The neuropeptide VIP regulates the expression of osteoclastogenic factors in osteoblasts

Osteoclast formation is controlled by stromal cells/osteoblasts expressing macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), crucial for osteoclast progenitor cell proliferation, survival and differentiation, and osteoprotegerin (OPG) that inhibits the interaction between RANKL and its receptor RANK. Recent data have strongly indicated that the nervous system plays an important role in bone biology. In the present study, the effects of the neuropeptide vasoactive intestinal peptide (VIP), present in peptidergic skeletal nerve fibers, on the expression of RANKL, OPG, and M-CSF in osteoblasts and stromal cells have been investigated. VIP and pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38), but not secretin, stimulated rankl mRNA expression in mouse calvarial osteoblasts. In contrast, VIP inhibited the mRNA expressions of opg and m-csf, effects shared by PACAP-38, but not by secretin. VIP did not affect rankl, opg, or m-csf mRNA expression in mouse bone marrow stromal cells (BMSCs). The effects by VIP on the mRNA expression of rankl, opg, and m-csf were all potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram. In addition, VIP robustly enhanced the phosphorylation of ERK and the stimulatory effect by VIP on rankl mRNA was inhibited by the MEK1/2 inhibitor PD98059. These observations demonstrate that activation of VPAC(2) receptors in osteoblasts enhances the RANKL/OPG ratio by mechanisms mediated by cyclic AMP and ERK pathways suggesting an important role for VIP in bone remodeling.     

Immature NK cells suppress dendritic cell functions during the development of leukemia in a mouse model

To analyze the mechanisms by which cancer cells escape from hosts' immune surveillance, we investigated the changes in immune status during the progression of leukemia induced by injecting mice with WEHI-3B cells. In the bone marrow (BM) of leukemic mice, only DX5(+)CD3(-) cells were continuously increased, despite the progression of leukemia. In addition, DX5(+)CD3(-) cells were rapidly increased in peripheral blood (PB) 20 days after inoculation. We also found that myeloid dendritic cells (DCs) expressing low levels of I-A(d) and having low allo-T cell stimulatory activity were markedly increased in PB and spleen. The increase in DX5(+) cells in BM was thought to be induced by soluble factors from leukemic cells. DX5(+) cells from leukemic mice were CD3(-), B220(-), Gr-1(-), CD14(-), CD94(-), Ly-49C/F(-), asialo GM1(+), CD25(+), CD122(+), Thy-1(bright), and c-kit(dim) and showed low killing activity against YAC-1 cells, suggesting that those DX5(+) cells were immature NK cells. NK cells from leukemic PB down-regulated the expression of I-A(d) on DCs, an effect mediated by TGF-beta. Moreover, these NK cells significantly suppressed the allo-T cell stimulatory activity of DCs, an effect requiring cell-to-cell contact between NK cells and DCs and thought to involve CD25. Importantly, NK cells from leukemic PB inhibited generation of autotumor-specific CTL induced by DCs in primary MLR or by DC immunization. In conclusion, we identified circulating immature NK cells with immunosuppressive activities. These cells may be important for understanding the involvement of the host immune system during the development of leukemia.     

Ox40 costimulation enhances the development of T cell responses induced by dendritic cells in vivo.

Dendritic cells (DCs) are bone marrow-derived APCs that display unique properties aimed at stimulating naive T cells. Several members of the TNF/TNFR families have been implicated in T cell functions. In this study, we examined the role that Ox40 costimulation might play on the ability of DCs to regulate CD4(+) and CD8(+) T cell responses in vivo. Administration of anti-mouse Ox40 mAb enhanced the Th response induced by immunization with Ag-pulsed DCs, and introduced a bias toward a Th1 immune response. However, anti-Ox40 treatment enhanced the production of Th2 cytokines in IFN-gamma(-/-) mice after immunization with Ag-pulsed DCs, suggesting that the production of IFN-gamma during the immune response could interfere with the development of Th2 lymphocytes induced by DCs. Coadministration of anti-Ox40 with DCs during Ag rechallenge enhanced both Th1 and Th2 responses induced during a primary immunization with DCs, and did not reverse an existing Th2 response. This suggests that Ox40 costimulation amplifies an ongoing immune response, regardless of Th differentiation potential. In an OVA-TCR class II-restricted adoptive transfer system, anti-Ox40 treatment greatly enhanced the level of cytokine secretion per Ag-specific CD4(+) T cell induced by immunization with DCs. In an OVA-TCR class I-restricted adoptive transfer system, administration of anti-Ox40 strongly enhanced expansion, IFN-gamma secretion, and cytotoxic activity of Ag-specific CD8(+) T cells induced by immunization with DCs. Thus, by enhancing immune responses induced by DCs in vivo, the Ox40 pathway might be a target for immune intervention in therapeutic settings that use DCs as Ag-delivery vehicles.     

