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The Journal of Immunology, 2006, 176: 2389-2396.
Copyright © 2006 by The American Association of Immunologists

B Cell-Specific Deficiency for Smad2 In Vivo Leads to Defects in TGF-beta-Directed IgA Switching and Changes in B Cell Fate1

Jörg Klein*, Wenjun Ju§, Jörg Heyer§, Britta Wittek2,{dagger}, Torsten Haneke{ddagger}, Petra Knaus3,{dagger}, Raju Kucherlapati, Erwin P. Böttinger§, Lars Nitschke4,5,* and Burkhard Kneitz4,5,{ddagger}

* Institute for Virology and Immunobiology, {dagger} Department of Physiological Chemistry II, Biocenter, and {ddagger} Department of Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg, Germany; § Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029; and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Smad2 is a member of the intracellular mediators that transduce signals from TGF-beta receptors and activin receptors. Targeted inactivation of Smad2 in mice leads to early lethality before gastrulation. It was shown previously that TGF-betaRII deficiency in vivo leads to defects in B cell homeostasis, Ag responsiveness, and IgA class switch recombination of B cells. To investigate the importance of Smad2-mediated signaling in B lymphocytes, we generated a B cell-specific inactivation of Smad2 in mice (bSmad2–/–). bSmad2–/– mice had normal B cell numbers in the spleen but showed a reduced population of marginal zone B cells. In contrast, B cells in Peyer’s patches and peritoneal B-1a cells of bSmad2–/– mice were increased in numbers. bSmad2–/– mice showed a reduced number of surface-IgA+ B cells and of IgA-secreting cells in Peyer’s patches, decreased levels of IgA in serum, and, after immunization with a T cell-dependent Ag, a reduced IgA response. Class switch recombination to IgA was impaired in Smad2-deficient B cells, when stimulated in vitro with LPS in the presence of TGF-beta. The growth-inhibitory effects of TGF-beta in LPS-stimulated B cells were not affected in Smad2-deficient B cells. In summary, our data indicate a crucial role of Smad2 in mediating signals for the TGF-beta-directed class switch to IgA and the induction of IgA responses in vivo. Other B cell functions like growth-inhibitory signaling, which are known to be regulated by signals via the TGF-betaR, are not affected in Smad2-deficient B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The TGF-beta superfamily consists of >40 ligands that function in diverse developmental processes in mammals. These ligands include three isoforms of the cytokine TGF-beta, several activins, and bone morphogenic proteins. TGF-beta is a multifunctional regulator of cellular differentiation, proliferation, and apoptosis (1). In the immune system, TGF-beta plays a central role as a cytokine that negatively regulates immune responses (2). This has been demonstrated by the generation of TGF-beta1 knockout mice, which die shortly after weaning as a result of a multifocal, inflammatory disease, with lymphocyte infiltrations in many organs and autoimmunity (3). TGF-beta family members initiate intracellular signaling by inducing the assembly of a heterotetrameric complex of two types of transmembrane receptors known as type I and type II receptors (TbetaR-I and TbetaR-II).6 Binding of TGF-beta activates the TbetaR-II, and the TbetaR-II, which is a Ser/Thr kinase, phosphorylates TbetaR-I. In turn, TbetaR-I transmits intracellular signals by phosphorylation of intracellular mediators, the Smad proteins (1, 2). In addition to TGF-beta, also other ligands such as activins, hepatocyte growth factor, and epidermal growth factor can bind to receptor serine-threonine kinases of the TGF-beta superfamily and transduce signals by activating Smad proteins (4).

Three types of Smad proteins exist, those which are activated by the receptors (R-Smads), a common Smad (Co-Smad; in mammals, Smad4), and inhibitory Smads. Inhibitory Smads are induced by TGF-beta and negatively regulate the signal by a feedback mechanism. Two activated R-Smads form a heterotrimeric complex with Smad4. This complex formation leads to translocation to the nucleus where the heterotrimer directly induces or represses TGF-beta target genes (2). The R-Smads, which are activated by TGF-beta signaling, are Smad2 and Smad3. Smad oligomers are able to bind to DNA with low affinity and specificity, and recruit transcriptional coactivators or corepressors. Smad3 and Smad4 have a DNA-binding activity through their N-terminal MH1 domain. Although Smad2 is highly homologous to Smad3, it does not have DNA binding activity due to a short region of 30 aa in the MH1 domain, encoded by exon 3 (2); however, a splice variant of Smad2 exists, which is capable of binding to DNA, similar to Smad3 (5). Whether direct binding of Smads to DNA is required for transcriptional activation of target genes is controversial. Although differentially controlled during development, both the R-Smads Smad2 and Smad3, as well as Smad4, are expressed in most cell types.

