Analysis of the activation profile of dendritic cells derived from the bone marrow of interleukin-12/interleukin-23-deficient mice

We have previously shown that macrophages from interleukin (IL)-12p40 gene knockout (IL-12/IL-23-/-) mice have a bias towards the M2 activation profile, spontaneously secreting large quantities of transforming growth factor-beta1 (TGF-beta1) and producing low levels of nitric oxide (NO) in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). To verify whether the activation profile of dendritic cells (DCs) is also influenced by the absence of IL-12/IL-23, bone marrow-derived DCs from IL-12/IL-23-/- and C57BL/6 mice were evaluated. At first we noticed that approximately 50% of the C57BL/6 DCs were dead after LPS-induced maturation, whereas the mortality of IL-12/IL-23-/- DCs was < 10%, a protective effect that diminished when recombinant IL-12 (rIL-12) was added during maturation. Similarly to macrophages, mature IL-12/IL-23-/- DCs (mDCs) produced higher levels of TGF-beta1 and lower levels of NO than C57BL/6 mDCs. NO release was IFN-gamma-dependent, as evidenced by the poor response of IFN-gamma-/- and IL-12/IL-23-/-IFN-gamma-/- mDCs. Nevertheless, IFN-gamma deficiency was not the sole reason for the weak NO response observed in the absence of IL-12/IL-23. The high level of TGF-beta1 secretion by IL-12/IL-23-/- mDCs could explain why exogenous IFN-gamma partially restored the NO production of IFN-gamma-/- mDCs, while IL-12/IL-23-/- IFN-gamma-/- mDCs remained unresponsive. We also showed that CD4+ T-cell proliferation was inhibited by C57BL/6 mDCs, but not by IL-12/IL-23-/- mDCs. IFN-gamma and NO appear to mediate this antiproliferative effect because this effect was not observed in the presence of mDCs from IFN-gamma-/- or IL-12/IL-23-/- IFN-gamma-/- mice and it was attenuated by aminoguanidine. We conclude that the presence of IL-12/IL-23 during LPS-induced maturation influences the activation profile of DCs by a mechanism that is, only in part, IFN-gamma dependent.     

Inhibition of human allergic T-helper type 2 immune responses by induced regulatory T cells requires the combination of interleukin-10-treated dendritic cells and transforming growth factor-β for their induction

BACKGROUND: In grass pollen-allergic individuals, T cell anergy can be induced by IL-10-treated dendritic cells (IL-10-DC) resulting in the suppression of T helper type 1 (Th1) as well as Th2 cells. This study was performed to analyse whether such IL-10-DC-treated T cells are able to act as regulatory T cells (Treg) suppressing the function of other T cells in the periphery. As transforming growth factor (TGF)-beta is also a potential inducer of Treg, we additionally analysed the inhibitory capacity of TGF-beta-treated T cells in this system. MATERIALS AND METHODS: Freshly isolated CD4+ or CD4+ CD25- T cells from grass pollen-allergic donors were stimulated with autologous mature monocyte-derived allergen-pulsed DC in the presence or absence of T cells previously cultured with IL-10-DC- and/or TGF-beta. RESULTS: Anergic T cells induced by allergen-pulsed IL-10-treated DC or allergen-pulsed DC and TGF-beta enhanced IL-10 production and strongly inhibited IFN-gamma production of freshly prepared peripheral CD4+ or CD4+ CD25- T cells while proliferation and Th2 cytokine production were only slightly reduced. The combination of allergen-pulsed IL-10-treated DC and TGF-beta had an additional effect leading to a significant suppression also of Th2 cytokine production and proliferation. Suppression was not antigen-specific and was mainly mediated by cell-to-cell contact and by the molecule-programmed death-1 and only partially by CTLA-4, TGF-beta and IL-10. CONCLUSION: These data demonstrate that regulatory T cells that also suppress Th2 cytokine production are induced by two signals: TGF-beta and IL-10-DC. This is of importance for the regulation of allergic immune responses and might be exploited for future therapeutic strategies for allergic diseases.     