Murine Th9 cells promote the survival of myeloid dendritic cells in cancer immunotherapy

Dendritic cells (DCs) are professional antigen-presenting cells to initiate immune responses, and DC survival time is important for affecting the strength of T-cell responses. Interleukin (IL)-9-producing T-helper (Th)-9 cells play an important role in anti-tumor immunity. However, it is unclear how Th9 cells communicate with DCs. In this study, we investigated whether murine Th9 cells affected the survival of myeloid DCs. DCs derived from bone marrow of C57BL/6 mice were cocultured with Th9 cells from OT-II mice using transwell, and the survival of DCs was examined. DCs cocultured with Th9 cells had longer survival and fewer apoptotic cells than DCs cultured alone in vitro. In melanoma B16-OVA tumor-bearing mice, DCs conditioned by Th9 cells lived longer and induced stronger anti-tumor response than control DCs did in vivo. Mechanistic studies revealed that IL-3 but not IL-9 secreted by Th9 cells was responsible for the prolonged survival of DCs. IL-3 upregulated the expression of anti-apoptotic protein Bcl-xL and activated p38, ERK and STAT5 signaling pathways in DCs. Taken together, our data provide the first evidence that Th9 cells can promote the survival of DCs through IL-3, and will be helpful for designing Th9 cell immunotherapy and more effective DC vaccine for human cancers.     

Ovariectomy-induced bone loss occurs independently of B cells

Estrogen withdrawal is associated with a significant expansion in B cell precursor and mature B cell populations. However, despite significant circumstantial evidence the role of B lineage cells in ovariectomy-induced bone loss in vivo is unclear. In vitro studies have demonstrated that mature B cells have the potential to both positively and negatively impact osteoclastogenesis by virtue of their capacity to secrete pro-osteoclastogenic cytokines including receptor activator of NFkappaB ligand (RANKL), as well as anti-osteoclastogenic cytokines such as osteoprotegerin (OPG) and transforming growth factor beta (TGFbeta). Although several studies have suggested that expansion of the B lineage following ovariectomy may play a key role in the etiology of ovariectomy-induced bone loss, in vivo studies to directly test this notion have yet to be conducted. In this study, we performed ovariectomy on microMT(-/-) mice which are specifically deficient in mature B cells. Analysis of bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) and micro-computed tomography (CT) demonstrate that mature B cell-deficient mice undergo an identical loss of bone mass relative to wild-type (WT) control mice. Our data demonstrate that mature B cells are not central mediators of ovariectomy-induced bone loss in vivo.     

Toll-like receptor 7 controls the anti-retroviral germinal center response

The development of vaccines that can enhance immunity to viral pathogens is an important goal. However, the innate molecular pathways that regulate the strength and quality of the immune response remain largely uncharacterized. To define the role of Toll-like receptor (TLR) signaling in control of a model retroviral pathogen, Friend virus (FV), I generated mice in which the TLR signaling adapter Myd88 was selectively deleted in dendritic cell (DC) or in B cell lineages. Deletion of Myd88 in DCs had little effect on immune control of FV, while B cell specific deletion of Myd88 caused a dramatic increase in viral infectious centers and a significantly reduced antibody response, indicating that B cell-intrinsic TLR signaling plays a crucial role, while TLR signaling in DCs is less important. I then identified the single-stranded RNA sensing protein TLR7 as being required for antibody-mediated control of FV by analyzing mice deficient in TLR7. Remarkably, B cells in infected TLR7-deficient mice upregulated CD69 and CD86 early in infection, but failed to develop into germinal center B cells. CD4 T cell responses were also attenuated in the absence of TLR7, but CD8 responses were TLR7 independent, suggesting the existence of additional pathways for detection of retroviral particles. Together these results demonstrate that the vertebrate immune system detects retroviruses in vivo via TLR7 and that this pathway regulates a key checkpoint controlling development of germinal center B cells.     

