Fluor., Relative fluorescence.

 
Defective in vitro LPS response in G{alpha}i2-/- mice

IL-10 expression is important for mucosal homeostasis, and B cells are a major producer of IL-10, particularly on activation with LPS. It is thus appealing that colitis susceptibility in G{alpha}i2-/- mice might involve impaired production of B cell-derived IL-10. To test this possibility in vitro, the LPS response was examined in G{alpha}i2-/- mice. Splenic CD43- cells (resting B cells) and peritoneal cells were isolated from wild-type and G{alpha}i2-/- mice, cultured with LPS or anti-CD40, and assayed for proliferation and cytokine production (IL-10). Whereas wild-type splenic B cells responded to LPS by both proliferation and IL-10 production, both responses were deficient in G{alpha}i2-/- mice (Fig. 5, F and G). As a positive control, wild-type and G{alpha}i2-/- B cells were similarly active for anti-CD40 proliferation (Fig. 5E). These results indicate that G{alpha}i2-/- resting B cells are selectively deficient in LPS-triggered proliferation and IL-10 production. This conclusion is concordant with the exclusive capacity of T2 and MZ cell subpopulations for LPS responsiveness, suggested by Oliver et al. (45), and directly demonstrated in a recent study.5 peritoneal cells, IL-10 production was only slightly reduced in G{alpha}i2-/- mice, despite a 5- to 10-fold reduction of peritoneal B cells (Fig. 5H). This suggests that non-B cells can be a major source of LPS-induced IL-10 production in this cellular compartment.

Intact TD and thymus-independent type II (TI-II) responses in G{alpha}i2-/- mice

B cell function in wild-type and G{alpha}i2-/- mice was compared in vivo for the TD and TI-II responses. FM B cells are the predominant responder to TD-Ag and produce IgG1, whereas MZ B cells respond to TI-II Ags and produce IgM and IgG3 (38). G{alpha}i2-/- mice are distinguished by an increased percent of FM and reduced MZ B cells. Accordingly, we predicted that G{alpha}i2-/- might express an elevated TD response, and reduced TI-II response. To test this, wild-type and G{alpha}i2-/- mice were immunized with TD (TNP-KLH) and TI-II (TNP-Ficoll) Ags, and TNP-specific IgG3, IgM, and IgG1 responses were analyzed by ELISA (Fig. 6). In the TI-II response (TNP-Ficoll), there was a statistically significant reduction in the IgM response, concordant with the relative deficit in B-1 and MZ B cells (Fig. 6B). However, this reduction was moderate, and there was no significant difference in the IgG3 (Fig. 6A). The modest phenotype may reflect the relatively preserved absolute numbers of MZ and B-1 cells in G{alpha}i2-/- mice. As expected, the TD response (IgG1 anti-TNP after TNP-KLH immunization) was comparable in wild-type and G{alpha}i2-/- mice (Fig. 6C), reflecting the preservation of both relative and absolute numbers of FM B cells.



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  		FIGURE 6. Intact TD and TI-II response in G{alpha}i2-/- mice. Wild-type (WT) and G{alpha}i2-/- mice were immunized i.p. with TNP-Ficoll (TI-II) (A and B) or TNP-KLH (TD) (C). Preimmune (Pre-im.), wk 1 and wk 2 serum was collected, and anti-TNP-specific responses for IgG3 (A), IgM (B), and IgG1 (C) were analyzed by ELISA on TNP-BSA. Two separate experiments are shown ({circ}, •, {square}, {blacksquare}). Each point (•, {blacksquare}, G{alpha}i2-/-; {circ}, {square}, wild-type) represents data from one mouse.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study revealed a previously unappreciated role for G{alpha}i2 in B cell development. G{alpha}i2-/- mice were impaired in the formation of splenic T2, MZ, and peritoneal B cells, accompanied by an expansion of FM B cells. Similar deficiencies in B cell development were observed in RAG2-/- mice reconstituted with G{alpha}i2-/- bone marrow, indicating that the developmental abnormality was a cis effect within the hemopoietic compartment. G{alpha}i2-/- mice have normal numbers of T1 B cells, suggesting that the G{alpha}i2-dependent developmental steps occur after bone marrow B lymphopoiesis.

G{alpha}i2 in FM vs MZ B cell development

The phenotypic disorder of B cells in G{alpha}i2-/- mice is the reciprocal change in FM and MZ/T2 B cells (expanded and impaired, respectively). One possible mechanism for this B cell development phenotype is BCR strength. Deficiencies in various positive regulators of BCR (46, 47, 48, 49, 50, 51, 52, 53) result in the reduction of FM B cells, and mice lacking certain negative regulators (50, 54, 55, 56) form more FM B cells and/or fewer MZ B cells.