Impaired maturation and function of dendritic cells by mycobacteria through IL-1beta

Dendritic cells (DC) are pivotal for initiation and regulation of innate and adaptive immune responses evoked by vaccination and natural infection. After infection, mycobacterial pathogens first encounter monocytes, which produce pro-inflammatory cytokines, including IL-1beta, TNF-alpha and IL-6. The role of these cytokines in DC maturation remains incompletely understood. Here, we show that maturation of DC from monocytes was impaired by pretreatment of monocytes with low doses of IL-1beta. Under these conditions, Mycobacterium leprae-infected DC failed to stimulate antigen-specific T cell responses. Expression of CD86 and CD83 and production of IL-12 in response to lipopolysaccharide and peptidoglycan were diminished. In contrast, these DC functions were not impaired by pretreatment with TNF-alpha, IL-6 or IL-10. When monocytes were infected with M. bovis Bacillus Calmette-Guérin, and subsequently differentiated to DC, the activity of these DC was suppressed as well. Thus, IL-1beta acts at early stages of differentiation of DC and impairs biological functions of DC at later stages. Therefore, production of IL-1beta by mycobacteria-infected antigen-presenting cells counteracts effective stimulation of innate and adaptive immune responses.     

IL-2 production by dendritic cells is not critical for the activation of cognate and innate effectors in draining lymph nodes

Dendritic cells (DC) are unique antigen-presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL-2 production by mouse DC. Previous reports demonstrated that IL-2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC-derived IL-2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone marrow-derived DC results in early IL-2 production unless IL-4 is introduced in DC cultures. Here we show that IL-2 produced by LPS-activated DC is dispensable for cognate T cell responses since IL-2 loss of function DC elicit OVA-specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL-2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL-2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.     

Interleukin-15 production by monocyte-derived dendritic cells and T cell proliferation in HIV-infected patients with discordant response to highly active antiretroviral therapy

A discordant response to highly active antiretroviral therapy (HAART) occurs when CD4 T cell counts are stable or increased over time despite persistently detectable HIV-RNA levels. In order to identify immunological factors affecting discordant treatment responses, a total of 27 HIV-infected patients were studied: (a) 10 naive patients (mean CD4+ = 101.5 cells/microl; mean HIV-RNA = 4.8 log10 copies/ml); (b) seven responder patients (mean CD4+ = 908.9 cells/microl); and (c) 10 discordant patients (mean CD4+ = 396.1 cells/microl; mean HIV-RNA = 5.4 log10 copies/ml). Five healthy blood donors were included as HIV-seronegative controls. The following parameters were evaluated: interleukin (IL)-15 production by monocyte-derived dendritic cells (MDDC) after stimulation with lypopolysaccaride (LPS) and Candida albicans; recall and HIV-1-specific antigen lymphocyte proliferation (LP). Increased levels of IL-15 production by MDDC after stimulation with LPS and C. albicans were found both in discordant patients and responder patients. Conversely, a strong reduction of IL-15 levels was observed in naive patients. Discordant patients developed positive LP responses to C. albicans and HIV-1 p24. LP in response to C. albicans and HIV-1 p24 was also positive in responder patients. Decreased LP response was found in naive patients. In conclusion, HIV-infected patients with discordant viro-immunological responses to HAART present increased levels of IL-15 production by MDDC and enhanced recall and HIV-1-specific antigen LP responses, suggesting an improvement in indices of immune function.     

