Flt3 ligand expands dendritic cell numbers in normal and malignant murine prostate

We have developed a murine model that facilitates the structural and functional analysis in vivo of dendritic cell (DC)-mediated phagocytosis of prostate epithelial cells. Recombinant human Flt3 ligand (rhFL) expands the number of dendritic cells in lymphoid and non-lymphoid tissues of mice. We show that rhFL also induced the ingress of dendritic cells into murine prostate, which involutes via epithelial apoptosis after surgical castration. Intact or castrated C57BL/6 and syngeneic transgenic adenocarcinoma of mouse prostate (TRAMP) mice were treated with rhFL or PBS control. Prostate and spleen were then studied by flow cytometry and immunohistochemistry.The number of prostatic CD11c+ and CD11b+ dendritic cells increased significantly in rhFL-treated mice compared with PBS-treated control mice and this effect was greatly augmented by castration of the mice. The immunophenotype of rhFL-mobilized prostatic cells was consistent with that of Langerhans cells (MHC class II+, CD11c+,CD11b+, DEC-205+, CD8 alpha-).MHC class II+ and CD11c+ dendritic cells that were present in the prostate glands of rhFL-treated and castrated C57BL/6 mice were intimately associated with TUNEL+ inclusions, which suggests that Langerhans-type dendritic cells in prostate participated in the clearance of apoptotic cells. Expression of MHC class II, CD54, CD80 and CD86 by prostatic dendritic cells was not up-regulated after castration and freshly isolated rhFL-induced prostate cells were unable to prime allogeneicT cells unless they were activated by culture either on plastic or with recombinant soluble CD40 ligand. Our data suggest that rhFL-mobilized prostatic dendritic cells resemble the functionally immature dendritic cells, which reside in peripheral tissues and contribute to the maintenance of peripheral tolerance.     

Comparison of the functional properties of murine dendritic cells generated in vivo with Flt3 ligand, GM-CSF and Flt3 ligand plus GM-SCF

Flt3 ligand (FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are important growth factors for dendritic cells (DC). Substantial numbers of DC can be generated in vivo following the administration of either factor. We sought to extend our knowledge of the functional properties of these cells including their ability to prime na?ve CD8(+) T cells. In addition, we compared the nature of the DC generated in vivo with the single cytokines to those generated with the combination of FL+polyethylene glycol-modified GM-CSF (pGM-CSF). Treatment with FL+pGM-CSF yielded greater numbers of both CD11b(low) and CD11b(high) DC than with either cytokine alone, and these DC were more efficient at antigen (Ag) capture. The FL+pGM-CSF-generated CD11b(low) DC lacked expression of CD8alpha. Following treatment with LPS in vivo, all DC subsets upregulated CD40, CD80, CD86, and MHC class II expression, but surprisingly Ag capture was not downregulated and some DC subsets retained expression of intracellular MHC class II vesicles. Thus, even after activation in vivo with LPS, DC retained Ag capture properties of immature DC, and Ag presentation/costimulation properties of mature DC. Though all DC subsets stimulated CD4(+) T cell proliferation equivalently, FL-generated DC were more efficient at priming Ag-specific CD8(+) cytolytic T cells than DC generated with either pGM-CSF alone or FL+pGM-CSF, and CD11b(high) DC were more efficient at priming CD8(+) T cells than CD11b(low) DC.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking

To gain ample numbers of dendritic cells (DCs) for investigation, or for immunotherapy, the culture of DC precursors from bone marrow in either GM-CSF and IL-4 (GM/IL4-DCs) or Flt3L (FL-DCs) has often been used. Despite their common use, the relationship of these culture-derived DCs to those in vivo, and their relative potential for use in immunotherapy, needs further elucidation. In this study we found that in contrast to FL-DCs, highly purified GM/IL4-DCs were larger and more granular, surface Mac-3(+), and were comprised of two populations (CD24(low)CD11b(high) and CD24(high)CD11b(low)). Functionally, although comparable in T cell activation, GM/IL4-DCs produced more inflammatory mediators including TNF-alpha, IL-10, CCL-2, and NO than FL-DCs upon TLR ligation. However, FL-DCs migrated more efficiently to draining lymph nodes after s.c. injection and produced a different profile of cytokines to GM/IL4-DCs. Developmentally, unlike GM/IL4-DCs, FL-DCs cannot be differentiated from CD11b(high)Ly6C(high)Ly6G(-) monocytes. Collectively, these data suggest that the GM/IL4-DCs are the equivalents of the TNF-alpha and inducible NO synthase producing DCs in vivo that emerge after inflammation whereas FL-DCs better represent the steady-state resident DCs. The differences between GM/IL4-DCs and FL-DCs have serious implications for DC-based immunotherapeutic strategies.     

