Goo-Young Seo

Mechanisms underlying TGF-β1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis

TGF‐β induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF‐β1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia‐inducible factor‐1α/β (HIF‐1α/β). To examine the transcriptional regulation of VEGF by TGF‐β1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF‐1α/β or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF‐β1, whereas cotransfection of HIF‐1α/β and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF‐1 and two Smad‐binding elements were critical for TGF‐β1‐induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF‐1α/β and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF‐β1 also increased the expression of fetal liver kinase‐1 (Flk‐1), a major VEGF receptor, and TGF‐β1 and VEGF stimulated pro‐matrix metalloproteinase 9 (MMP‐9) and active‐MMP‐9 expression, respectively. The results from the present study indicate that TGF‐β1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP‐9, and Flk‐1.

TGF-β1 and IFN-γ stimulate mouse macrophages to express BAFF via different signaling pathways

B cell‐activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells and stimulates the proliferation, differentiation, and survival of B cells and their Ig production. In the present study, we examined the pathways by which TGF‐β1 and IFN‐γ induce BAFF expression to see if TGF‐β1 and IFN‐γ regulate B cell differentiation via macrophages. We found that TGF‐β1 stimulated mouse macrophages to express BAFF and that a typical TGF‐β signaling pathway was involved. Thus, Smad3 and Smad4 promoted BAFF promoter activity, and Smad7 inhibited it, and the BAFF promoter was shown to contain three Smad‐binding elements. Importantly, TGF‐β1 enhanced the expression of membrane‐bound and soluble forms of BAFF. IFN‐γ further augmented TGF‐β1‐induced BAFF expression. IFN‐γ caused phosphorylation of CREB, and overexpression of CREB increased IFN‐γ‐induced BAFF promoter activity. Furthermore, H89, a protein kinase A (PKA) inhibitor, abrogated the promoter activity. Neither Stat1α (a well‐known transducing molecule of IFN‐γ) nor AG490 (a JAK inhibitor) affected BAFF expression in response to IFN‐γ. Taken together, these results demonstrate that TGF‐β1 and IFN‐γ up‐regulate BAFF expression through independent mechanisms, i.e., mainly Smad3/4 and PKA/CREB, respectively.

Retinoic acid, acting as a highly specific IgA isotype switch factor, cooperates with TGF‐β1 to enhance the overall IgA response

The present study demonstrates that RA has activity of an IgA switch factor and is more specific than TGF‐β1. RA independently caused only IgA switching, whereas TGF‐β1 caused IgA and IgG2b switching. We found that RA increased IgA production and that this was a result of its ability to increase the frequency of IgA‐secreting B cell clones. Increased IgA production was accompanied by an increase of GLTα. RA activity was abrogated by an antagonist of the RAR. Additionally, RA affected intestinal IgA production in mice. Surprisingly, RA, in combination with TGF‐β1, notably enhanced not only IgA production and GLTα expression but also CCR9 and α4β7 expression on B cells. These results suggest that RA selectively induces IgA isotype switching through RAR and that RA and TGF‐β have important effects on the overall gut IgA antibody response.

IL-21 ensures TGF-β1-induced IgA isotype expression in mouse Peyer’s patches

It is well established that TGF‐β1 induces IgA and IgG2b class‐switching recombination in murine B cells. In the present study, we assessed the activity of IL‐21 along with TGF‐β1 in Ig synthesis by murine spleen B cells. IL‐21 showed antiproliferative activity on LPS‐activated splenic B cells, comparable with that of TGF‐β1. IL‐21 alone had little effect on IgA secretion and decreased other isotypes. Likewise, IL‐21 also did not alter the TGF‐β1‐induced IgA synthesis and concurrently diminished the syntheses of IgM and IgG2a, which were repressed by TGF‐β1. Unexpectedly, IL‐21 inhibited the TGF‐β1‐induced IgG2b production. This IL‐21 effect was examined using B cells from IL‐21R knockout mice, where the IgA production profile was paralleled by that seen in wild‐type B cells. However, the inhibitory effect of IL‐21 on TGF‐β1‐induced IgG2b synthesis was not seen in the IL‐21R−/− mouse, suggesting that IL‐21 causes TGF‐β1‐stimulated B cells to decrease IgG2b synthesis. Expression patterns of Ig germ‐line α(GLα)/GLγ2b transcripts under the influence of TGF‐β1 and IL‐21 were paralleled by IgA/IgG2b secretion. This was also observed in the activities of GLα and GLγ2b promoters. These results indicate that IL‐21 decreases IgG2b secretion mainly through inhibition of GLγ2b transcription and is ultimately associated with selective IgA secretion induced by TGF‐β1. Our results showed that IL‐21 was expressed in greater magnitude in Peyers patches (PP) than in spleen. These results suggest that IL‐21 has an important effect on selective IgA+ B cell commitment in PP.

