Delta-like 1 is essential for the maintenance of marginal zone B cells in normal mice but not in autoimmune mice

Notch2 and Delta-like 1 (Dll1) have been implicated in the development of marginal zone B (MZB) cells. In the present study, we characterized the expression and function of mouse Notch receptors and ligands in the spleen by using newly generated mAbs. Although Notch2 was expressed on both B and T cells in the spleen, the highest expression was observed on precursors of marginal zone B and MZB cells. Dll1 was expressed on macrophage and erythroblasts in the red pulp, but not on B cells or marginal zone macrophage. Administration of a blocking mAb against Dll1 not only blocked the development of MZB cells in juvenile mice but also gradually depleted the pre-established MZB cells in adult mice, indicating a critical role for Dll1 in the maintenance of MZB cells in the spleen of normal mice. Interestingly, Dll1 was not necessary for the maintenance of MZB cells in lupus-prone (NZB x NZW) F1 mice particularly after the onset of the disease, suggesting that the Dll1 independence may be a feature of dysregulated MZB cells producing auto-antibodies.     

Notch-RBP-J signaling is involved in cell fate determination of marginal zone B cells

RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria.     

Expression of Delta-like 1 in the splenic non-hematopoietic cells is essential for marginal zone B cell development

The Notch ligand Delta-like 1 (Dll1) is critical for the generation of marginal zone (MZ) B cells in the spleen. However, the precise mechanism underlying the differentiation of MZB cells is unclear. To determine whether hematopoietic cells or non-hematopoietic cells provides the Dll1-mediated signals to primitive hematopoietic cells, we transplanted lineage(-)c-kit(+)Sca-1(+) (KSL) bone marrow cells derived from wild-type (Dll1(+/+)) GFP-transgenic mice into lethally irradiated Dll1 conditional knockout (cKO) mice. After transplantation, we examined the kinetics of hematopoietic reconstitution and found that although the frequency of stem/progenitor subsets and of more mature lymphoid, myeloid, and erythroid lineages were normal, the donor-derived hematopoietic cells failed to differentiate into MZB cells. We further demonstrated that while the splenic stromal cells of wild-type mice expressed Dll1 molecule, the splenic stromal cells of recipient Dll1 cKO mice deleted the expression of Dll1. These results suggesting that the expression of Dll1 in splenic non-hematopoietic stromal cells, but not hematopoietic cells, is essential for the development of MZB cells.     

Effects of Delta1 and Jagged1 on early human hematopoiesis: correlation with expression of notch signaling-related genes in CD34+ cells

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34(+) CD38(+) cells. Jagged1 increases the number of bipotent colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34(+) cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1-CD34(+) cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34(+) CD38(+) cells as they develop in Jagged1- or Delta1-stromal cell environments, which appear to reflect sequential maturational stages of CD34(+) cells into distinct cell lineages.     

Marginal zone B-cell depletion impairs murine host defense against Borrelia burgdorferi infection

Marginal zone B (MZB) cells are a B-cell subset that produces T-cell-independent antibodies to blood-borne antigens. In this study, we examined the effects of MZB cell depletion on the immune response to the Lyme disease spirochete Borrelia burgdorferi, an extracellular pathogen for which T-cell-independent antibody is an important host defense. MZB cell depletion of C3H/HeJ mice using monoclonal antibody to LFA-1 and alpha(4)beta(1) integrins reduced B. burgdorferi-specific immunoglobulin M (IgM) titers, enhanced pathogen burden, and led to more severe arthritis assessed within the first 2 weeks of infection. In addition, MZB cell-depleted mice had reduced levels of B. burgdorferi-specific IgG, which correlated with diminished splenic CD4+ T-cell-activation, proliferation, and cytokine production. Passive transfer of immune mouse serum from infected control mice into infected MZB cell-depleted mice reduced pathogen burden but did not alter the expression of T-cell activation markers on splenic CD4+ T cells. These findings demonstrate that MZB cells not only are a source of pathogen-specific IgM important for limiting spirochete burden and pathology but also play a prominent role in the priming of splenic T-cell responses to a blood-borne pathogen.     