Conditional expression of Wnt4 during chondrogenesis leads to dwarfism in mice

Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26(floxneoWnt4). Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26(floxneoWnt4); Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26(floxneoWnt4); Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF). These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype.     

Interferon expression in the testes of transgenic mice leads to sterility

A plasmid containing the mouse interferon-alpha 1 gene under control of the mouse metallothionein-I promoter was used for the construction of transgenic mice. Four transgenic mice (two males and two females) were obtained containing 1 to over 10 copies of the introduced DNA. Both males appeared to be sterile. One of the female mice founded a transgenic strain in which the foreign DNA was transmitted to her offspring in a Mendelian fashion. In this strain most male animals are sterile or turn sterile with time. Northern blot analysis of several tissues of these animals shows that expression of the introduced interferon gene occurs only in the testis. In some of the animals biologically active interferon could also be detected in testes homogenates. Histological examination of testis tissue shows an ongoing degeneration of spermatogenic cells leading to calcium deposits and complete atrophy of the seminiferous tubules.     

Flt3 ligand preferentially increases the number of functionally active myeloid dendritic cells in the lungs of mice

In the present study, we investigated the effects of in vivo Flt3L administration on the generation, phenotype, and function of lung dendritic cells (DCs) to evaluate whether Flt3L favors the expansion and maturation of a particular DC subset. Injection of Flt3L into mice resulted in an increased number of CD11c-expressing lung DCs, preferentially in the alveolar septa. FACS analysis allowed us to quantify a 19-fold increase in the absolute numbers of CD11c-positive, CD45R/B220 negative DCs in the lungs of Flt3L-treated mice over vehicle-treated mice. Further analysis revealed a 90-fold increase in the absolute number of myeloid DCs (CD11c positive, CD45R/B220 negative, and CD11b positive) and only a 3-fold increase of lymphoid DCs (CD11c positive, CD45R/B220 negative, and CD11b negative) from the lungs of Flt3L-treated mice over vehicle-treated mice. Flt3L-treated lung DCs were more mature than vehicle-treated lung DCs as demonstrated by a significantly higher percentage of cells expressing MHC class II, CD86, and CD40. Freshly isolated Flt3L lung DCs were not fully mature, because after an overnight culture they continued to increase accessory molecule expression. Functionally, Flt3L-treated lung DCs were more efficient than vehicle-treated DCs at stimulating naive T cell proliferation. Our data show that administration of Flt3L favors the expansion of myeloid lung DCs over lymphoid DCs and enhanced their ability to stimulate naive lymphocytes.     

Conditional cell ablation by tight control of caspase-3 dimerization in transgenic mice

Studying the effects of the loss of a specific cell type is a powerful approach in biology. Here we present a method based on the controlled activation of the apoptotic machinery. We expressed a modified caspase-3-containing chemical inducer of dimerization (CID)-binding sites in the livers of transgenic mice. In the absence of CID, no liver injury was detectable, underlining the absence of leakage in our system. In contrast, injection of the CID produced activation of the chimeric caspase-3, which led to a dose-dependent pure hepatocyte ablation with subsequent regeneration. This method is effective in both growing and nongrowing cells, and is therefore applicable to a wide range of cells and tissues. Moreover, because apoptosis has been described in numerous pathological circumstances, this system is useful for generating mouse models of human disorders as well as for studying the recovery or regeneration of tissues after cell loss.     

Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion

In mice, immunoregulatory APCs express the dendritic cell (DC) marker CD11c, and one or more distinctive markers (CD8alpha, B220, DX5). In this study, we show that expression of the tryptophan-degrading enzyme indoleamine 2,3 dioxygenase (IDO) is selectively induced in specific splenic DC subsets when mice were exposed to the synthetic immunomodulatory reagent CTLA4-Ig. CTLA4-Ig did not induce IDO expression in macrophages or lymphoid cells. Induction of IDO completely blocked clonal expansion of T cells from TCR transgenic mice following adoptive transfer, whereas CTLA4-Ig treatment did not block T cell clonal expansion in IDO-deficient recipients. Thus, IDO expression is an inducible feature of specific subsets of DCs, and provides a potential mechanistic explanation for their T cell regulatory properties.     

Split tolerance in peripheral B cell subsets in mice expressing a low level of Igkappa-reactive ligand

Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed kappa-macroself Ag, a synthetic superantigen reactive to Igkappa. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature kappa+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of kappa+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,kappa. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.     

Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.     

Improved hematopoietic stem cell engraftment following ex vivo expansion of murine marrow cells with SCF and Flt3L

BACKGROUND: In vitro incubation of murine BM cells with IL-3, IL-6, IL-11 and SCF induces expansion of HPC but fails to preserve 'engraftability' in comparison with normal untreated marrow cells. We studied how culturing marrow cells for 48 and 72 h with a combination of the cytokines SCF and Flt3L influences cell expansion and engraftability. METHODS: Competitive repopulation of lethally irradiated C57BL/6 mice was used to examine engraftability of ex vivo cytokine-expanded Ptprc chimeric BM. A methylcellulose in vitro assay was used to determine the expansion of substitute progenitors. RESULTS: Both cytokine combinations successfully expanded progenitor populations when assayed in methylcellulose culture in vitro. After 72 h, the colony numbers of the expansion cultures increased 61% with IL-3, IL-6, IL-11 and SCF stimulation and 96% with SCF and Flt3L stimulation. Engraftment of competitively transplanted cells, cultured with IL-3, IL-6, IL-11 and SCF, consistently dropped to levels below 16%. However, 48 h culture with SCF and Flt3L resulted in 53.5+/-1.6% engraftment at 17 days and 64+/-3.7% engraftment at 19 weeks post-transplantation. Extending the cytokine exposure to 72 h resulted in 70+/-4.4% short-term engraftment at 17 days, and 64+/-2.4% engraftment at 19 weeks post-transplantation. DISCUSSION: The data demonstrate the ability of SCF and Flt3L cytokine-stimulated BM cells to maintain short- and long-term engraftability. We conclude that these cytokines play a crucial role in maintaining engraftment of hematopoietic progenitors.     

外周血T细胞亚群在小鼠结核感染中的意义

目的探讨外周血T细胞亚群在小鼠结核感染中的意义,并与加用免疫调节剂组对比研究。方法60只小鼠随机分为3组,每组20只。结核感染组:小鼠经血管注射结核分枝杆菌(H37RV)0.05 ml,建立小鼠结核模型。生理盐水组:用0.05 ml生理盐水替代H37RV处理。母牛分枝杆菌菌苗组:按结核感染组同样剂量和方法复制小鼠结核模型,H37RV感染后第3、10、17天分别肌注22.5μg母牛分枝杆菌菌苗。感染4周后,取外周血用流式细胞仪检测T细胞亚群。结果感染组外周血T细胞亚群比率显著降低,母牛分枝杆菌菌苗组小鼠外周血T细胞亚群比率显著高于感染组。结论外周血T细胞亚群数量与结核感染的病情密切相关,调节小鼠结核模型的T细胞亚群免疫功能可使结核感染的病情发展得到一定程度的的控制。     

Aminopeptidase P isozyme expression in human tissues and peripheral blood mononuclear cell fractions

