Murine Splenic Dendritic Cells Research Articles

IL-35Ig-expressing dendritic cells induce tolerance via Arginase 1.

The cytokine interleukin IL‐35 is known to exert strong immunosuppressive functions. Indoleamine 2,3‐dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL‐35 and the activity of those enzymes. We transfected a single chain IL‐35Ig gene construct in murine splenic DCs (DC 35) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL‐35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC 35, and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed‐type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC 35 was associated with the detection of CD25+ CD39+, rather than Foxp3+, regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC 35 appears to be an early event in IL‐35Ig–mediated immunosuppression.

Sustained cross-presentation capacity of murine splenic dendritic cell subsets in vivo.

An exclusive feature of dendritic cells (DCs) is their ability to cross‐present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen‐antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α+ DCs showed sustained MHC class I‐peptide specific CD8+ T‐cell activation for more than 4 days. CD8α− DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α− DCs were able to present antigen in MHC class II to specific CD4+ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T‐cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T‐cell activation and have distinct roles in antigen presentation to specific T cells in vivo.

Galectin-9 Produced by Intestinal Epithelial Cells Enhances Aldehyde Dehydrogenase Activity in Dendritic Cells in a PI3K- and p38-Dependent Manner

Intestinal epithelial cells (IEC) drive regulatory T cell (T_reg) responses by promoting the differentiation of aldehyde dehydrogenase (ALDH)-expressing CD103⁺ dendritic cells (DC). Apical stimulation of TLR9 by CpG DNA on IEC supports galectin-9 expression by IEC, which is promoted by short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (GF). While galectin-9 can induce the maturation of monocyte-derived DC (moDC), the contribution of galectin-9 on the induction of ALDH activity in DC is not known. To this end, DC were stimulated with galectin-9, and ALDH activity and the expression of CD103 were assessed. ALDH activity was increased by moDC exposed to galectin-9, while the expression of CD103 remained unaltered. Galectin-9 secreted by IEC apically exposed to CpG DNA and GF enhanced ALDH activity, but not CD103 expression by moDC, which was abrogated upon galectin-9 neutralization. Similar observations were found in murine GM-CSF-cultured bone marrow-derived DC (BMDC). Using Flt3L-cultured BMDC and ex vivo murine splenic DC, it was observed that galectin-9 only enhanced ALDH activity in the presence of GM-CSF in CD103^- cells. The induction of ALDH activity in BMDC was dependent on p38 and PI3K signaling. These data indicate a novel role for galectin-9 in modulating innate immunity by inducing ALDH activity in DC.

Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen.

Macrophages (MΦ) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and MΦs in immunity against Mycobacterium bovis Bacillus Calmette-Guérin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic MΦs compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in MΦs were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic MΦs were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic MΦs participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation.

CD8− Dendritic Cells and Macrophages Cross-Present Poly(D,L-lactate-co-glycolate) Acid Microsphere-Encapsulated Antigen In Vivo

The analysis of cell types involved in cross-priming of particulate Ag is essential to understand and improve immunotherapies using microparticles. In this study, we show that murine splenic dendritic cells (DCs) as well as macrophages (MΦs) are able to efficiently endocytose poly(D,L-lactate-co-glycolate) acid (PLGA) microspheres (MS) and to cross-present encapsulated Ags in the context of MHC class I molecules in vitro. A comparison of purified CD8(+) and CD8(-) DCs indicated that both DC subtypes are able to present OVA-derived epitopes on MHC class I and II in vitro. To determine the contribution of DCs and MΦs to cross-priming of PLGA MS in vivo, DCs were depleted in transgenic CD11c-DTR mice, and MΦs were depleted by clodronate liposomes in wild-type mice before immunizing mice with OVA-encapsulated MS. Our results show that the depletion of DCs or MΦs alone only led to minor differences in the OVA-specific immune responses. However, simultaneous depletion of DCs and MΦs caused a strong reduction of primed effector cells, indicating a redundancy of both cell populations for the priming of PLGA MS-encapsulated Ag. Finally, we analyzed PLGA MS trafficking to draining lymph nodes after s.c. injection. It was evident that fluorescent particles accumulated within draining lymph nodes over time. Further analysis of PLGA MS-positive lymphatic cells revealed that mainly CD8(-) DCs and MΦs contained MS. Moreover, immune responses in BATF3 knockout mice lacking CD8(+) DCs were normal. The results presented in this work strongly suggest that in vivo cross-priming of PLGA MS-encapsulated Ag is performed by CD8(-) DCs and MΦs.