The role of defects in the TGF-beta pathway in tumorigenesis in humans or mouse models is well established (1, 6). Smad4 was found to be deleted in ~50% of pancreatic carcinomas, Smad3 was reported to be drastically reduced in pediatric T cell acute lymphoblastic leukemia (7), and Smad2 is frequently mutated in several types of cancer (8), but a general role of Smads as tumor suppressor genes is controversial.

Because TGF-beta is a central regulator of the immune system, it is important to dissect the biological role of various Smad proteins in lymphocytes in vivo. However, Smad-deficient mice often show an embryonic lethal phenotype, for instance, Smad2- and Smad4-deficient mice both show lethality before embryonic gastrulation (9, 10, 11, 12). Smad3-deficient mice die between 1 and 10 mo (13, 14), although one study reported normal survival (15). Smad3-deficient mice showed immune dysfunction, characterized by T cells with an activated phenotype, impaired neutrophil chemotaxis, and impaired mucosal immunity leading to chronic intestinal inflammation (13). This suggested a crucial role for Smad3 in several cell types of the immune system.

In B lymphocytes, TGF-beta1 has been implicated in inhibiting proliferation and Ab secretion, affecting B cell differentiation and survival, and in inducing the differentiation to IgA-secreting plasma cells (3, 16, 17). Accordingly, mice that lacked the TbetaR-II in B cells through conditional mutagenesis showed B cell hyperresponsiveness, an enlarged population of B1 cells, increased Ab production, and a selective defect in IgA Abs (18). This established a crucial role for TGF-beta regulation of B cell responses in vivo. The intracellular mechanisms how the regulation of these various B cell responses are mediated via TGF-beta and Smad signaling have not been elucidated so far. We approached this question by establishing a B cell-specific inactivation of Smad2 using a loxP-flanked Smad2 gene and CD19-Cre mice (19). Mice that lacked Smad2 in B cells showed a normal B cell development but had an increased number of B1 cells and B cells in the Peyer’s patches (PP). The switch to IgA was impaired, both in vivo, as well as in vitro. Interestingly, proliferation of Smad2-deficient B cells was still inhibited by TGF-beta1, similar to control B cells. Our mouse model allows for the first time a study of the in vivo role of SMAD2 in B lymphocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of bSmad –/– mice

Smad2flox/flox mice carrying a floxed exon2 were generated as described elsewhere (20). Smad2flox/flox mice on a mixed genetic background (C57BL/6J x 129SV) were intercrossed with heterozygous CD19Cre mice (CD19Cre/+) on a C57BL/6J background (19). The presence of the Smad2flox allele was detected with the primers W127 5'-TTCCATCATCCTTCATGCAAT-3' and W101 5'-CTTGTGGCAAATGCCCTTAT-3', resulting in a 451-bp product, whereas the wild-type allele results in a 271-bp product. The detection of the CD19 Cre locus was performed as described (19) by PCR.

Southern blot

Genomic DNA was isolated from thymocytes, total splenocytes, or B cells. B cells were purified by MACS isolation using anti-B220 magnetic beads (Miltenyi Biotec). DNA was digested with relevant restriction enzymes, separated by agarose gel electrophoresis, and transformed to nylon membranes (Amersham Biosciences) by capillary blot under denaturing conditions. For hybridization, a DIG-labeled PCR product spanning the indicated region was used as a probe and visualized using the ECL detection system.

Western blot

Thymocytes and resting or activated B lymphocytes were homogenized in lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1% Triton X-100, and a protease inhibitor mixture. The protein lysate was isolated by electrophoresis in a 7.5% polyacrylamide gel and blotted onto nitrocellulose membranes. Smad2 or C-terminal phosphorylated Smad2 was detected by a anti-Smad2-specific mAb (Cell Signaling Technology) or an anti-phospho- Smad2 mAb (21) and visualized using the ECL detection system. Primary Abs were removed by incubation the membrane in stripping buffer (5 mM phosphate buffer, 2% SDS, and 0.014% 2-ME) for 30 min at 60°C.

Flow cytometry analysis

Cells from spleen, PP, and peritoneal cavity (PC) were obtained from bSmad2+/+ (floxed control) and bSmad2–/– (B cell-specific knockout) mice in the age of 8–12 wk. Abs used for staining of splenic cells were anti-IgM-PE, anti-B220-biotin, anti-CD21-FITC, and anti-CD23-PE, and for staining of peritoneal B cell populations were IgM-PE and CD5-biotin, and for PP stainings were B220-bio and anti-IgA-FITC (all BD Pharmingen). Biotin-labeled Abs were revealed by streptavidin-CyChrome (BD Pharmingen). Cell surface marker expression was analyzed using a four-color flow cytometer (FACSCalibur) and CellQuest software (BD Biosciences).

Determination of serum Abs and immunization

Determination of Ig serum titers and immunizations of 10- to 12-wk-old bSmad2+/+ and bSmad2–/– mice with trinitrophenyl (TNP)-OVA were performed as described (22).