Human dendritic cells transfected with allergen-DNA stimulate specific immunoglobulin G4 but not specific immunoglobulin E production of autologous B cells from atopic individuals in vitro

Atopic/allergic diseases are characterized by T helper 2 (Th2)-dominated immune responses resulting in immunoglobulin E (IgE) production. DNA-based immunotherapies have been shown to shift the immune response towards Th1 in animal models. In further studies we showed that human dendritic cells (DC) transfected with allergen-DNA are able to stimulate autologous CD4(+) T cells from atopic individuals to produce Th1 instead of Th2 cytokines and to activate interferon-gamma (IFN-gamma)-producing CD8(+) T cells. The aim of this study was to analyse whether DC transfected with allergen-DNA are also able to influence immunoglobulin production of B cells from atopic donors. For this purpose, human monocyte-derived DC from grass-pollen allergic donors were transfected with an adenovirus encoding the allergen Phleum pratense 1 and cocultured with B cells, autologous CD4(+) T cells, and CD40 ligand-transfected L-cells. B cells receiving help from CD4(+) T cells stimulated with allergen-transfected dendritic cells produced more allergen-specific IgG4 compared to stimulation with allergen protein pulsed DC or medium, while total IgG4 production was not affected. In contrast, specific IgE production was not enhanced by stimulation with allergen-DNA transfected DC compared to medium and inhibited compared to allergen protein-pulsed DC with similar effects on total IgE production in vitro. Allergen-DNA transfected dendritic cells are able to direct the human allergic immune response from Th2-dominance towards Th1 and Tc1 also resulting in decreased IgE and increased IgG4 production.     

Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-γ production by T helper 1 cells

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-γ (IFN-γ), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell-mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, Northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-γ bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 bymurine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral blood mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-γ production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.     

Deregulated cytokine network and defective Th1 immune response in multiple myeloma

Intracellular cytokine production by peripheral blood mononuclear cells (PBMC) was analysed in 51 patients with multiple myeloma (MM), 22 with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy subjects, as a parameter of immunological dysfunction in MM. An increased proportion of T cells and HLA-DR+ cells producing IL-6 was observed in MM patients with active disease (at diagnosis and relapsing) compared with patients in remission and with MGUS, whereas no difference of IFN-gamma+, IL-2+ PBMC between patients and controls was evident. Determination of serum cytokine levels demonstrated that the imbalanced IL-6 production by T cells and the defective anti-tumour Th1 cell activity were related to elevated levels of IL-6 and IL-12. In vitro studies of PHA- and anti-CD3/anti-CD28 MoAbs stimulation of PBMC demonstrated the ability of lymphocytes from MM patients to differentiate towards the Th1 subset in the presence of rIL-12. By contrast, addition of exogenous rIL-6 impaired IFN-gamma production by rIL-12-prompted T cells. Inhibition of Th1 polarization of the immune response by IL-6 was direct on T cells and not mediated by dendritic cells (DC). Evaluation of the ability of MM-derived DC to stimulate cell proliferation of allogenic T lymphocytes and produce IL-12 in vitro, in fact, suggested that MM-derived DC were functionally active. Taken as a whole, these results indicate that a deregulated cytokine network occurs in active MM. They also suggest that increased IL-6 production by peripheral T lymphocytes contributes to the immune dysfunction observed in MM, and enables tumour cells to escape immune surveillance by preventing the anti-tumour Th1 immune response.     

Levamisole enhances immune response by affecting the activation and maturation of human monocyte-derived dendritic cells

The primary aim of this study was to evaluate the role of natural killer (NK) cells on antigen-specific adaptive immune responses. After analysing the mechanism of impaired adaptive immune responses of NK-depleted mice, an immune interventional approach was developed to restore adaptive immunity in NK-depleted mice. NK cells were depleted from mice by administration of anti-asialo GM1 antibody (100 mul/mouse), twice, at an interval of 48 h. Hepatitis B surface antigen (HBsAg) was administered intraperitoneally to normal C57BL/6 mice (control mice) and NK-depleted mice. The levels of antibody to HBsAg (anti-HBs) in the sera and HBsAg-specific lymphocytes in the spleen were assessed. The functions of T lymphocytes, B lymphocytes and dendritic cells (DCs) were evaluated in vitro. HBsAg-pulsed DCs were prepared by culturing spleen DCs with HBsAg for 48 h and administered once to NK-depleted mice. The levels of anti-HBs in the sera and HBsAg-specific lymphocytes were significantly lower in NK-depleted mice compared with control mice (P < 0.05). The functions of T and B lymphocytes were similar between control mice and NK-depleted mice. However, the functions of spleen DC and liver DC were significantly lower in NK-depleted mice compared with control mice (P < 0.05). Administration of HBsAg-pulsed DCs, but not HBsAg, induced HBsAg-specific humoral and cellular immune responses in NK-depleted mice. Our study suggests that cross-talk between NK cells and DCs regulates the magnitude of adaptive immunity. In addition, antigen-pulsed immunogenic DCs represent potent immune modulator even if subjects with diminished innate immunity.     