CCR7-Dependent Stimulation of Survival in Dendritic Cells Involves Inhibition of GSK3β

Chemokine receptor CCR7 regulates chemotaxis and survival in mature dendritic cells (DCs). We studied the role of glycogen synthase kinase-3beta (GSK3beta) in the regulation of CCR7-dependent survival. We show that GSK3beta behaves as a proapoptotic regulator in cultured monocyte-derived human DCs and murine splenic DCs in vitro, and in lymph node DCs in vivo. In keeping with its prosurvival role, stimulation of CCR7 induced phosphorylation/inhibition of GSK3beta, which was mediated by the prosurvival regulator Akt1, but it was independent of ERK1/2, a key regulator of chemotaxis. Stimulation of CCR7 also induced translocation of two transcription-factor targets of Akt, prosurvival NF-kappaB and proapoptotic FOXO1, to the nucleus and cytosol, respectively, resulting in DCs with a phenotype more resistant to apoptotic stimuli. We analyzed if GSK3beta was able to modulate the mobilizations of these transcription factors. Using pharmacological inhibitors, small interfering RNA, and a construct encoding constitutively active GSK3beta, we show that active GSK3beta fosters and hampers the translocations to the nucleus of FOXO and NF-kappaB, respectively. Inhibition of GSK3beta resulted in the degradation of the NF-kappaB inhibitor IkappaB, indicating a mechanism whereby GSK3 can control the translocation of NF-kappaB to the nucleus. GSK3beta and FOXO interacted in vivo, suggesting that this transcription factor could be a substrate of GSK3. The results provide a novel mechanism whereby active GSK3beta contributes to regulate apoptosis in DCs. They also suggest that upon stimulation of CCR7, Akt-mediated phosphorylation/inhibition of GSK3beta may be required to allow complete translocations of FOXO and NF-kappaB that confer DCs an extended survival.

Characterisation of myeloid receptor expression and interferon alpha/beta production in murine plasmacytoid dendritic cells by flow cytomtery

This is a flow cytometric study of expression of a diverse set of myeloid receptors on murine splenic plasmacytoid dendritic cells (pDCs) and the description of a FACS based assay for measurement of interferon (IFN)α/β. We have extended the known repertoire of PRR expressed on murine pDCs with the novel observation that they express Dectin-2 and contain intracellular MR. In addition, this is the first report of F4/80 and CD200 on murine pDCs. We have confirmed the observation by others that murine pDCs express CD200R, the lectin Dectin-1 and the scavenger receptor CD36. This report also details a flow cytometry-based protocol to measure the production of murine IFNα/β by splenic pDC. Briefly, splenocytes can be stimulated with virus or a TLR9 agonist and IFNα/β production by pDCs is detected following intracellular staining. pDCs are specifically identified by 120G8 staining at 6 h after stimulation with inactivated influenza virus, however the specificity of 120G8 for pDCs is reduced at times later than 12 h. This assay is suitable for use with splenocytes from some mouse strains (129/SvEv), but not others (C57BL/6J), probably due to C57BL6J producing insufficient amounts of IFN following stimulation to be detected by intracellular staining. However, IFN production by C57BL/6J splenocytes is readily detectable by bioassay. In addition to being more sensitive than intracellular staining, the bioassay is also more sensitive than an IFNα ELISA. The comparable sensitivities of these assays are often a critical determinant of the choice of assay and are an important consideration in experimental design.