ELISPOT assay

Spleen and PP cells from bSmad2+/+ and bSmad2–/– mice of the same age were prepared. The ELISPOT assay was performed in triplicate. Plates were coated with goat-anti-mouse IgG or with goat anti-mouse IgA (both from Southern Biotechnology), and cells were added at four different concentrations. After overnight incubation, the cells were washed away, and the spots were revealed with goat anti-mouse IgG or IgA Abs that were alkaline phosphatase labeled (from Southern Biotechnology).

In vitro assay for class switch recombination

Splenic B cells were purified from bSmad–/– and bSmad+/+ mice and stimulated in the presence of 10 µg/ml LPS (Sigma-Aldrich) with or without 5 ng/ml TGFbeta (R&D Systems) for 24 h. Total RNA was extracted using TRIzol (Invitrogen Life Technologies) according to the manufacturer’s instructions. cDNA was synthesized with Superscript II (Invitrogen Life Technologies) using 2.5 mg of total RNA for reverse transcription (RT-PCR) in a 50-µl volume reaction. PCR conditions and primers to amplify the {alpha}CT transcripts were used as described (23).

Lymphocyte purification and proliferation assay

Single-cell suspension was prepared from spleens or PP of 8- to 12-wk-old mice. B cells were purifed by MACS isolation using anti-B220 magnetic beads (Miltenyi Biotec). For proliferation assays, B cells (1 x 106/ml) were cultured in triplicate in RPMI 1640 medium with 5% FCS in 96-well flat-bottom plates with 10 µg/ml LPS (Sigma-Aldrich) with or without 5 ng/ml TGFbeta (R&D Systems). Cultures were cultivated for 72 h, with 0.25 µCi of [3H]thymidine (Amersham Biosciences) added per well for the final 12 h of culture. The incorporated radioactivity was measured with a beta plate harvester.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To generate conditional B cell-specific Smad2 inactivation in mice, a previously generated mouse line with a loxP-flanked ("flox") exon 2 of the Smad2 gene (20) was crossed with CD19cre mice. CD19-cre mice induce efficient B lineage-specific deletion of loxP-flanked target sequences (19). The resulting mice with the genotype Smad2flox/flox/CD19cre/+ are called bSmad2–/–, whereas control mice which are Smad2flox/flox/ CD19+/+ are called bSmad+/+ in the following. To determine the efficiency of Smad2 gene deletion in B cells of bSmad2–/– mice, a Southern blot analysis was performed on purified B cells, thymocytes, and total spleen cells (Fig. 1A). Efficient Cre-mediated deletion was demonstrated by the nearly homogeneous representation of the Smad2{Delta}ex2 allele in B cells of bSmad2–/– mice. No deletion was detected in thymocytes of bSmad2–/– mice. To determine also the loss of the Smad2 protein in B cells, Western blot experiments were performed with purified B cells from both bSmad2+/+ and bSmad2–/– mice and thymocytes of both types of mice. The loss of Smad2 protein was specific in B cells (Fig. 1B). Purified splenic T cells from bSmad2–/– mice showed no deletion of Smad2 protein (data not shown). Also, in purified splenic B cells, which were stimulated with LPS plus TGF-beta1 for 2 h, a strongly reduced level of SMAD2 protein, as detected with anti-Smad2 Ab, was found (Fig. 1C). Additionally, a phospho-Smad2-specific Ab did not detect any phosphorylated form of full-length Smad2 in B cells upon stimulation with LPS plus TGF-beta1, confirming the loss of Smad2-mediated TGF-betaR activity. These results revealed that the Smad2 gene is efficiently inactivated in B cells of bSmad2–/– mice, caused by a loss of the full-length Smad2 protein in these cells.


Figure 1
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  		FIGURE 1. Efficient B cell-specific deletion of Smad2 in bSmad2–/– mice. A, A Southern blot of thymocytes (Thy), purified splenic B cells, or total splenocytes (Spl) is shown. Genomic DNA is EcoRI digested and probed with a probe which binds 3' to the floxed exon 2 of the Smad2 gene. The probe detects a 2.6-kb fragment of the floxed gene (Smad2 flox #) and a 4.8-kb # of the deleted gene (Smad2{Delta} #). The almost exclusive 4.8-kb band in purified B cells shows the specific Cre-mediated deletion in B cells. B, A Western blot of purified B cells or thymocytes from bSmad2+/+ or bSmad2–/– mice, stained with anti-Smad2 or Erk-2 as a loading control. C, Purified B cells from bSmad2+/+ or bSmad2–/– mice are stimulated for 2 h with 10 µg/ml LPS plus 5 ng/ml TGF-beta1 and stained with anti-Smad2 or anti-phospho-Smad2 Abs. Both B and C show the almost complete lack of the Smad2 protein.