Lymphocyte development and function in the absence of retinoic acid-related orphan receptor alpha

The orphan nuclear receptor, retinoid acid-related orphan receptor (ROR)alpha, is essential for the development of cerebellar Purkinje cells and bone tissue. RORalpha may also play a critical role in lymphocyte development and function because staggerer mice, a natural mutant strain with a disrupted expression of RORalpha, have reduced thymic and splenic cellularity. In this report, we analyzed the role of RORalpha in lymphocyte development by examining lymphoid compartments in RORalpha(-/-) mice and Rag-2(-/-) mice reconstituted with RORalpha(-/-) bone marrow. We found that T and B cell development was severely defective in RORalpha(-/-) mice, but not in Rag-2(-/-)/RORalpha(-/-) chimeric mice. We also analyzed cellular and humoral immune responses in Rag-2(-/-)/RORalpha(-/-) chimeric mice. Our results show that serum IgG levels were elevated in Rag-2(-/-)/RORalpha(-/-) chimeric mice after immunization with a T-dependent Ag compared with control chimeras. IFN-gamma production by RORalpha(-/-) CD8(+) T cells after TCR stimulation was also increased. Furthermore, RORalpha(-/-) mast cells and macrophages produced an increased amount of TNF-alpha and IL-6 upon activation. These results indicate that RORalpha indirectly regulates lymphocyte development by providing an appropriate microenvironment and controls immune responses by negatively regulating cytokine production in innate immune cells and lymphocytes.     

Bradykinin B2 Receptors of dendritic cells, acting as sensors of kinins proteolytically released by Trypanosoma cruzi, are critical for the development of protective type-1 responses

Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B2 receptors (B2R). Here we report that C57BL/6.B2R-/- mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B2R+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B2R-/- heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-gamma-producing CD4+ and CD8+ T cells in the spleen of B2R-/- and wild-type mice. However, production of IFN-gamma by effector T cells isolated from B2R-/- heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-gamma-producing (CD4+CD44+ and CD8+CD44+) T cells both in the spleen and heart while B2R-/- mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B2R-/- mice was linked to upregulated secretion of IL-17 and TNF-alpha by antigen-responsive CD4+ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c+ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B2R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c+ DCs isolated from B2R+/+ spleen, but not by DCs from B2R-/- mice. Notably, adoptive transfer of B2R+/+ CD11c+ DCs (intravenously) into B2R-/- mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed TH17 responses. Collectively, our results demonstrate that activation of B2R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection.     

Role of B7 in T cell tolerance

The induction of effective immune responses requires costimulation by B7 molecules, and Ag recognition without B7 is thought to result in no response or tolerance. We compared T cell responses in vivo to the same Ag presented either by mature dendritic cells (DCs) or as self, in the presence or absence of B7. We show that Ag presentation by mature B7-1/2-deficient DCs fails to elicit an effector T cell response but does not induce tolerance. In contrast, using a newly developed adoptive transfer system, we show that naive OVA-specific DO11 CD4+ T cells become anergic upon encounter with a soluble form of OVA, in the presence or absence of B7. However, tolerance in DO11 cells transferred into soluble OVA transgenic recipients can be broken by immunization with Ag-pulsed DCs only in B7-deficient mice and not in wild-type mice, suggesting a role of B7 in maintaining tolerance in the presence of strong immunogenic signals. Comparing two double-transgenic models--expressing either a soluble or a tissue Ag--we further show that B7 is not only essential for the active induction of regulatory T cells in the thymus, but also for their maintenance in the periphery. Thus, the obligatory role of B7 molecules paradoxically is to promote effective T cell priming and contain effector responses when self-Ags are presented as foreign.     