Aminopeptidase P (APP) isoforms specifically remove the N-terminal amino acid from peptides that have a proline residue in the second position. The mRNA levels of three different isoforms, each coded by a different gene, were determined in 16 human tissues and in peripheral blood mononuclear cell (PBMC) fractions by RT-PCR. The cytosolic isoform, APP1, and the cell surface membrane-bound isoform, APP2, are expressed in all of the human tissues and PBMC fractions examined. The very high expression of APP2 mRNA in kidney compared to other tissues was confirmed by enzyme activity measurements. Among the PBMC fractions, APP2 expression is highest in resting CD8(+) T cells, but decreases in these cells following their activation with phytohemagglutinin; in contrast, expression of APP2 increases in CD4(+) T cells upon activation. The third isoform, APP3, is a hypothetical protein identified by nucleotide sequencing. A detailed analysis of its amino acid sequence confirmed that the protein is an aminopeptidase P-like enzyme with greater similarity to Escherichia coli APP than to either APP1 or APP2. Two splice variants of APP3 exist, one of which is predicted to have a mitochondrial localization (APP3m) while the other is cytosolic (APP3c). Both forms are variably expressed in all of the human tissues and PBMC fractions examined.     

Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice

To control G protein signaling in vivo, we have modified G protein-coupled receptors to respond exclusively to synthetic small molecule agonists and not to their natural agonist(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). A prototype RASSL (Ro1) based on the Gi-coupled K opioid receptor was expressed in transgenic mice under the control of the tetracycline transactivator (tet) system. Activation of Ro1 expressed in the heart decreased heart rate by up to 80%, an expected effect of increased Gi signaling. Maximal heart rate changes occurred in less than 1 min, demonstrating the speed of this inducible signaling system. This Ro1-mediated slowing of heart rate was also subject to desensitization, which lasted more than 24 h. Both the initial effect on heart rate and the desensitization occurred, even though Ro1 is derived from a human opioid receptor not normally involved in heart rate control. In addition, the tet system was used to induce Ro1 expression in hepatocytes and salivary gland, where Gi signaling is known to control physiologic events such as proliferation and secretion. These studies demonstrate that a RASSL can be inducibly expressed in several mouse tissues and used in vivo to activate G protein signaling in a controllable fashion.     

Radiation-induced increase in plasma Flt3 ligand concentration in mice: evidence for the implication of several cell types

Circulating T lymphocytes were proposed as the main producer of Flt3 ligand. However, during aplasia, there is a drastic reduction in the number of T lymphocytes, while plasma Flt3 ligand concentration is increased. This contradiction prompted us to compare variations in plasma Flt3 ligand during radiation-induced aplasia in BALB/c mice and in T-lymphocyte-deficient NOD-SCID mice to delineate the role of T lymphocytes in the increase in Flt3 ligand concentration. The results showed that plasma Flt3 ligand concentration was increased similarly in the two strains of mice, and that Flt3 ligand concentration was negatively correlated to the number of residual hematopoietic progenitors. Moreover, the Flt3 ligand mRNA expression and Flt3 ligand protein concentration were similar in the two strains of mice in all organs tested, i.e. thymus, spleen, bone marrow, liver, brain and blood cells. These results confirm that Flt3 ligand concentration in the blood is a reflection of bone marrow function and that T lymphocytes are not the main regulator of Flt3 ligand variations during aplasia.     

Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice

Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior.     

Altered expression after expansion of a v-erbA transgene in transgenic mice

Repetitive DNA is known to undergo size variations based on an increase or decrease in the number of monomer units. We describe here the spontaneous expansion of an experimentally introduced tandem array of repeats in a transgenic mouse consisting of the human -actin promoter fused to the viral oncogene v-erbA and an SV40 polyadenylation signal (hAP/v-erbA). The expansion of the transgene was identified during routine screening of transgenic offspring for heterozygosity or homozygosity in one founder line. The control heterozygote genome consisted of the hAP/v-erbA transgene organized as a tandem array approximately five monomer units at a single chromosomal locus. The number of units increased to 20–21 copies, and germline transmission of the expanded units was stable for at least two generations. The majority of animals carrying the expanded monomer units retained their RNA expression patterns; however, some of these animals had drastically reduced expression levels. We discuss the possibility that expansion of repeated units may provide a mechanism by which the expression of a deleterious transgene is reduced.