These precedents indicate that weak BCR signaling favors the formation of MZ B cells instead of FM B cells. In the context of G{alpha}i2-/- mice, we therefore considered the prediction that G{alpha}i2 activity reduces the amplitude of BCR signaling. In agreement with this prediction, pharmacologic inactivation of G{alpha}i2 activity with PT indeed augmented BCR signaling (intracellular calcium mobilization), and BCR signaling was elevated in FM B cells from G{alpha}i2-/- vs wild-type mice. Although PT is known to preserve BCR signaling (assayed by phosphoinositide breakdown) (57), this apparent role of G{alpha}i in attenuating BCR signaling is a novel observation that deserves analysis with regard to biochemical cross-talk with BCR signaling. It also implies that there is sufficient G-coupled receptor ligation of FM cells in situ to permit detection of this G{alpha}i2-dependent difference after cell isolation.

Curiously, G{alpha}i2 genotype had no effect on BCR signaling in MZ and T2 cells. T2 cells are the probable stage at which FM vs MZ choice occurs; terefore, the signal strength model would predict that these cells would display augmented BCR signaling in G{alpha}i2-/- mice. One explanation is that in contrast to FM cells, MZ and T2 cells encounter minimal activation via G{alpha}i2-coupled receptors in situ. This may be due to differences in levels of pertinent ligands in follicular vs nonfollicular splenic microenvironments or to B cell subset-specific differences G-coupled receptor expression (40).

Other factors influenced by G{alpha}i2 may also play a role in intrasplenic B cell development. Gi-coupled chemokine receptor function is required for microanatomic homing of B cells and a helper T cell subset (58, 59, 60, 61), and such a process with these or other chemokine receptors may impair the organization of an effective environment for T2 to MZ differentiation. Aberrant T cell function in G{alpha}i2-/- mice may also disturb the relative success of FM vs MZ cells. In G{alpha}i2-/- mice or PT-treated mice, T cells are distinguished by increased capacity for TCR-mediated activation during thymocyte development and peripheral function, relaxed costimulatory requirement, augmented mature and memory populations in the thymic and peripheral compartments, and increased IFN-{gamma} production (1, 9, 10, 12). T cell function, particularly through B-T interactions at the immunologic synapse, is an important factor in FM B cell expansion and retention (62, 63, 64). In contrast, although MZ and B-1 cells are also responsive to B-T interaction, T-independent mechanisms play a large role in the dynamics of FM B cell populations (27, 28, 38). Accordingly, augmented TCR signaling and expanded memory T cell populations in G{alpha}i2-/- mice might selectively expand the FM B cell population, leaving a relative reduction in the T2 and MZ B cells. Such activity of the expanded, activated T cell population might also explain B cell hyperplasia in the native G{alpha}i2-/- mice.

G{alpha}i2 in peritoneal B-1 B cell development

Our results suggest that G{alpha}i2 is also important for the development and/or recruitment of B-1a and B-1b cells from the peritoneal cavity. In G{alpha}i2-/- bone marrow chimeras, there is a reduction in the total number of B cells in the peritoneal cavity, with equivalent drops in both B-1a and B-1b cells. More importantly, in native G{alpha}i2-/- mice, peritoneal B-1a B cells are essentially absent, whereas B-1b B cells are expanded above levels observed in wild-type mice. This phenotype is consistent with genetic studies supporting a positive role for BCR signaling in development of B-1a B cells, as recently reviewed (65). Specifically, B-1a B cells are reduced in mice deficient in various positive regulators of BCR signaling (46, 47, 48, 49, 53, 66, 67, 68, 69), and B-1a (56) or B-1b (27, 70, 71) are augmented in mice deficient in certain negative regulators. Thus, tuning of BCR signaling by G{alpha}i2 may titrate development of B-1a vs B-1b B cells.

G{alpha}i2 might affect formation of the B-1 B cells by other mechanisms, such as defective recruitment of B-1a or B-1b B cells into the peritoneal cavity. This idea is appealing because most chemokine receptors are linked to G{alpha}i proteins, and null mutations of certain chemokines and chemokine receptor signaling components disturb formation or function of these populations (72). The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA Ab-secreting cells (73) (thought to be of B-1a origin), and CXCL13 may be critical for peritoneal B-1 B cell development (74). Alymphoplasia-type NF{kappa}B-inducing kinase causes defects in secondary lymphoid tissue chemokine receptor signaling and homing of peritoneal cells to the intestine-associated lymphatic tissue system (75). phospholipase C {gamma}2, phospholipase C{gamma}3, and PI3-kinase are necessary for mediating G{alpha}i chemoattractant signal transduction (76). In the present study, the discordant effect of G{alpha}i-/- on B-1a and B-1b cells makes it unlikely that a predominant effect of G{alpha}i occurs at the level of body cavity B-1 B cell trafficking.