The stimulation of CD8 + T cells by dendritic cells pulsed with polyketal microparticles containing ion-paired protein antigen and poly(inosinic acid)–poly(cytidylic acid)

New adjuvants and delivery strategies are needed to optimize the ability of protein-based vaccines to elicit CD8 + T cell responses. We have developed a model vaccine formulation containing ovalbumin (OVA) and the double-stranded RNA analog poly(inosinic acid)–poly(cytidylic acid) (poly(I:C)), a TLR3 agonist. OVA and poly(I:C) were each ion-paired to cetyltrimethylammonium bromide (CTAB) to produce hydrophobic complexes, which were co-encapsulated in pH-sensitive polyketal (PK3) microparticles (1–3 μm) using a single emulsion method. Loading levels ranged from 13.6 to 18.8 μg/mg OVA and 4.8 to 10.3 μg/mg poly(I:C). Murine splenic dendritic cells (DCs) pulsed with PK3-OVA–poly(I:C) microparticles, at antigen doses of 0.01 and 0.1 μg/mL, induced a higher percentage of IFNγ-producing CD8 + T cells than DCs treated with PK3-OVA particles or soluble OVA/poly(I:C). A higher antigen dose (1 μg/mL) was less effective, which can be attributed to CTAB toxicity. At the lowest antigen dose (0.01 μg/mL), PK3-OVA–poly(I:C) microparticles also enhanced TNF-α and IL-2 production in CD8 + T cells. These data demonstrate the potential of polyketal microparticles in formulating effective CD8 + T cell-inducing vaccines comprising protein antigens and dsRNA adjuvants.

Anti-tumor activity of Acinetobacter baumannii outer membrane protein A on dendritic cell-based immunotherapy against murine melanoma

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a major surface protein that is an important pathogen-associated molecular pattern. Based on our previous findings that AbOmpA induced the phenotypic maturation of dendritic cells (DCs) and drove the Th1 immune response in vitro, we investigated the therapeutic efficacy of AbOmpA-pulsed DC vaccines in a murine melanoma model. The surface expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex class I and II molecules was higher in DCs pulsed with AbOmpA alone or with a combination of B16F10 cell lysates than that of DCs pulsed with B16F10 cell lysates. AbOmpA stimulated the maturation of murine splenic DCs in vivo. In a therapeutic model of murine melanoma, AbOmpA-pulsed DCs significantly retarded tumor growth and improved the survival of tumor-bearing mice. AbOmpA-pulsed DCs significantly enhanced CD8+, interleukin-2+ T cells and CD4+, interferon-gamma+ T cells in tumor-bearing mice. These results provide evidence that AbOmpA may be therapeutically useful in adjuvant DC immunotherapy against poorly immunogenic melanoma without tumor-specific antigens.

Isolation and characterization of dendritic cells from common marmosets for preclinical cell therapy studies

Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c(+) cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.

Novel Plant Virus-Based Vaccine Induces Protective Cytotoxic T-Lymphocyte-Mediated Antiviral Immunity through Dendritic Cell Maturation

Currently used vaccines protect mainly through the production of neutralizing antibodies. However, antibodies confer little or no protection for a majority of chronic viral infections that require active involvement of cytotoxic T lymphocytes (CTLs). Virus-like particles (VLPs) have been shown to be efficient inducers of cell-mediated immune responses, but administration of an adjuvant is generally required. We recently reported the generation of a novel VLP system exploiting the self-assembly property of the papaya mosaic virus (PapMV) coat protein. We show here that uptake of PapMV-like particles by murine splenic dendritic cells (DCs) in vivo leads to their maturation, suggesting that they possess intrinsic adjuvant-like properties. DCs pulsed with PapMV-like particles displaying the lymphocytic choriomeningitis virus (LCMV) p33 immunodominant CTL epitope (PapMV-p33) efficiently process and cross-present the viral epitope to p33-specific transgenic T cells. Importantly, the CTL epitope is also properly processed and presented in vivo, since immunization of p33-specific T-cell receptor transgenic mice with PapMV-p33 induces the activation of large numbers of specific CTLs. C57BL/6 mice immunized with PapMV-p33 VLPs in the absence of adjuvant develop p33-specific effector CTLs that rapidly expand following LCMV challenge and protect vaccinated mice against LCMV infection in a dose-dependent manner. These results demonstrate the efficiency of this novel plant virus-based vaccination platform in inducing DC maturation leading to protective CTL responses.