Macrophage-derived BAFF induces AID expression through the p38MAPK/CREB and JNK/AP-1 pathways

BAFF is expressed primarily by macrophages and DCs. BAFF stimulates the differentiation and survival of B cells and induces Ig production. We have demonstrated previously that murine macrophages treated with TGF‐β1 or IFN‐γ express membrane‐bound and soluble forms of BAFF. The ability of these two forms of BAFF to induce expression of AID, which plays a critical role in Ig CSR in B cells, was investigated. Both forms of BAFF, derived from macrophages activated by IFN‐γ or TGF‐β1, can increase AID expression. Subsequent analysis of BAFF signaling suggested that BAFF induces AID through BCMA, a BAFF‐receptor, and p38MAPK and CREB act as intermediates in AID expression. In addition, JNK and AP‐1 have similar activities. Our findings suggest that macrophage‐derived BAFF stimulates B cells to express AID through BCMA and at least two different pathways, including the p38MAPK/CREB and the JNK/AP‐1 pathways.

Retinoic acid acts as a selective human IgA switch factor

Retinoic acid (RA) is known to have several functions that lead to a potent mucosal IgA response. Nevertheless, its exact role in human IgA synthesis has yet to be elucidated. Thus, we investigated the role of RA in promoting IgA isotype switching in human B cells. We found that RA increased IgA production and the expression of germ-line IgA1 and IgA2 transcripts (GLTα1 and GLTα2). This induction occurred alongside an increase in the frequency of IgA1-secreting B cell clones, as assessed by limiting dilution analysis. Under the same conditions, RA did not increase IgM and IgG production. Am80, an agonist of RA receptor α (RARα), increased IgA production. In addition, RA activity was abrogated by LE540, an antagonist of RAR, suggesting that the RAR pathway is involved in RA-induced IgA production. Taken together, these results indicate that RA induces IgA isotype switching mainly through RARα in human B cells.

Alum Directly Modulates Murine B Lymphocytes to Produce IgG1 Isotype

Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and GLTγ1 expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production.

APRIL stimulates NF-κB-mediated HoxC4 induction for AID expression in mouse B cells.

Activation-induced cytidine deaminase (AID) plays a key role in B cell immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM). We have previously reported that the highly conserved homeodomain HoxC4 transcription factor binds to the Aicda (AID gene) promoter to induce AID expression. Here, we investigated the regulation of HoxC4 transcription by a proliferation-inducing ligand (APRIL) and B cell-activating factor belonging to the TNF family (BAFF) in mouse B cells. APRIL substantially increased both HoxC4 and AID expression, whereas BAFF induced the expression of AID but not HoxC4. To elucidate the underlying mechanisms, we constructed a HoxC4 gene promoter reporter vector and analyzed the promoter induction after APRIL stimulation. APRIL enhanced the HoxC4 promoter activity by 2.3-fold, and this increase disappeared when the second putative NF-κB-binding promoter element (NBE2) was mutated. Based on ChIP assays, we found that NF-κB bound to the HoxC4 promoter NBE2 region. Furthermore, the overexpression of NF-κB augmented the APRIL-induced HoxC4 promoter activity, while the expression of dominant negative-IκBα suppressed it. Taken together, our findings suggest that NF-κB mediates APRIL-induced HoxC4 transcription.

The transcription factor NR4A3 controls CD103+ dendritic cell migration

The transcription factor NR4A3 (also known as NOR-1) is a member of the Nr4a family of nuclear receptors and is expressed in myeloid and lymphoid cells. Here, we have shown that Nr4a3 is essential for the migration of CD103+ dendritic cells (DCs) to lymph nodes (LNs). Nr4a3-deficient mice had very few CD103+ migratory DCs (mDCs) present in LNs, and mixed-chimera studies revealed that this migratory defect was cell intrinsic. We further found that CD103+ DCs from Nr4a3-deficient mice displayed a marked loss of surface expression of the chemokine CCR7. This defect in CCR7 expression was confined to CD103+ DCs, as CCR7 expression on T lymphocytes was unaffected. Moreover, CCR7 was not induced on CD103+ DCs from Nr4a3-deficient mice in response to either administration of the TLR7 agonist R848 or infection with Citrobacter rodentium in vivo. The transcription factor FOXO1 has been shown to regulate CCR7 expression. We found that FOXO1 protein was reduced in Nr4a3-deficient DCs through an AKT-dependent mechanism. Further, we found a requirement for NR4A3 in the maintenance of homeostatic mitochondrial function in CD103+ DCs, although this is likely independent of the NR4A3/FOXO1/CCR7 axis in the regulation of DC migration. Thus, NR4A3 plays an important role in the regulation of CD103+ mDCs by regulating CCR7-dependent cell migration.

Lactoferrin Combined with Retinoic Acid Stimulates B1 Cells to Express IgA Isotype and Gut-homing Molecules.

It is well established that TGF-β1 and retinoic acid (RA) cause IgA isotype switching in mice. We recently found that lactoferrin (LF) also has an activity of IgA isotype switching in spleen B cells. The present study explored the effect of LF on the Ig production by mouse peritoneal B cells. LF, like TGF-β1, substantially increased IgA production in peritoneal B1 cells but little in peritoneal B2 cells. In contrast, LF increased IgG2b production in peritoneal B2 cells much more strongly than in peritoneal B1 cells. LF in combination with RA further enhanced the IgA production and, interestingly, this enhancement was restricted to IgA isotype and B1 cells. Similarly, the combination of the two molecules also led to expression of gut homing molecules α4β7 and CCR9 on peritoneal B1 cells, but not on peritoneal B2 cells. Thus, these results indicate that LF and RA can contribute to gut IgA response through stimulating IgA isotype switching and expression of gut-homing molecules in peritoneal B1 cells.