负载滋养层细胞抗原对小鼠树突状细胞表型及功能的影响

目的 研究负载滋养层细胞抗原对小鼠髓源性树突状细胞(DC)分化成熟过程的影响,获得致耐受性DC.方法 体外使用粒细胞巨细胞集落刺激因子(GM-CSF)诱导小鼠骨髓细胞定向分化、经LPS刺激获得成熟DC;通过外胎盘锥组织块培养法获得滋养层细胞,制备可溶性抗原,加入DC培养体系.流式细胞术检测DC表面共刺激分子及MHC-Ⅱ的表达,ELISA法检测DC分泌IL-10和IL-12的浓度,混合淋巴细胞培养评估 DC刺激同种T细胞增殖、活化的功能.结果 成熟DC表型为CD40high CD80highCD86highMHC-Ⅱhigh,分泌大量的IL-12和极少量的IL-10 ,体外能有效刺激T细胞的增殖;负载滋养层细胞抗原的DC表型为CD40midCD80lo wCD86lowMHC-Ⅱlow,在分泌大量IL-12的同时IL-10也明显升高,不能有效刺激T细胞增殖,并使T细胞分泌细胞因子呈现明显Th2偏倚.结论 负载滋养层细胞抗原后的DC表面共刺激分子及MHC-Ⅱ表达降低,刺激T细胞增殖能力下降;其自分泌和促使T细胞旁分泌的细胞因子呈现Th2偏倚,是一种耐受性DC.     

Type-1 polarized nature of mouse liver CD8alpha- and CD8alpha+ dendritic cells: tissue-dependent differences offset CD8alpha-related dendritic cell heterogeneity

Interleukin-12 p70 (IL-12p70) is a major dendritic cell (DC)-produced cytokine known to support type-1 T helper (Th1) cells and inflammatory-type immunity. While the ability of DC to produce bioactive IL-12p70 depends on both the DC subtype and the microenvironmental conditions of DC development, the relative contribution of each of these factors remains unclear. Here, we report that in contrast to spleen CD8alpha+ and CD8alpha- DC that show strong differences in their respective IL-12p70-producing capacities, CD8alpha+ and CD8alpha- DC isolated from the liver, a non-lymphoid organ, both efficiently produce IL-12p70 in amounts comparable to spleen CD8alpha+ DC. The IL-12p70-producing capacity CD8alpha+ and CD8alpha- DC from either location is greatly increased following their overnight culture in the presence of granulocyte-macrophage colony-stimulating factor. The elevated production of IL-12p70 by short-term cultured DC correlates with their enhanced expression of CD40 and other costimulatory molecules, and elevated T cell-stimulatory capacity. These data indicate that low IL-12-producing capacity is not an intrinsic property of the CDalpha8- DC subtype, and support the hypothesis that factors such as the site of DC development and maturation stage play a dominant role in defining DC function.     

