Cutting edge: merocytic dendritic cells break T cell tolerance to beta cell antigens in nonobese diabetic mouse diabetes.

In type 1 diabetes, the breach of central and peripheral tolerance results in autoreactive T cells that destroy insulin-producing, pancreatic beta cells. In this study, we identify a critical subpopulation of dendritic cells responsible for mediating both the cross-presentation of islet Ags to CD8(+) T cells and the direct presentation of beta cell Ags to CD4(+) T cells. These cells, termed merocytic dendritic cells (mcDCs), are more numerous in the NOD mouse and, when Ag-loaded, rescue CD8(+) T cells from peripheral anergy and deletion while stimulating islet-reactive CD4(+) T cells. When purified from the pancreatic lymph nodes of overtly diabetic NOD mice, mcDCs break peripheral T cell tolerance to beta cells in vivo and induce rapid onset type 1 diabetes in the young NOD mouse. Thus, the mcDC subset appears to represent the long-sought APC responsible for breaking peripheral tolerance to beta cell Ags in vivo.

Wnt2 regulates progenitor proliferation in the developing ventral midbrain.

Wnts are secreted, lipidated proteins that regulate multiple aspects of brain development, including dopaminergic neuron development. In this study, we perform the first purification and signaling analysis of Wnt2 and define the function of Wnt2 in ventral midbrain precursor cultures, as well as in Wnt2-null mice in vivo. We found that purified Wnt2 induces the phosphorylation of both Lrp5/6 and Dvl-2/3, and activates beta-catenin in SN4741 dopaminergic cells. Moreover, purified Wnt2 increases progenitor proliferation, and the number of dopaminergic neurons in ventral midbrain precursor cultures. In agreement with these findings, analysis of the ventral midbrain of developing Wnt2-null mice revealed a decrease in progenitor proliferation and neurogenesis that lead to a decrease in the number of postmitotic precursors and dopaminergic neurons. Collectively, our observations identify Wnt2 as a novel regulator of dopaminergic progenitors and dopaminergic neuron development.

WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature.

Macrophages have a critical role in inflammatory and immune responses through their ability to recognize and engulf apoptotic cells. Here we show that macrophages initiate a cell-death programme in target cells by activating the canonical WNT pathway. We show in mice that macrophage WNT7b is a short-range paracrine signal required for WNT-pathway responses and programmed cell death in the vascular endothelial cells of the temporary hyaloid vessels of the developing eye. These findings indicate that macrophages can use WNT ligands to influence cell-fate decisions--including cell death--in adjacent cells, and raise the possibility that they do so in many different cellular contexts.

Nrarp coordinates endothelial Notch and Wnt signaling to control vessel density in angiogenesis.

When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections.

The countervailing actions of myeloid and plasmacytoid dendritic cells control autoimmune diabetes in the nonobese diabetic mouse.

Islet Ag-specific CD4(+) T cells receive antigenic stimulation from MHC class II-expressing APCs. Herein, we delineate the direct in vivo necessity for distinct subsets of macrophages and dendritic cells (DC) in type 1 diabetes mellitus of the NOD mouse by using diphtheria toxin-mediated cell ablation. The ablation of macrophages had no impact on islet Ag presentation or on the induction of insulitis or diabetes in either transfer or spontaneous models. However, the ablation of CD11b(+)CD11c(+) DC led to the loss of T cell activation, insulitis, and diabetes mediated by CD4(+) T cells. When the specific myeloid DC subset was "added-back" to mice lacking total DC, insulitis and diabetes were restored. Interestingly, when NOD mice were allowed to progress to the insulitis phase, the ablation of DC led to accelerated insulitis. This accelerated insulitis was mediated by the loss of plasmacytoid DC (pDC). When pDC were returned to depleted mice, the localized regulation of insulitis was restored. The loss of pDC in the pancreas itself was accompanied by the localized loss of IDO and the acceleration of insulitis. Thus, CD11c(+)CD11b(+) DC and pDC have countervailing actions in NOD diabetes, with myeloid DC providing critical antigenic stimulation to naive CD4(+) T cells and pDC providing regulatory control of CD4(+) T cell function in the target tissue.

NKT cells and IFN-gamma establish the regulatory environment for the control of diabetogenic T cells in the nonobese diabetic mouse.

In type 1 diabetes mellitus (T1DM), T cell-mediated destruction of insulin-producing pancreatic beta cells leads to the acute onset of hyperglycemia. The nonobese diabetic mouse model of human T1DM reveals that T cells capable of inducing diabetes can escape normal central tolerance, and can cause T1DM if left unchecked. However, several regulatory T cell subsets can temper autoaggressive T cells, although it remains undetermined when and how, and by which subset, homeostatic control of diabetogenic T cells is normally achieved in vivo. Using a cotransfer model, we find that NKT cells efficiently dampen the action of diabetogenic CD4+ T cells, and do so in an indirect manner by modifying the host environment. Moreover, the NKT cell-containing population modifies the host via production of IFN-gamma that is necessary for driving the inhibition of diabetogenic T cells in vivo.

Characterization of murine cathepsin W and its role in cell-mediated cytotoxicity.

Cathepsin W is a member of the papain-like family of cysteine proteases. In this report, we have isolated the cDNA for murine CtsW (mCtsW) from a splenocyte library. The deduced 371-amino-acid sequence shares 68% identity with human CtsW and includes the conserved catalytic triad cysteine, histidine, and asparagine found in all members of this family. In addition to the fulllength form of mCtsW, we have isolated an alternatively spliced form of the mRNA that lacks a complete catalytic triad. An S1 nuclease protection assay and a Western blot analysis showed that mCtsW is mainly restricted to the CD8(+) T cell and natural killer cell compartments. In addition, we confirmed that, like its human homologue, mCtsW is localized mainly to the endoplasmic reticulum and its expression is up-regulated upon activation. We also characterized the mCtsW locus using bacterial artificial chromosome clones. The gene consists of 10 coding exons and 9 introns spanning 3.2 kb. To elucidate the physiologic role of this protease, we generated mice deficient in mCtsW. Our data establish that mCtsW is not required for cytotoxic lymphocyte-induced target cell death in vitro. In addition, mCtsW deficiency does not alter the susceptibility of cytotoxic lymphocytes to suicide or fratricide after degranulation. Thus, mCtsW does not have a unique role in target cell apoptosis or cytotoxic cell survival in vitro.