Low-affinity, Smith antigen-specific B cells are tolerized by dendritic cells and macrophages

Polyclonal B cell activation promotes immunity without the loss of tolerance. Our data show that during activation of the innate immune system, B cell tolerance to Smith Ag Sm is maintained by dendritic cells (DCs) and macrophages (MPhi). TLR4-activated myeloid DCs and MPhi, but not plasmacytoid or lymphoid DCs, repressed autoreactive B cells through the secretion of soluble mediators, including IL-6. Although IL-6 promotes plasma cell differentiation of B cells acutely stimulated by Ag, we show that it repressed cells that were chronically exposed to self-Ag. This mechanism of tolerance was not limited to Smith Ag-specific B cells as hen egg lysozyme- and p-azophenylarsonate-specific B cells were similarly affected. Our data define a tolerogenic role for MPhi and DCs in regulating autoreactive B cells during activation of the innate immune system.     

B cells control the migration of a subset of dendritic cells into B cell follicles via CXC chemokine ligand 13 in a lymphotoxin-dependent fashion

Certain classes of dendritic cells (DCs) meet rare cognate Ag-specific T and B cells inside primary B cell follicles for the development of germinal centers. However, the mechanisms underlying this coordination are still undefined. Cysteine-rich (CR) domain of the mannose receptor (CR-Fc)(+) DCs are a newly discovered subset of DCs that migrate rapidly into the primary lymphoid follicles from marginal zone after immunization. In this work, we uncover the key role of B cells in the establishment of a microenvironment that allows these DCs to be in the B cell area in a lymphotoxin (LT)-dependent fashion. CR-Fc(+) DCs are absent from the spleens of both LTbetaR- and LTalpha-deficient mice, suggesting that signaling by membrane LT is required for the presence of CR-Fc(+) DCs in the spleen. Interestingly, analysis of mutant mice that lack T, B, or NK cells demonstrates that B cell-derived membrane LT is essential for the unique localization of CR-Fc(+) DCs in the spleen. Using bone marrow transfer and ligand-blocking approaches, we provide evidence that B cell-derived LT acts indirectly on CR-Fc(+) DCs through LTbetaR(+) stromal cells. In analogous fashion to certain Ag-activated T and B cells, CR-Fc(+) DCs, expressing CXCR5, localize to primary lymphoid follicles in response to CXC ligand 13 (B lymphocyte chemoattractant). Together, we propose that B cells play a central role in establishing the chemotactic gradient that attracts not only Ag-activated T and B cells but also Ag-carrying CR-Fc(+) DCs. In turn, CR-Fc(+) DCs and T cells home to B cell follicles to interact with B cells in the developing germinal center.     

IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation

Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130(F759/F759)) or STAT3 (gp130(FxxQ/FxxQ)), and combined gp130 and IL-6 defects (gp130(F759/F759)/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130(F759/F759) mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.     

Biology of RANK,RANKL, and osteoprotegerin

The discovery of the receptor activator of nuclear factor-kappaB ligand (RANKL)/RANK/osteoprotegerin (OPG) system and its role in the regulation of bone resorption exemplifies how both serendipity and a logic-based approach can identify factors that regulate cell function. Before this discovery in the mid to late 1990s, it had long been recognized that osteoclast formation was regulated by factors expressed by osteoblast/stromal cells, but it had not been anticipated that members of the tumor necrosis factor superfamily of ligands and receptors would be involved or that the factors involved would have extensive functions beyond bone remodeling. RANKL/RANK signaling regulates the formation of multinucleated osteoclasts from their precursors as well as their activation and survival in normal bone remodeling and in a variety of pathologic conditions. OPG protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from binding to its receptor, RANK. Thus, RANKL/OPG ratio is an important determinant of bone mass and skeletal integrity. Genetic studies in mice indicate that RANKL/RANK signaling is also required for lymph node formation and mammary gland lactational hyperplasia, and that OPG also protects arteries from medial calcification. Thus, these tumor necrosis factor superfamily members have important functions outside bone. Although our understanding of the mechanisms whereby they regulate osteoclast formation has advanced rapidly during the past 10 years, many questions remain about their roles in health and disease. Here we review our current understanding of the role of the RANKL/RANK/OPG system in bone and other tissues.     