Fetal BM‐derived mesenchymal stem cells promote the expansion of human Th17 cells,but inhibit the production of Th1 cells

Th type 17 (Th17) cells have been identified as a proinflammatory T‐cell subset. Here, we investigated the regulation of human Th17 cells by fetal BM‐derived mesenchymal stem cells (FBM‐MSC). We cocultured FBM‐MSC with human PBMC or CD4⁺ T cells from healthy donors. FBM‐MSC significantly suppressed the proliferation of CD4⁺ T cells stimulated by PHA and recombinant IL‐2. Significantly higher levels of IL‐17 were observed in FBM‐MSC cocultured with either PBMC or CD4⁺ T cells than that in PBMC cultured alone or CD4⁺ T cells cultured alone. Flow cytometry analysis showed that the percentage of Th17 cells in coculture of FBM‐MSC and CD4⁺ T cells was significantly higher than that in CD4⁺ T‐cell cultured alone. FBM‐MSC did not express IL‐17 protein. Consistent with the augmentation of Th17 cells, significantly higher levels of IL‐6 and IL‐1 were observed in coculture of FBM‐MSC and CD4⁺ T cells than that in CD4⁺ T‐cell culture, while the levels of IL‐23 were similar between FBM‐MSC + PBMC coculture and PBMC alone, or FBM‐MSC + CD4⁺ T‐cell and CD4⁺ T‐cell alone. The presence of FBM‐MSC decreased the percentage of Th1 cells, but minimally affected the expansion of CD4⁺CD25⁺ T cells. In conclusion, our data demonstrate for the first time that FBM‐MSC promote the expansion of Th17 cells and decrease IFN‐γ‐producing Th1 cells. These data suggest that IL‐6 and IL‐1, instead of IL‐23, may be partly involved in the expansion of Th17 cells.     

Selective maturation of dendritic cells by Nippostrongylus brasiliensis-secreted proteins drives Th2 immune responses

Helminth infections at mucosal and tissue sites strongly polarize towards Th2 immune responses, following pathways which have yet to be elucidated. We investigated whether dendritic cells (DC) exposed to gastrointestinal nematodes induce Th2 differentiation and, if so, whether this outcome reflects the absence of DC activation (the default hypothesis). We studied secreted proteins from the parasite Nippostrongylus brasiliensis, which induce Th2 development in vivo without live infection. Murine bone marrow-derived DC pulsed with N. brasiliensis excretory/secretory antigen (NES) can, on transfer to naive recipients, prime mice for Th2 responsiveness. Heat inactivation of NES abolishes both its ability to drive Th2 responses in vivo and its capacity to stimulate DC for Th2 induction. NES, but not heat-inactivated NES, up-regulates DC maturation markers associated with Th2 promotion (CD86 and OX40L), with little change to CD80 and MHC class II. Moreover, DC exposed to NES readily produce IL-6 and IL-12p40, but not IL-12p70. LPS induced high IL-12p70 levels, except in DC that had been pre-incubated with NES. These data contradict the default hypothesis, demonstrating that a helminth product (NES) actively matures DC, selectively up-regulating CD86 and OX40L together with IL-6 production, while blocking IL-12p70 responsiveness in a manner consistent with Th2 generation in vivo.     

Effect of interleukin-10 on dendritic cell maturation and function

The main function of dendritic cells (DC) is to induce the differentiation of naive T lymphocytes into helper cells producing a large array of lymphokines, including interleukin (IL)-2; interferon-γ (IFN-γ), IL-4, IL-5 and IL-10. The potent immunostimulatory properties of DC develop during a process of maturation that occurs spontaneously in vitro. Since IL-10 has been shown to inhibit Th1 responses, we determined its effect on DC maturation and accessory function. Our data show that DC that have undergone maturation in vitro in the presence of IL-10, have an impaired capacity to induce a Th1-type response in vivo, leading to the development of Th2 lymphocytes. Their inability to promote the synthesis of IFN-γ seems to correlate with a decreased production of IL-12, an heterodimeric cytokine necessary for optimal generation of Th1-type cells. These results suggest that IL-10 skews the Th1/Th2 balance to Th2 in vivo by selectively blocking IL-12 synthesis by the antigen-presenting cells that play a role of adjuvant of the primary immune response. The cytokines present in the environment at the presentation step may, therefore, determine the class of the immune response induced by DC in vivo, i.e. Th0 Th1 and/or Th2.     