 
To study the role of Smad2 in the development and differentiation of B cells, cell suspensions from bone marrow, spleen, and PP, and from the PC were analyzed by flow cytometry. In the bone marrow, there were no differences in the total number of bone marrow cells between bSmad2–/– and bSmad2+/+ mice. Also, the relative frequency of pro-B cells, pre-B cells, immature B cells, and mature B cells was unchanged (data not shown). Splenocyte numbers as well as the number of total T cells and B cells in the spleen of bSmad2–/– mice were normal (Fig. 2A). However, the total cell number of PP cells was ~3-fold increased (Fig. 2A). For the spleen, stainings were performed to distinguish the immature, transitional, and mature B cell populations. With anti-IgM vs anti-CD21 staining, we found a significant reduction of transitional T2 B cells in bSmad2–/– mice, whereas the populations of transitional T1 and mature (M) B cells were unaffected (Fig. 2B). The reduction of T2 B cells was also confirmed with an anti-IgM/anti-IgD staining (data not shown). The population of IgMhighCD21high cells is composed of both T2 and marginal zone (MZ) B cells; therefore, the population of MZ was analyzed by a separate staining. Fig. 2C shows B220-gated cells stained with anti-CD23 and anti-CD21. A significant reduction of MZ B cells was found in bSmad2–/– mice, when compared with control bSmad2+/+ mice. This reduction of MZ B cells was confirmed with an anti-CD1d/B220 staining (not shown). Because the total B cell numbers are the same between bSmad2–/– and bSmad2+/+ mice (Fig. 2A), this relative reduction of MZ B cells is also reflected in absolute numbers.


Figure 2
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  		FIGURE 2. bSmad2–/– mice have increased numbers of PP cells, a reduced MZ B cell population, and an increased number of B1a cells. A, Total cell numbers are shown. There is no significant difference for splenocytes (spl.), splenic B cells (spl. B), or splenic T cells (spl. T) or PC cells (PC). PP cell numbers are increased in bSmad2–/– mice, shown also on the right for individual mice. B, Splenocytes are analyzed by flow cytometry to separate T1, T2, MZ, and M B cells. Cellular percentages are given (±SD). C, B220+-gated splenocytes are shown in a staining that separates MZ from follicular (FO) B cells. D, Peritoneal lavage cells are shown. CD5lowIgM+ cells are B1a cells. CD5–IgM+ cells are conventional B2 cells plus the minor population of B1b cells. E, B220+-gated PP cells are shown. Percentage ± SD is given for a group of three to five animals. Significant changes are shown as follows: *, p < 0.05; and ***, p < 0.001 in Student’s t test.

 
In the PC, the unconventional B1 cells were analyzed. Similar to TbetaR-II-deficient mice, the population of B1a cells of the PC was significantly increased in bSmad2–/– mice (Fig. 2D) (18). We also noted a relative decrease of T cells (CD5high, IgM–) in the PC of bSmad2–/– mice. In absolute numbers, the population of B1a cells is ~2-fold increased in bSmad2–/–, compared with bSmad2+/+ mice. To address the question of which population of cells is expanded in the PP, cells were analyzed by flow cytometry. Fig. 2E shows the IgM and IgD expression of B220+-gated PP cells. Notably, there is a population of IgM–IgD– cells that is relatively increased. These cells are higher in forward scatter than normal lymphocytes (not shown) and are probably activated B cells that have down-regulated surface IgM and IgD.

Because TGFbeta is a cytokine implicated in regulating the class switch to IgA (17), the immune status and immune responses were studied in Smad2-deficient mice. bSmad2–/– mice had significantly lowered levels of IgG1, IgG3, and IgA in the serum (Fig. 3A; reduction, ~2-fold each). To determine the level of IgA secreted in the gut, IgA levels were measured in the feces. A similar 2-fold reduction as in the serum was detected in bSmad2–/– mice (not shown). Next, bSmad2–/– and bSmad2+/+ mice were immunized with TNP-OVA to measure the thymus-dependent Ig response. There was a similar primary and secondary TNP-specific IgG1 response (the mainly produced isotype; Fig. 3B) and a similar TNP-specific IgM, IgG3, IgG2b, or IgE response (not shown) in bSmad2–/– and control mice. However, the TNP-specific IgA response of bSmad2–/– mice was strongly reduced, both after the primary and secondary immunization (Fig. 3B; reduction, 5- to 10-fold). To analyze whether the impairment of IgA production was also reflected on the cellular level, we determined the number of Ab-secreting cells in naive, unimmunized mice by ELISPOT assay. The number of IgM-secreting cells was increased in spleen and normal in PP of bSmad2–/– and bSmad2+/+ mice. Although IgA-secreting cells were present in equal numbers in the spleen, bSmad2–/– mice had ~5-fold decreased number of IgA Ab-secreting cells in the PP, compared with control mice (Fig. 3C). Also, we found a significant reduction of switched surface IgA+ B cells in PP of bSmad2–/– mice (Fig. 3D).