B220− murine splenic natural killer dendritic cells (NKDC) can acquire MHC II expression and become potent stimulators of T cells (36.14)

Abstract Introduction: NKDC (CD3−NK1.1+CD11c+B220+/−) are novel immune cells with characteristics of NK cells and DC. As NKDC exhibit broad cell surface expression of B220, we explored the functional and phenotypic differences between B220+ and B220− NKDC. Methods: Phenotype analysis was performed on wild-type (WT) and Flt3L expanded B6 splenic NKDC. WT NKDC were FACS purified based on B220 expression and used in various assays. T cell activity was measured by mixed leukocyte reaction (MLR). Supernatant IFN-γ levels were measured with a cytometric bead array. TLR9 ligand CpG (ODN 1826) and IL-18 were added to some wells. NK cells and DC were used as controls. Results: B220+ NKDC accounted for 33% of total NKDC, were larger in size, and expressed a higher level of MHC II by flow cytometry. After 24 hr culture in media alone, however, B220− NKDC gained high expression of MHC II and other co-stimulatory molecules (MHC I, CD40, CD80, CD86). Additionally, Flt3L expansion, which activates NKDC, led to a similar high level expression of MHC II on B220+ and B220− NKDC. When cultured in IL-18 and CpG for 72 hr, B220+ NKDC secreted higher levels of IFN-γ (73.3±7.4 vs. 50.1±4.1 ng/ml). Compared to B220+ NKDC, B220− NKDC caused lower allostimulation of T cells in an MLR. However, 2 hr activation with CpG prior to culture lead to an increase (and equalization) in allostimulatory capacity of both NKDC subtypes. Conclusion: B220− NKDC are an immature subtype of NKDC that possess the capacity to gain MHC II expression and become potent stimulators of allogeneic T cells.

Aging Effects on Dendritic Cells after Total Body Irradiation in Mice

Background: It is still obscure how dendritic cells (DCs) can orchestrate whole immune reactions according to the host age. We studied changes of murine splenic DCs after total body irradiation (TBI), with regards to age. Methods: Young (8∼14 wk) and old (12∼16 mo) C57Bl/6 mice were irradiated with a dose of 1,100 cGy and were assessed 6 h later for phenotypic and functional changes of the DCs. The mean fluorescence intensities and cytokine producing cell proportions were analyz ed with the student's t-test. Results: Interleukin-12 (IL-12), interferon (IFNγ) and tumor necrosis factor (TNFα) producing classical DCs (cDCs) were more numerous in the young untreated mice than in the old mice. However, the number of these cells decreased in the young mice and increased in the old mice after TBI. IL-12, IFNγ and TNFα producing plasmacytoid DCs (pDCs) were more frequent in the old mice than in the young mice before TBI both mice showed an increased frequency of cells producing these cytokines after TBI. Overall, the highest numbers of cDCs and pDCs producing IL-12, IFNγ and TNFα were present in the old mice after TBI. In both the cDC and pDC populations, the old mice had a higher frequency of IL-10+ cells prior to TBI. After irradiation, the young mice had a higher frequency of IL-10+ cells. Conclusion: With TBI, the DCs showed dramatic differences between young and old mice. Young mice turned to an immuno-suppressive response whereas the old mice changed to an immuno-stimulation of DCs after TBI. From these dramatic aging effects, we hope to explain the different frequencies and severities of acute GvHD after allogeneic hematopoietic stem cell transplantation according to host age.