The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level

BACKGROUND: The proteolytic activity of the house dust mite allergen Der p 1 has recently been shown to bias Th cell subset development in favour of Th2. Apart from its direct effect on T cells, it is conceivable that the proteolytic activity of Der p 1 may induce the generation of dendritic cells (DCs) that favour a Th2 response. OBJECTIVE: To study the effect of the proteolytic activity of Der p 1 on DC functions; namely cell surface phenotype, IL-12 production and ability to favour a Th2 response. METHODS: We have generated immature DCs from peripheral blood monocytes, matured them with LPS in the presence of either proteolytically active or inactive Der p 1 and compared their functions using flow cytometric analysis. RESULTS: Here we demonstrate for the first time that DCs that have been matured in the presence of proteolytically active Der p 1 produce significantly less IL-12, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. The suppression of IL-12 production was due to the cleavage of CD40 by the proteolytic activity of Der p 1, hence rendering the DCs less responsive to stimulation through the CD40L-CD40 pathway. Furthermore, we demonstrate that DCs that have been matured in the presence of proteolytically active Der p 1 induce the production of significantly less IFN-gamma and more IL-4 by CD4 T cells, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. CONCLUSIONS: Collectively, our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in directing DCs to induce Th2 subset development.     

IL‐12‐polarized Th1 cells produce GM‐CSF and induce EAE independent of IL‐23

CD4⁺ T‐helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL‐23 is universally required for the acquisition of pathogenic properties by myelin‐reactive T cells. We show that IL‐12‐modulated Th1 cells readily produce IFN‐γ and GM‐CSF in the CNS of mice and induce a severe form of EAE via an IL‐23‐independent pathway. Th1‐mediated EAE is characterized by monocyte‐rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL‐23‐modulated, stable Th17 cells induce EAE with a relatively mild course via an IL‐12‐independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4⁺ T‐helper‐cell subsets and polarizing factors.     

Questioning the Th1/Th2 paradigm in reproduction: peripheral levels of IL-12 are down-regulated in miscarriage patients

PROBLEM: It has been postulated that a T helper (Th)1 response is associated with pregnancy failure, whereas a Th2 response contributes to pregnancy maintenance. However, this Thl/Th2 dichotomy has recently been hypothesized to be an oversimplification. To prove this novel hypothesis, we investigated the levels of the Th1-inducer cytokine interleukin (IL)-12 in immunocompetent cells of patients with normal pregnancies (NP) and spontaneous abortion (SA). METHODS: Presence of intracellular IL-12 was evaluated in CD8+ and CD56-blood and decidual lymphocytes as well as in monocytes and granulocytes by flow cytometry from NP and SA individuals. IL-12 serum levels were measured by enzyme-linked immunosorbent assay (ELISA). We further investigated the effect of recombinant human (rh) IL-12 on the production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha in peripheral leukocytes ex vivo. RESULTS: In patients suffering from SA we observed lower percentages of IL-12 in lymphocytes, monocytes and granulocytes derived from peripheral blood and decidua, compared with women with normally progressing pregnancies. No differences could be observed when evaluating the levels of IL-12 in the granulocyte population. The IL-12 serum levels were below the ELISA sensitivity limit. Ex vivo stimulation of the peripheral blood cells with increasing doses of IL-12 resulted in a significant decrease of IFN-gamma+, whereas levels of TNF-alpha+ in lymphocytes were unaffected. CONCLUSIONS: The classical Th1/Th2 paradigm appears to be insufficient to exclusively explain the causes of pregnancy loss. Our current results render us to requestion the role of Th1 cytokines during pregnancy and suggest some protective function of the Th1-inducer cytokine IL-12.     

Altered phenotype and function of blood dendritic cells in multiple sclerosis are modulated by IFN-beta and IL-10.

Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-gamma and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-gamma by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-beta and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-beta and IL-10 significantly suppressed IFN-gamma production by MNCs. IFN-beta in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-beta and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.