Osteoprotegerin reduces the serum level of receptor activator of NF-kappaB ligand derived from osteoblasts

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF-kappaB ligand (RANKL). We previously reported that OPG deficiency elevated the circulating level of RANKL in mice. Using OPG(-/-) mice, we investigated whether OPG is involved in the shedding of RANKL by cells expressing RANKL. Osteoblasts and activated T cells in culture released a large amount of RANKL in the absence of OPG. OPG or a soluble form of receptor activator of NF-kappaB (the receptor of RANKL) suppressed the release of RANKL from those cells. OPG- and T cell-double-deficient mice showed an elevated serum RANKL level equivalent to that of OPG(-/-) mice, indicating that circulating RANKL is mainly derived from bone. The serum level of RANKL in OPG(-/-) mice was increased by ovariectomy or administration of 1alpha,25-dihydroxyvitamin D(3). Expression of RANKL mRNA in bone, but not thymus or spleen, was increased in wild-type and OPG(-/-) mice by 1alpha,25-dihydroxyvitamin D(3). These results suggest that OPG suppresses the shedding of RANKL from osteoblasts and that the serum RANKL in OPG(-/-) mice exactly reflects the state of bone resorption.     

H2-O expression in primary dendritic cells

H2-O is a nonpolymorphic class II molecule whose biological role remains to be determined. H2-O modulates H2-M function, and it has been generally believed to be expressed only in B lymphocytes and thymic medullary epithelial cells, but not in dendritic cells (DCs). In this study, we report identification of H2-O expression in primary murine DCs. Similar to B cells, H2-O is associated with H2-M in DCs, and its expression is differentially regulated in DC subsets as well as during cell maturation and activation. Primary bone marrow DCs and plasmacytoid DCs in the spleen and lymph nodes express MHC class II and H2-M, but not the inhibitor H2-O. In contrast, myeloid DCs in secondary lymphoid organs express both H2-M and H2-O. In CD8alphaalpha(+) DCs, the ratio of H2-O to H2-M is higher than in CD8alphaalpha(-) DCs. In DCs generated from GM-CSF- and IL-4-conditioned bone marrow cultures, H2-O expression is not detected regardless of the maturation status of the cells. Administration of LPS induces in vivo activation of myeloid DCs, and this activation is associated with down-regulation of H2-O expression. Primary splenic DCs from H2-O(-/-) and H2-O(+/+) mice present exogenous protein Ags to T cell hybridomas similarly well, but H2-O(-/-) DCs induce stronger allogeneic CD4 T cell response than the H2-O(+/+) DCs in mixed leukocyte reactions. Our results suggest that H2-O has a broader role than previously appreciated in regulating Ag presentation.     

GPR54 deficiency reduces the Treg population and aggravates experimental autoimmune encephalomyelitis in mice

GPR54 is highly expressed in the central nervous system and plays a crucial role in pubertal development. However, GRP54 is also expressed in the immune system, implying possible immunoregulatory functions. Here we investigated the role of GPR54 in T cell and immune tolerance. GPR54 deficiency led to an enlarged thymus, an increased number of thymocytes, and altered thymic micro-architecture starting around puberty, indicating GPR54 function in T-cell development through its regulatory effect on the gonadal system. However, flow cytometry revealed a significant reduction in the peripheral regulatory T cell population and a moderate decrease in CD4 single-positive thymocytes in prepubertal Gpr54~(-/-) mice. These phenotypes were confirmed in chimeric mice with GPR54 deficient bone marrow-derived cells. In addition, we found elevated T cell activation in peripheral and thymic T cells in Gpr54~(-/-) mice. When intact mice were immunized with myelin oligodendrocyte glycoprotein, a more severe experimental autoimmune encephalomyelitis(EAE) developed in the Gpr54~(-/-) mice. Interestingly, aggravated EAE disease was also manifested in castrated and bone marrow chimeric Gpr54~(-/-) mice compared to the respective wild-type control,suggesting a defect in self-tolerance resulting from GPR54 deletion through a mechanism that bypassed sex hormones. These findings demonstrate a novel role for GPR54 in regulating self-tolerant immunity in a sex hormone independent manner.