The efficient isolation of murine splenic dendritic cells and their cytochemical features

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.

Cryptococcus neoformans Glycoantigens Are Captured by Multiple Lectin Receptors and Presented by Dendritic Cells

Cell-mediated immune responses to glycoantigens have been largely uncharacterized. Protective T cell responses to the pathogenic yeast Cryptococcus neoformans are dependent on heavily mannosylated Ags termed mannoproteins. In the work presented, the innate immune response to mannoprotein was determined. Purified murine splenic dendritic cells (DC), B cells, and macrophages were used to stimulate mannoprotein-specific T cells. Only DC were capable of any measurable stimulation. Depletion of DC resulted in the abrogation of the T cell response. Human and murine DC rapidly captured fluorescent-labeled mannoprotein by a mannose receptor-mediated process. Using transfected cell lines, the type II C-type lectin receptor DC-specific ICAM-3-grabbing nonintegrin (CD209) was determined to have affinity for mannoprotein. Taken together with prior work demonstrating that mannoprotein was captured by the macrophage mannose receptor (CD206), these data suggest that multiple mannose receptors on DC recognize mannoprotein. Pulsing experiments demonstrated that DC captured sufficient mannoprotein over 2 h to account for 50% of total stimulation. Capture appeared dependent on mannose receptors, as competitive mannosylated inhibitors and calcium chelators each interfered with T cell stimulation. By confocal microscopy, intracellular mannoprotein trafficked to an endo-lysosomal compartment in DC, and at later time points extended into tubules in a similar fashion to the degradation marker DQ-OVA. Mannoprotein colocalized intracellularly with CD206 and CD209. These data suggest that DC provide the crucial link between innate and adaptive immune responses to C. neoformans via a process that is dependent upon the efficient uptake of mannoprotein by mannose receptors.

Recombinant Cholera Toxin B Subunit Activates Dendritic Cells and Enhances Antitumor Immunity

Activation of dendritic cells (DC) is crucial for priming of cytotoxic T lymphocytes (CTL), which have a critical role in tumor immunity, and it is considered that adjuvants are necessary for activation of DC and for enhancement of cellular immunity. In this study, we examined an adjuvant capacity of recombinant cholera toxin B subunit (rCTB), which is non-toxic subunit of cholera toxin, on maturation of murine splenic DC. After the in vitro incubation of DC with rCTB, the expression of MHC class II and B7-2 on DC was upregulated and the secretion of IL-12 from DC was enhanced. In addition, larger DC with longer dendrites were observed. These data suggest that rCTB induced DC maturation. Subsequently, we examined the induction of tumor immunity by rCTB-treated DC by employing Meth A tumor cells in mice. Pretreatment with subcutaneous injection of rCTB-treated DC pulsed with Meth A tumor lysate inhibited the growth of the tumor cells depending on the number of DC. Moreover, intratumoral injection of rCTB-treated DC pulsed with tumor lysate had therapeutic effect against established Meth A tumor. Immunization with DC activated by rCTB and the tumor lysate increased number of CTL precursor recognizing Meth A tumor. The antitumor immune response was significantly inhibited in CD8+ T cell-depleted mice, although substantial antitumor effect was observed in CD4+ T cell-depleted mice. These results indicated that rCTB acts as an adjuvant to enhance antitumor immunity through DC maturation and that CD8+ T cells play a dominant role in the tumor immunity. Being considered to be safe, rCTB may be useful as an effective adjuvant to raise immunity for a tumor in clinical application.

Human papillomavirus type-16 virus-like particles activate complementary defense responses in key dendritic cell subpopulations.