Figure 3
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  		FIGURE 3. bSmad2–/– mice show impaired IgA production in vivo. A, Total serum Ig levels. Reductions are seen for IgG1, IgG3, and IgA in bSmad2–/– mice. B, Mice were immunized with TNP-OVA at day 0 and day 14 (as indicated by arrows). TNP-specific IgG1 and IgA were determined in ELISA. TNP-specific IgA production in bSmad2–/– mice was strongly impaired. C, Total Ab-forming cells of unimmunized mice were determined in spleen and PP. The number of IgA-secreting cells in PP was lower in bSmad2–/– mice. D, PP cells expressing surface IgA. FACS panels on the left show percentages of all PP cells in lymphocyte gate. Bar diagram on the right shows percentage of surface IgA+ cells of all B220+ cells. bSmad2–/– mice show a lower percentage of surface IgA+ B cells in PP. A similar analysis of splenic B cells showed no significant difference: bSmad2+/+, 1.1% (±0.2%) IgA+ cells; bSmad2–/–, 1.5% (±0.6%) IgA+ cells. Significant differences are shown as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001 in Student’s t test.

 
To determine whether class switching to IgA is affected in bSmad2–/– mice, we performed an in vitro experiment in which we stimulated splenic B cells with LPS with or without TGFbeta1 for 2 days. After this period, RNA was extracted and transcripts from excised circular DNA, resulting from class switching to the IgA locus were detected by RT-PCR. The specific transcript ({alpha}CT) (23) was detectable only after LPS/TGFbeta1 stimulation of bSmad2+/+ B cells, but not at all in bSmad2–/– B cells (Fig. 4). This shows, at least under these in vitro conditions, that class switch recombination to IgA of bSmad2–/– B cells to IgA is severely impaired in response to TGFbeta1.


Figure 4
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  		FIGURE 4. bSmad2–/– B cells show impaired class switch to IgA in vitro. Purified splenic B cells of bSmad2+/+ mice or bSmad2–/– mice were stimulated with LPS with or without TGF-beta1. RT-PCR was performed to detect transcripts derived from circular excised DNA during class switch recombination to IgA ({alpha}CT). Two {alpha}CT-specific bands of 458 and 318 bp were detected only in bSmad2+/+, but not in bSmad2–/–, B cells after LPS/TGF-beta1 stimulation.

 
TGFbeta is known to suppress B cell proliferation (2); therefore, we studied the effect of TGFbeta on LPS-induced B cell proliferation by thymidine incorporation. Purified B cells derived from spleens or PP were stimulated with LPS. The proliferative responses of bSmad2–/– splenic B cells to LPS was similar, whereas the LPS response of bSmad2–/– PP cells was higher, compared with control mice. Importantly, in cells from both types of mice and from both organs, TGFbeta inhibited proliferation equally well (to ~50%) (Fig. 5A). To analyze the mechanism of these differences in proliferation, we determined the fraction of cells driven into blast formation and into cell death by a FACS assay. Fig. 5B shows that the higher proliferation of PP cells from bSmad2–/– mice is caused by a higher level of blast formation (gate R3). This may be due to the preactivated phenotype of these cells (Fig. 2E). The effect of TGF-beta in both types of mice is a block in LPS blast formation (R3), as well as a higher rate of cell death/apoptosis induction (R1) (Fig. 5B). Similar results were found for splenic B cells (not shown). We conclude from this in vitro experiment that the TGFbeta-mediated inhibition of proliferation is not affected in Smad2-deficient B cells. To analyze the function of Smad2 in the development of B cell lymphomas, we monitored survival and tumorigenesis of bSmad2–/– (n = 25) and bSmad2+/+ (n = 28) mice for a period of 18 mo. We could not detect a significant difference in death rate or development of lymphomas between both mouse populations (data not shown), indicating no function of Smad2 as a tumor suppressor gene in lymphomagenesis of B cells.