Both Notch1 and its ligands in B cells promote antibody production

Notch1 signaling regulates B and T lymphocyte development and also in vitro promotes antibody secretion upon B cell activation. However, it is still unclear about the role of Notch1 in antibody production upon in vitro and in vivo mixture lymphocytes activation. We first showed that Notch1 expressed in LPS-activated CD19^hi B cells and CD19^cre mediated Notch1 knock-down in LPS-activated B cells. Furthermore, we demonstrated that Notch1 knock-down in B cells reduced antibody production in LPS-stimulated B cells but did not affect antibody production in LPS-stimulated splenocytes and in experimental allergic encephalomyelitis (EAE) mice. Importantly, Notch1 ligands Dll1 and Jag1 expressed in B cells and pre-coated Notch1 protein promotes Notch1-knocked down B cells to produce antibody in LPS-stimulated B cells suggesting that Notch1 in other cells may promote antibody production by binding its ligands Dll1 and Jag1 in B cells. Together, our results suggest that both Notch1 and its ligands in B cells play an important role in antibody production.     

Putative role of marginal zone B cells in pathophysiological processes

The maintenance of inner integrity of an organism is founded on the proper performance of two immunity branches, innate and adaptive immune responses. Recently, it became apparent that subset of splenic B cells named marginal zone B cells (MZB cells) exhibits unique developmental and functional features that bridge these two immunity branches. Strategically positioned at the site where blood and lymph are filtered, MZB cells represent a population of sentinels that rapidly proliferate and differentiate into IgM plasmablast cells when encountered with blood-borne, thymus-independent (TI) Ags. Moreover, MZB cells have intrinsic capability to induce potent CD4⁺ helper T cell response and cytokine production upon stimulation with soluble antigens. Due to their ability to overcome a time gap prior the establishment of the full adaptive response towards pathogens, MZB cells connect and direct innate and adaptive immunity. An additional interesting characteristic of MZB cells is capacity to function as regulatory cells in autoimmune processes. MZB cells may also contribute to the control of autoimmunity via the induction of tolerance by apoptotic cells. Importantly, in the clear association with inflammation and autoimmunity, MZB cells may transform into MALT lymphoma, representing a concurrence point for the infection, immunity and malignancy. This paper presents an insight into the complex biology of marginal zone B cells and their role in intertwining and directing innate and adaptive immune processes at the physiological and pathological level.     

SWAP-70 controls formation of the splenic marginal zone through regulating T1B-cell differentiation

T1 and T2 transitional B cells are precursors for marginal zone B cells (MZB), which surround splenic follicles. MZB are essential for marginal zone formation, are central to the innate immune response, and contribute to adaptive immunity. Differentiation, migration, and homing of MZB and their precursors remain to be fully understood. We show that SWAP-70, a RhoGTPase-interacting and F-actin-binding protein with functions in cell polarization, migration, and adhesion regulates MZB development and marginal zone formation. The percentage of MZB in spleen of Swap70(-/-) mice was reduced to about one-third of that found in WT mice. Swap70(-/-) T1 cells accumulated in integrin ligand(high) regions of the splenic red pulp and failed to efficiently develop into T2 cells. Adoptive transfer and mixed BM chimera experiments demonstrated this to be a B-cell intrinsic phenotype. T-cell-independent antibody production was not impaired, however, and thus suggests that this process does not require correct homing of MZB precursors. B-cell adhesion through α(L)β(2) and α(4)β(1) integrins was hyper-activated in vitro and on tissue sections, and S1P-stimulated chemokinesis of MZB was reduced in the absence of SWAP-70. Thus, SWAP-70 acts as a regulator of the adhesion process, particularly important for differentiation control of B-cell precursors and their contribution to splenic tissue formation.     