LPS responsiveness and colitis susceptibility in G{alpha}i2-/- mice

Resting B cells from G{alpha}i2-/- mice are deficient in LPS-triggered proliferation and IL-10 production. Differential LPS responsiveness of B cell subpopulations was observed by Oliver et al. (45) and recently was shown to be the exclusive property of T2 and MZ cell subpopulations (38). Thus, the deficient LPS response in G{alpha}i2-/- mice is attributable to the lower percentage of T2 and MZ found in these mice, which presumably is also true in vivo. The intestine is enriched for LPS-producing bacteria, and LPS is a potent stimulator IL-10 production by B cells (23, 24, 25). From our results, we propose that the intestine normally bears B-1a and MZ B cells, which through tonic LPS stimulation from enteric microflora produces IL-10 for normal mucosal homeostasis. In contrast, in G{alpha}i2-/- mice, these IL-10-producing cells are diminished, and therefore insufficient mucosal IL-10 is produced to maintain normal immune homeostasis, resulting in colitis.

Collectively, our data implicate B cells as a novel factor in colitis susceptibility in these mice. The precedents for regulatory B cell subpopulations in autoimmune disease have been recently reviewed (77). In vitro studies have pointed to B cell subsets with distinct cytokine expression profiles modulating T cells (78). Selective chemokines recruit B cell subsets, such as the recent evidence for CXCL13 function in peritoneal B-1 B cell development (74). In this regard, it is important that mice bearing a null mutations for the {gamma} or {delta} subunits of PI3-kinase have a selective deficit in MZ development and B-1a function (44, 76, 79). PI3-kinase is a critical signaling transducer for Gi-dependent G-coupled protein receptors, indicating that these mice may represent a phenocopy of G{alpha}i2-/- mice. A protective role for B cells in colitis is known (21), and an important mechanistic study has demonstrated that IL-10 production by mucosal B cells is a critical element of this interaction (26). These B cells were distinguished by mucosal localization and surface phenotype (CD1dhigh-CD23high-CD21intermediate) distinctive from the MZ, T2, or B-1 B cell populations presented in this study. A protective role for IL-10-expressing B cells is also emerging in other immune-mediated inflammatory diseases (80, 81). The mechanism of this protective role certainly requires more investigation; because MZ B cells are particularly efficient at Ag presentation, the possible influence of these cells on the type of T cells that are activated in the wild-type and Gai2-/- environment may be instructive. Future studies will involve assessing the potential developmental relationship between the different IL-10 programmed B cell subpopulations, and the identification of the G protein-coupled receptor and ligands involved in this developmental program for immunoregulatory B cells.


   Acknowledgments
 
We thank Drs. Lutz Birnbaumer for G{alpha}i2-/- mice, William McBride for RAG2-/- mice, Stephan Krutzik for LPS reagents, and Dagoberto Grenet and Tiffany Huang for generation and maintenance of the G{alpha}i2-/- mice. We are grateful to Drs. K. Dorshkind, G. Cheng, L. Goodglick, and members of the Braun laboratory for advice and critical reading of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK46763 and DK43026, the University of California, Los Angeles, Clinical and Fundamental Immunology Training Grant (AI 07126-23), the Jonsson Comprehensive Cancer Center, and the Crohn’s and Colitis Foundation of America. Flow cytometry was performed in the University of California, Los Angeles, Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility, supported by National Institute of Health Awards CA-16042 and AI-28697. Back

2 H.D. and B.W. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jonathan Braun, Department of Pathology and Laboratory Medicine, CHS 13-222, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095-1732. E-mail address: jbraun{at}mednet.ucla.edu Back

4 Abbreviations used in this paper: G{alpha}i, G protein {alpha} inhibitory subunit; IBD, inflammatory bowel disease; RAG2-/- mice; recombination-activating gene 2-deficient mice; T1, transitional B cells of type 1; T2, transitional B cell of type 2; MZ, marginal zone; FM, follicular; PT, pertussis toxin; BCR, B cell receptor; KLH, keyhole limpet hemocyanin; TNP, trinitrophenyl; TD, thymus-dependent; TI-II, thymus-independent type II; PI3-kinase, phosphoinositide 3-kinase. Back

5 B. Wei, T. T. Su, H. Dalwadi, T. Huang, R. P. Stephan, D. J. Rawlings, and J. Braun. Endogenous gut microflora and Toll-like receptor recognition drive the generation of innate B cells. Submitted for publication. Back

Received for publication March 7, 2002. Accepted for publication December 10, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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