Figure 5
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  		FIGURE 5. bSmad2–/– B cells show no impairment in TGF-beta1-mediated inhibition of proliferation. A, Purified B cells from spleens or PP of bSmad2+/+ mice or bSmad2–/– mice were stimulated with LPS with or without TGF-beta1 for 48 h, and the proliferation was measured by [3H]thymidine incorporation. TGF-beta1 did inhibit the proliferation in cells from both types of mice. Significant differences were found (**, p < 0.01; and ***, p < 0.001 in Student’s t test) for LPS responses of bSmad2+/+ mice and bSmad2–/– mice. TGF-beta1 plus LPS treatment caused significantly lower proliferation (***, p < 0.001) than LPS treatment in both organs, in both types of mice. B, FACS analysis of PP cells stimulated with LPS with or without TGF-beta1 for 48 h. Forward vs side scatter shows percentage of apoptotic/dead cells (R1), cells of the normal size of lymphocytes (R2), and blast cells (R3). Before stimulation, there were <3% of cells in R1 and <4% in R3. Significant changes (*, p < 0.05) in R3 were detected in LPS responses between bSmad2+/+ mice and bSmad2–/– mice. The number of cells in R1 and R3 are significantly different between LPS and LPS plus TGF-beta1 treatment (**, p < 0.01; and ***, p < 0.001, for both types of mice).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have generated a B cell-specific Smad2-deficient mouse line and have demonstrated the efficient and B cell-specific deletion of full-length Smad2 in bSmad2–/– mice both at the DNA as well as at the protein level. Loss of full-length SMAD2 protein was also obtained in hepatocytes of the Smad2flox/flox mice, when crossed with Albumin-Cre transgenic mice (20). In hepatocytes of Albumin-Cre/Smad2flox/flox mice, a weakly expressed, truncated Smad2 protein with an N-terminal deletion of the first 240 aa was observed. This truncated Smad2 protein is identical with the Smad2{Delta}1–240 protein identified previously in Smad2 knockout mouse embryonic fibroblasts (24). The truncated Smad2 protein could be weakly phosphorylated by TGF-beta1 in hepatocytes of Albumin-Cre/Smad2flox/flox mice, but importantly, it did not show any transcriptional coactivator activity (20). Although we did not detect the phosphorylated truncated Smad2 protein in B cells of bSmad2–/– mice using a mAb binding to the C-terminal region of Smad2 (data not shown), we cannot exclude the possibility that it is also expressed at a very low level. But because it would likely be inactive, we conclude that the Cre-mediated deletion of exon2 in our Smad2flox/flox mice creates a functional inactivation of Smad2 in B cells of bSmad2–/– mice.

Our analysis of bSmad2–/– mice reveals several distinct roles of this R-Smad in B cells. Smad2 was shown to be important for the induction of IgA responses. Also, it seems to regulate maturation or maintenance of peripheral B cells, because there was an increased population of B1a cells in the PC, an increased number of PP B cells, and a changed composition of marginal vs follicular B cells in the spleen. Of note, there are some phenotypes observed in TGF-beta1-deficient or TbetaRII B cell-deficient mice that were not detected in bSmad2–/– mice. bSmad2–/– mice did not show a general B cell hyperresponsiveness, and, related to this, TGF-beta1 could still inhibit the proliferative response of B cells.

TGF-beta1 seems to be a crucial cytokine for induction of IgA responses. It has been shown that TGF-beta1 synergizes with other factors such as IL-5 or IL-2 for IgA production from murine LPS-stimulated B cells in vitro (17). In vivo studies showed a clear dependence of IgA responses on a functional TGF-beta1/TbetaR pair. TbetaRII-B cell-deficient mice had almost absent IgA levels in the serum, and IgA was not inducible by T cell-dependent Ags (18). IgA production was also partially blocked in TGF-beta1-deficient mice (3). Because TGF-beta1 mainly induces the R-Smads Smad2 and Smad3, a reduction of IgA would also be expected in Smad2- or Smad3-deficient mice. Interestingly, Smad3-deficient mice did not show an impaired in vivo IgA response as determined by the normal number of IgA+ plasma cells in intestine, spleen, and lymph nodes (13). However, Smad3-deficient mice die in the age of 1–8 mo due to chronic intestinal inflammation and highly activated T cells; therefore, a B cell-specific function of Smad3 in regulation of IgA response is hard to study in these mice. In contrast, bSmad2–/– mice show a clear impairment in IgA production. The total serum level of IgA is only mildly reduced. However, Ag-specific IgA production is strongly impaired. This is isotype specific, because a normal Ag-specific response is seen for all other Ig isotypes. Smad2 seems to have a clear role as R-Smad induced by TGF-beta1 in regulation of IgA production. The phenotype of bSmad2–/– mice is milder than the one of TbetaRII B cell-deficient mice (18). This is unlikely to be caused by a less efficient Smad2 gene deletion than TbetaRII gene deletion in the respective conditional knockout mice, because the same Cre expression locus (CD19-cre) was used. More likely, the loss of Smad2 function in regulation of IgA responses may be partially compensated for by other R-Smads, such as Smad3, or a Smad-independent TGF-beta signal pathway may exist in B cells (25, 26).