Lsc regulates marginal-zone B cell migration and adhesion and is required for the IgM T-dependent antibody response

The humoral immune response to protein antigens is composed of a rapid low-affinity IgM antibody response followed by an IgG response exhibiting higher affinity. Here, we demonstrate that Lsc, a protein that regulates G protein-coupled-receptor signaling and RhoA activation, is required by B lymphocytes for the antigen-specific IgM antibody response to a protein antigen. We further show that in lsc(-/-) mice, MZB cells are selectively affected such that naive and in vivo-activated MZB cells migrate toward sphingosine-1-phosphate at increased proportions but release inefficiently from integrin ligands. Consequently, lsc(-/-) MZB cells do not traffick appropriately in an immune response and do not contribute to the TD antibody response. These data demonstrate that Lsc regulates the migration and adhesion of MZB cells, and this regulation appears to be required for these cells to contribute to the antibody response to TD antigens.     

IkappaBalpha is required for marginal zone B cell lineage development

Inactivation of members of the nuclear factor-kappaB (NF-kappaB) family results in the decrease or defect of marginal zone B (MZB) cells. It is not known which inhibitors of the NF-kappaB family (IkappaB) are required for MZB cell development. Here, we show that mice with B cell-specific inactivation of the main NF-kappaB inhibitor IkappaBalpha have a marked decrease of MZB cells and their presumed precursors. They exhibited increased mortality rates after blood-borne bacterial infection, indicating the importance of MZB cells for bacterial clearance. In contrast, response to T cell-dependent and -independent antigens resulted only in minor changes in immunoglobulin production. Our data demonstrate the importance of the intact NF-kappaB/IkappaBalpha pathway for proper MZB cell development.     

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.     

Specific over-expression of deltex and a new Kelch-like protein in human germinal center B cells

Ig gene hypermutation was originally described as the molecular process underlying B cell affinity maturation following a T-dependent immune response. Somatic hypermutation is also used in some species such as sheep, to generate diversity during formation of the primary antibody repertoire. In sheep, B cells mutate their Ig receptor during antigen-independent development in the lymphoid follicles of ileal Peyer's patches, but this process is arrested when these same B cells are cultured in vitro. We have used these differences between in vivo and in vitro B cell development to perform a cDNA subtraction between these two cell populations, in order to search for genes that might be involved in the hypermutation process. We describe in this paper the characterization of two genes, highly expressed in sheep ileal Peyer's patch B cells and also in centroblasts of human tonsils: deltex (Drosophila) homolog 1 (DTX1), which is related to the Notch pathway and a new Kelch-like protein, KLHL6. The putative role of these proteins, which are more likely involved in the germinal center B cell differentiation pathway than in the hypermutation mechanism per se, is discussed.     

Reduced notch activity is associated with an impaired marginal zone B cell development and function in Sly1 mutant mice

MZ B cells represent a distinct lineage of naive B lymphocytes, apart from FO B cells and peritoneal B1 cells, and mediate humoral immune responses against blood-borne type 2 T-independent antigens. Regulation of MZ B cell development involves the Notch receptor signaling, the intensity of B cell receptor signals, and cell compartmentalization by adhesion and chemokine receptors. Our previous work showed that gene-targeted mice expressing a truncated form of the putative signaling adapter protein SLy1 exhibit reduced numbers of a splenic B cell population enriched in MZ B cells. Here, we demonstrate that Sly1(d/d) mice exhibit a partial, but selective, block in the transition from pre-MZ to mature MZ B cells. Development of both T1 and T2 precursor subsets and FO B cells was normal in Sly1(d/d) mice. Consistent with the loss of MZ B cells, the production of antigen-specific IgM antibodies following immunization with pneumococcal polysaccharides was severely impaired in Sly1(d/d) mice. Importantly, expression of the Notch signaling mediator RBP-J and the Notch target genes Hes-1 and Hes-5 was markedly reduced in MZ but not FO B cells of Sly1(d/d) mice. In contrast, B cell receptor signaling, expression and function of LFA-1 and alpha4-integrins, and expression of chemokine receptors appeared intact in Sly1(d/d) cells. Collectively, these results provide strong evidence that SLy1 is important for the generation and function of MZ B cells and suggest a novel link between SLy1 and the activity of the Notch pathway in the development of MZ B cells.     

Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response

The lymphoid organs contain specialized microanatomic structures composed of lymphoid, myeloid and stromal cells that are vital to the generation of an effective adaptive immune response. Although the existence of these specialized structures has been known for over a century, the developmental signals that generate them and the specific roles of these structures in the immune response have remained largely elusive. Because of their position adjacent to the marginal sinuses, marginal zone B (MZB) cells are amongst the first population of cells seen by blood born antigens and are presumed to have a critical role in host defense against bacterial pathogens. Here we demonstrate that a deficiency of the tyrosine kinase (Pyk-2) results in a cell autonomous defect of MZB cell production. In response to repetitive polysaccharide antigens (T-independent type II (TI-II)) Pyk-2-deficient mice displayed marked suppression of IgM, IgG3 and IgG2a production. Furthermore, complement receptor engagement proved necessary for the specific targeting of polysaccharide antigens to MZB cells. These results suggest how innate immune responses mediated through complement coupling are translated into an adaptive response by MZB cells, and provide a potential mechanism for the T cell independence of humoral responses to polysaccharide antigens.     

Notch signaling is activated by TLR stimulation and regulates macrophage functions

Notch signaling is a well-conserved pathway involved in cell fate decisions, proliferation and apoptosis. We report on the involvement of Notch signaling in regulating gene expression in activated macrophages. Toll-like receptors (TLR) agonists such as bacterial lipopeptide, polyI:C, lipopolysaccharide and unmethylated CpG DNA all induced up-regulation of Notch1 in primary and macrophage-like cell lines. Notch1 up-regulation was dependent on the MyD88 pathway when stimulated through TLR2, but not TLR4. Activated Notch1 and expression of the Notch target genes, Hes1 and Deltex, were detected in activated macrophages, suggesting that Notch signaling was activated upon stimulation. Inhibiting processing of Notch receptor by gamma-secretase using a gamma-secretase inhibitor (GSI), the expression of Notch1 was down-regulated to basal levels. This treatment significantly modulated expression of TNF-alpha, IL-6, and IL-10. In addition, the amount of nitric oxide produced was significantly lower and the expression of MHC class II was up-regulated in GSI-treated cells. Treatment with GSI or silencing Notch1 resulted in decreased translocation of NF-kappaBp50 into nucleus upon stimulation. Taken together, stimulation of macrophages through the TLR signaling cascade triggered activation of Notch signaling, which in turn regulated gene expression patterns involved in pro-inflammatory responses, through activation of NF-kappaB.     

Regulation of marginal zone B cell development by MINT,a suppressor of Notch/RBP-J signaling pathway

We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless.     

Reciprocal generation of Th1/Th17 and T(reg) cells by B1 and B2 B cells

Regulatory T (T(reg)) cells are indispensable for maintaining peripheral tolerance, whereas T helper (Th)1 and Th17 cells induce inflammation and tissue destruction. Using Foxp3-GFP knock-in mice, we report a novel regulatory role for B cell subsets in influencing the differentiation of T(reg) versus Th1/Th17 cells. Peritoneal B1 cells strongly promoted T cell proliferation and cytokine secretion when presenting nominal or allogeneic antigens, as compared to conventional follicular B (B2) cells. However, peritoneal B1 cells largely failed to convert naive Foxp3(-)CD4(+) T cells into Foxp3(+) T(reg) cells in the presence of TGF-beta and IL-2, in marked contrast to conventional B2 cells, which excelled in T(reg) conversion. Interestingly, under the same T(reg) conversion conditions, peritoneal B1 cells preferentially promoted Th1 and Th17 cell differentiation. Blockade of CD86 but not CD80 costimulation markedly enhanced T(reg) cell induction by B1 cells. Thus, B cell antigen presentation function is inversely correlated with de novo T(reg) cell induction for these B cell subsets. Our findings suggest that B1 and B2 cell subsets play distinct roles in immune regulation by promoting reciprocal differentiation of T cell lineages.