Human papillomavirus type-16 (HPV16) L1 virus-like particles (VLPs) activate dendritic cells (DCs) and induce protective immunity. In this study, we demonstrate, using global gene expression analysis, that HPV16 VLPs produce quite distinct innate responses in murine splenic DC subpopulations. While HPV16 VLPs increase transcription of IFN-gamma and numerous Th1-related cytokines and chemokines in CD8alpha(+)CD11c(+) DCs, CD4(+)CD11c(+) DCs up-regulate only type I IFN and a different set of Th2-associated cytokines and chemokines. Type I IFN, but not IFN-gamma, potentiates humoral immunity, notably production of VLP-specific IgG2a. However, HPV16 VLP-stimulated IL-12 production by CD8alpha(+)CD11c(+) DCs is augmented by autocrine IFN-gamma signaling. Thus, before adaptive immunity, HPV16 VLPs signal complementary defense responses in key DC subpopulations, indicating specialized DC lineages with predetermined polarization.

Measles virus interacts with human SLAM receptor on dendritic cells to cause immunosuppression

Measles virus (MV) infects dendritic cells (DCs) resulting in immunosuppression. Human DCs express two MV receptors: CD46 and human signaling lymphocyte activation molecule (hSLAM); thus, the role played by either alone is unclear. Because wild-type (wt) MV uses hSLAM receptor preferentially, we dissected the molecular basis of MV–DC interaction and resultant immunosuppression through the hSLAM receptor by creating transgenic (tg) mice expressing hSLAM on DCs. After infection with wt MV, murine splenic DCs expressing hSLAM receptor had less B7-1, B7-2, CD40, MHC class I, and MHC class II molecules on their surfaces and displayed an increased rate of apoptosis when compared to uninfected DCs. Further, MV-infected DCs failed to stimulate allogeneic T cells and inhibited mitogen-dependent T-cell proliferation. Individual expression of human SLAM, interferon α/β receptor, tumor necrosis factor-α, and lymphotoxin-α or β from T cells was not required for MV-infected DCs to inhibit the proliferation of T cells.

Morphological and cytochemical characteristics of purified murine splenic dendritic cells.

Dendritic cells function as the main cellular population responsible for professional antigen presentation and hence for induction of primary immune responses. Although they are present in virtually every tissue, nevertheless their number is usually so low that it makes their isolation for studies very difficult. In this study, we purified dendritic cells from mouse spleen by a three-step enrichment method and evaluated morphological and cytochemical characteristics of isolated cells. We showed that isolated dendritic cells from mouse spleen had all lobulated nuclei with multiple cytoplasmic projections and their morphological features changed after an overnight incubation. It was also shown that typical dendritic cells lacked both Myeloperoxidase (MPO) and Non Specific Esterase (NSE) activity. In conclusion, for reaching a reasonable purity in isolation of dendritic cells from lymphoid tissues, many enrichment steps should be taken, and for determining the purity of isolated cells, we recommend that a combination of morphological and cytochemical studies be used.

Prostaglandin D2 inhibits the production of interleukin-12 in murine dendritic cells through multiple signaling pathways.

Prostaglandin (PG) D(2), and its metabolites, are known to be important mediators during acute and chronic inflammation. However, their functions during the early phases of the immune response are poorly documented. In the present study, we show that PGD(2 )inhibits, in a dose-dependent manner, the CD40- and LPS-induced secretion of the Th1-driving factor IL-12 by murine splenic dendritic cells (DC), the most potent antigen-presenting cells. The inhibition of IL-12 production is mediated only in part by the cell surface G alpha s protein-coupled D prostanoid receptor (termed DP1) but not by the G alpha i protein-coupled DP receptor, DP2. We show that recruitment of DP1 in DC results in the activation of a cyclic AMP/protein kinase A pathway that is partially responsible for the inhibition of IL-12 production. We also suggest that the DP1-independent effects exerted by PGD(2) on IL-12 production may be due to the action of ist PGJ(2), but not PGF(2)alpha, metabolites. Electrophoretic mobility shift assays demonstrated that PGD(2) affects NF-kappa B activation through (the) DP1-independent pathway(s). Together these data suggest that PGD(2), by interacting with DP1 and by binding to other target cellular proteins, may regulate immune responses by affecting IL-12 production in DC.