At what stage of B cell activation does Smad2 control IgA production? We have shown that in vitro-stimulated bSmad2–/– B cells have a defect in class switch to IgA in response to TGF-beta1. The impairment of class switching was shown by the lack of specific C{alpha} transcripts resulting from excised circular DNA. This suggests a TGF-beta1-induced Smad2 activation as a prerequisite for the process of switching to IgA. Ex vivo, we found normal numbers of IgA-secreting plasma cells in the spleen, but a 5-fold reduction in PP of bSmad2–/– mice. Similarly, surface IgA+ B cells were clearly reduced in the PP of bSmad2–/– mice, but a similar reduction was not detectable in the small population of IgA+ B cells of the spleen. This may reflect organ-specific challenge of B cells with pathogens and organ-specific involvement of TGF-beta1. In accordance with this, stroma cells of the gut-associated lymphoid tissue (GALT), to which PP belongs, are known to secrete TGF-beta (27). Although serum IgA is derived from B cells activated in germinal centers and requires T cell help, IgA can also be produced from plasma cells in the lamina propria (LP) of the intestine. There is evidence that IgA-secreting plasma cells in the intestine are derived from B1 cells of the PC (28). It has also been shown that IgA+ B cells from PP home to the LP of the intestine (29). How the intestinal IgA secretion from plasma cells in the LP is affected by the Smad2 deficiency still needs to be specifically addressed. When IgA levels in the feces, expected to result from plasma cells secreting IgA in the gut, were analyzed, we found a similar reduction as in the serum of bSmad2–/– mice.

PP B cells are found in higher numbers in bSmad2–/– mice than in control mice. PPs of the GALT are specialized compartments in which Ags are delivered via epithelial M cells from the intestinal lumen to lymphocytes and macrophages. Apparently, the local TGF-beta production by stroma cells of the GALT (27) suppresses expansion and activation of PP B cells. Most likely due to defective TbetaR signaling in bSmad2–/– mice, the pool of PP B cells is expanded and has an activated phenotype (higher side scatter). It is interesting that even this higher population of PP B cells is still not able to produce the same amount of IgA as in control mice. Exactly the same phenotype was found in TbetaRII-B cell-deficient mice (18), showing that also for this regulation Smad2 is the crucial downstream R-Smad.

Although TbetaRII-B cell-deficient mice showed a B cell hyperresponsiveness resulting in elevated Ig serum levels of all classes apart from IgA (18), this was not seen in bSmad2–/– mice. The total Ig serum levels were not elevated; neither was the Ag-specific Ig response to a T cell-dependent Ag. In contrast, total IgG1 and IgG3 levels were even 2-fold lower in bSmad2–/– than in bSmad2+/+ mice. The mechanism of this reduction is unknown, but it may result from a defect in postswitch B cell differentiation or plasma cell survival. The normal or decreased Ig levels in bSmad2–/– mice suggest that the TbetaRII-mediated inhibition of Ig secretion of other isotypes than IgA is Smad-2 independent. This finding is significant because the bSmad2–/– mice share many common phenotypes with TbetaRII-B cell-deficient mice, suggesting that Smad2 is a crucial downstream transcription factor of this type II receptor, but here a phenotypic discrepancy is seen.

Although B cell development and maturation was not grossly impaired, specific changes were noted in bSmad2–/– mice. Apart from PP B cells, these changes affected the B1a cell population, a minor B cell population in adult mice, established early in ontogeny and found in adult mice mainly in the peritoneal and pleural cavities (30). The increase of the pool of B1a cells may indicate a normally negative regulation by TGF-beta signaling controlling the maintenance, self-renewal, or survival of this pool. B1 cells are highly sensitive to changes in BCR signaling, with stronger signaling leading often to their expansion. Therefore, the increased number of B1a cells could be caused by defective TGF-beta induced signals in bSmad2–/– mice. The same phenotype, a selective expansion of B1 cells, but not of the conventional B2 cells, was also noted in TbetaRII-B cell-deficient mice (18). This emphasizes that Smad2 is the crucial downstream transcription factor in TGF-beta-dependent regulation of the B1 cell pool.

In the spleen, a selective reduction of MZ B cell numbers was observed in bSmad2–/– mice. MZ B cells are a specialized B cell subset in the spleen, where they participate in T cell-independent Ab responses, especially to bloodborne microbial particulate Ags (31). The pool of MZ B cells is sensitive to changes of chemokine-receptor signaling, expression of adhesion molecules, but also to the strength of BCR-derived signals (31). Stronger B cell signaling seems to change the B cell fate in the spleen to a higher proportion of mature, and a reduced population of MZ B cells (32, 33). The reduced population of MZ B cells, unchanged numbers of mature B cells in bSmad2–/– mice, and in parallel an increased B1 cell population, could indicate enhanced BCR signaling due to a lack of TGF-beta-mediated inhibition. In support of this model, recent data have shown that TGF-beta can directly affect lymphocyte signaling. TGF-beta can inhibit phosphorylation of the Tec kinase Itk and Ca2+ mobilization in activated CD4+ T cells (34). In B cells, inhibitors of BCR signaling such as CD72 and SHIP-1 are induced by TbetaR signaling (35). Additional experiments have to be done to study the role of Smad2 in this process.

