Activation of human neonatal monocyte-derived dendritic cells by lipopolysaccharide down-regulates birch allergen-induced Th2 differentiation

Epidemiological studies describe an inverse association between the level of environmental endotoxin exposure during infancy and the prevalence of allergic disease in children. To study the effect of lipopolysaccharide (LPS) and lipopeptide Pam3Cys signaling via Toll-like receptor (TLR)4 and TLR2 on dendritic cells (DC), respectively, on birch allergen-induced T cell differentiation, cord blood monocyte-derived DC were exposed to birch allergen extract alone or in combination with LPS or Pam3Cys and thereafter co-cultured with naive autologous T cells. We demonstrate that birch allergen alone induced high levels of IL-13 from neonatal T cells, whereas the production of IL-5 and IFN-gamma was modest. Stimulation of DC with birch allergen together with LPS but not Pam3Cys resulted in a decreased IL-13 production by T cells compared to birch allergen alone. Furthermore, birch allergen together with LPS induced increased up-regulation of activation markers expressed on the surface and production of cytokines from DC relative to stimulation with birch allergen alone. Finally, birch allergen partially suppressed both LPS- and Pam3Cys-induced DC maturation. Our results indicate that concomitant TLR4 stimulation during the initial phase of immune activation to birch allergen in infants may inhibit the development of a T helper 2-type response.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Mouse neutrophils are professional antigen-presenting cells programmed to instruct Th1 and Th17 T-cell differentiation

Neutrophils play a major role in the innate immune system and are normally considered to be short-lived effector cells that exert anti-microbial activity and sometimes immunopathology. Here, we show that these cells possess an additional function as professional antigen-presenting cells capable of priming a T(h)1- and T(h)17-acquired immune response. Using flow cytometry, fluorescence microscopy and western blotting, we show that mouse neutrophils express MHC class II and co-stimulatory molecules CD80 and CD86 after T-cell co-incubation. Neutrophils pulsed with ovalbumin (OVA) process and present peptide antigen to OVA-specific T cells in an MHC class II-dependent manner. Importantly, we demonstrate that neutrophils can prime antigen-specific T(h)1 and T(h)17 immune responses even without the addition of exogenous cytokines to cell cultures.     

CD14+ antigen-presenting cells in human dermis are less mature than their CD1a+ counterparts

We recently demonstrated that three antigen-presenting cell (APC) subsets exist in the healthy human dermis, CD14(+) and CD1a(+) dermal APCs and migratory dermal Langerhans cells. Here, we extend these findings by defining CD208 as an exclusive marker of migratory dermal Langerhans cells, confirming that migratory dermal Langerhans cells (CD1a(high) CD207(+) CD208(+)) and CD1a(+) dermal APCs (CD1a(mid) CD207(-) CD208(-)) are two distinct APC populations. Using flow cytometry and multicolor fluorescence immunohistochemistry, we demonstrated that there were striking differences between CD1a(+) and CD14(+) dermal APCs in their expression of pattern recognition receptors and maturation markers. Expression of Toll-like receptor (TLR) 2, CD206 and CD209 was largely restricted to CD14(+) dermal APCs. Consistent with these observations, most CD14(+) dermal APCs expressed an immature phenotype when compared with CD1a(+) dermal APCs, which expressed high levels of the maturation marker CD83 on their cell surface. However, a subset of CD14(+) dermal APCs also expressed cell-surface CD83, associated with a loss of cell-surface TLR2, suggesting that they have the capacity to mature. CD14(+) dermal APCs are therefore the dominant cutaneous APC population capable of sensing ligands recognized by CD206, CD209 and TLR2 and subsequently may have the potential to mature. CD68 expression was largely restricted to a subset of CD14(+) dermal APCs, while both CD14(+) and CD1a(+) dermal APCs expressed CD11b and CD11c. These findings have important implications for understanding cutaneous immune responses in humans and for the optimization of vaccine delivery via the skin.     

CD83 monocyte-derived dendritic cells are present in human decidua and progesterone induces their differentiation in vitro

PROBLEM: Dendritic cells (DCs) play an important role in antigen presentation and immunoregulation. Modifications of the immune response during pregnancy require the participation of DC. The aim of this study was to follow-up the changes of DCs in human decidua and their correlations to progesterone (Pg) concentrations. METHODS OF STUDY: Blood leukocytes were isolated from human decidua and analyzed by flow cytometry for the expression of HLA-DR and CD83 markers. Blood monocytes were cultured to differentiate and mature to DCs in the presence of Pg, estradiol or testosterone and analyzed by flow cytometry. RESULTS: The percentage of mature DCs is increased in human decidua during the 1st trimester. When Pg is added to DCs cultured in vitro the percentage of cells expressing both markers increased in specific and dose-dependent manner. CONCLUSIONS: Pg increases the numbers of monocytic DCs locally and it may be speculated that these cells help the Th1/Th2 switching in pregnancy.     