TGF-beta is an important inhibitor of cellular proliferation, and this has also been demonstrated in lymphocytes. In epithelial cells, TGF-beta causes a G1 cell cycle arrest by inhibiting cyclin-dependent kinase via induction of the inhibitors p15INK4b and p21Cip1 (2). The p21Cip1 gene is activated by Smad3 and Smad4 in cooperation with the transcription factor Sp1. Sp1 interacts with Smad2 and Smad3, as has been shown in keratinocytes (36). In epithelial cells, TGF-beta also mediates by Smad3 the repression of the growth promoting gene c-myc (37). In T lymphocytes, it has been shown that TGF-beta induces growth arrest by regulation of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 (38). Both in murine B cell lines, as well as in LPS-stimulated B cells, up-regulation of p27Kip1 plays a functional role in TGF-beta-induced growth arrest (39, 40).

Loss of these cell cycle regulatory functions by mutations in components of the TGF-beta signaling pathway are discussed to be responsible for tumorigenesis of various cancer types. Our results revealed that loss of Smad2 does not lead to increased malignant transformation of B cells in our mouse model, indicating that Smad2 plays no major role as a tumor suppressor gene in B cells lymphomagenesis. It is known that loss of the TGF-beta pathway in cancer cells is most commonly found at later stages in carcinogenic progression (6) and that the expression of Smad3 is frequently dysregulated in pediatric T cell acute lymphoblastic leukemias (7). Therefore, it is possible that epigenetic defects or loss of Smad2 at later stages of cancer progression might be involved in B cell lymphomagenesis. Nevertheless, the TGF-beta1-mediated inhibition of mitotic responses is intact in bSmad2–/– mice, and no change to control B cells could be observed. In Smad3-deficient mice, TGF-beta1 also inhibits B cell proliferation normally, whereas inhibition of T cell proliferation is affected (13, 15). These results are surprising and could mean that inhibition of proliferation in B cells is Smad independent. Interestingly, TGF-beta1 is also known to induce Smad-independent pathways leading to growth arrest and apoptosis in other cell types (25, 26). However, it is not clear whether a heterotrimeric complex, involving both R-Smads Smad2 and Smad3, together with Smad4, is needed to function as transcriptional regulator in the nucleus, or whether the loss of one R-Smad can be compensated for by the other R-Smad. A way to address this question would be to generate mice with Smad2/Smad3 double-deficient B cells. In any case, Smad2 seems to be dispensable for inhibition of B cell proliferation.

In summary, the phenotype of the B cell-specific Smad2 knockout mouse shows that this R-Smad is a crucial downstream signaling protein in TGF-beta receptor signaling in B cells. In general, the phenotype of in bSmad2–/– mice was comparable to that of TbetaRII-deficient mice, but milder. This suggests an important role for other Smads, which may compensate for the loss of Smad2, or for other signaling pathways induced by TbetaRII in B cells. In particular, Smad2 plays an important role in Ag-specific IgA generation, and this seems to involve TGF-beta-dependent regulation of IgA switching. bSmad2–/– mice did not show a general B cell hyperresponsiveness. TGF-beta-mediated inhibition of B cell proliferation was still functional in bSmad2–/– B cells. However, the populations of B1 cells and PP B cells were increased. This work shows that Smad2 plays a specific, nonredundant role in mouse B cell immunity.


   Acknowledgments
 
We thank Carolin Dix and Martina Döhler for expert technical help.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft (Ni 549/4-1 and GK639) and Wilhelm-Sanderstiftung. Back

2 Current address: Heinrich-Pette-Institute, University of Hamburg, D-20206 Hamburg, Germany. Back

3 Current address: Institute for Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany. Back

4 L.N. and B.K. contributed equally to this work. Back

5 Address correspondence and reprint requests to Dr. Lars Nitschke, Department of Genetics, University of Erlangen, Staudtstrasse 5, 91058 Erlangen, Germany, E-mail address: nitschke{at}biologie.uni-erlangen.de or Dr. Burkhard Kneitz, Department of Urology, University of Würzburg, Oberdürrbacher Strasse 6, 97080 Würzburg, Germany, E-mail address: kneitz_b{at}klinik.uni-wuerzburg.de Back

6 Abbreviations used in this paper: TbetaR-I, type I transmembrane receptor; PP, Peyer’s patch; PC, peritoneal cavity; MZ, marginal zone; M, mature; T1, transitional type 1; T2, transitional type 2; TNP, trinitrophenyl; GALT, gut-associated lymphoid tissue; LP, lamina propria. Back

Received for publication June 22, 2005. Accepted for publication November 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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