Mice vaccinated with allergen-pulsed myeloid dendritic cells are not protected from developing allergen-induced Th2 responses

BACKGROUND: Dendritic cells (DC) play a decisive role in the induction of allergen-induced Th1 and Th2 responses. Since the induction of allergen-specific Th1 responses has shown to inhibit allergen-induced Th2-type inflammation, in this study we investigated whether manipulated myeloid-derived DC pulsed with the specific allergen would predominantly induce allergen-specific Th1 responses thereby reducing the development of Th2 responses. METHODS: Murine bone marrow (BM)-DC were generated and pulsed with ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG-ODN). Langerhans cells (LC) were also isolated and pulsed in vitro with OVA. Subsequently, mice were vaccinated intravenously with either CpG/OVA-pulsed BM-DC or OVA-pulsed LC, and the protocol to induce OVA-specific Th2 responses using OVA/alum sensitization was initiated. Airway inflammation and OVA-specific serum antibody levels were evaluated 6 days after the intranasal challenge with OVA. RESULTS: The application ofCpG/OVA-pulsed BM-DC was unable to reduce airway eosinophilia and inflammation in OVA/alum-immunized mice. OVA-specific IgG1 or IgE serum levels were also not reduced. The experiments using LC pulsed with OVA yielded similar results. However, mice vaccinated with CpG/OVA-pulsed BM-DC had greatly enhanced levels of serum OVA-specific IgG2a, suggesting the induction of allergen-specific Th1 responses in vivo. Moreover, allergen-induced mast cell degranulation was decreased using this approach. CONCLUSIONS: Taken together, our results demonstrated that the vaccination with OVA-pulsed BM-DC matured with CpG-ODN or OVA-pulsed LC did not result in a reduction in allergen-specific Th2 responses in a murine model of severe atopic asthma. Other DC-based vaccination strategies should be evaluated in order to prevent the development of allergic disorders.     

The primary responses of murine neonatal lymph node CD4+ cells are Th2-skewed and are sufficient for the development of Th2-biased memory

Exposure of neonatal mice to antigen often results in Th2-biased responses in later life. Examples of this Th2 tendency are (a) secondary antibody responses dominated by the Th2-associated IgG1 isotype and (b) Th2-mediated tolerance to alloantigens. We previously reported that neonates develop primary Th1 and Th2 function in the lymph nodes but exclusive Th2 primary splenic responses. Here, we have tested whether the Th2 bias of adults initially immunized as neonates is due to the early, primary Th2 polarization in the spleen. Surprisingly, removal of the spleen at birth had no affect on either IgG1-dominant secondary responses or the development of tolerance to alloantigens. Thus, neonatal lymph nodes are sufficient to generate Th2-biased function following neonatal antigen exposure. To understand how this could arise, we examined the primary Th1/Th2 responses of CD4+ lymph node cells. Unlike the balanced Th1/Th2 responses seen with total lymph node cells, the primary responses of isolated CD4+ cells were skewed to IL-4 producing function. These results suggest that the early development of Th2-dominant responses by lymph node CD4+ cells contributes substantially to the subsequent development of Th2-dominant memory in neonates.     

Interleukin-10 enhances the CD14-dependent phagocytosis of bacteria and apoptotic cells by human monocytes

Monocytes are centrally involved in both specific and nonspecific immunity by secretion of regulatory immune mediators, phagocytosis, and presentation of antigens. Recent work has shown that monocytes can phagocytose bacteria independently from Fc gamma, complement, and scavenger receptors via a CD14-mediated process. Furthermore, incorporation of cells undergoing apoptosis is also mediated by CD14. In this study we investigated the regulation of monocytic CD14-dependent phagocytosis by the immunoregulatory cytokines interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1). In this study an in vitro human whole-blood assay was used to test regulation of CD14-dependent phagocytosis of fluorescence-labeled E. coli by IL-10, IFN-gamma, and TGF-beta1 in monocytes from healthy donors. Phagocytosis by monocytes from a patient with paroxysmal nocturnal hemoglobinuria (PNH) and its regulation by IL-10 was also investigated. Finally, regulation of monocytic incorporation of apoptotic Jurkat cells by IL-10 was analyzed. For the CD14 blockade, murine anti-CD14 IgG2a antibody RMO52 was used. We observed that IL-10, suggested to be a monocyte-deactivating cytokine, strongly increased the monocytic CD14-dependent phagocytosis of E. coli. In contrast, IFN-gamma and TGF-beta1 depressed monocytic CD14 incorporation of E. coli. Compatible with this, IL-10 upregulated CD14 expression on monocytes, whereas IFN-gamma and TGF-beta1 downregulated its expression. IL-10 also increased the monocytic CD14-dependent and -independent phagocytosis of apoptotic cells. As expected, IL-10 strongly increased the CD14-independent phagocytosis but had no influence on the CD14-dependent phagocytosis of monocytes from a PNH patient. In conclusion, our data support a general role of IL-10 for activating monocytic scavenger functions, which are at least partly mediated by CD14. This is in line with the fact that IL-10 promotes the development of monocytes to macrophages. The contrasting effects of IL-10 and IFN-gamma on monocytic CD14-dependent phagocytosis may reflect a further mechanism counterbalancing antigen-presentation and nonimmunogenic scavenging of bacterial and cellular debris. TGF-beta, however, may be an inhibitor of both systems.     

Soluble lipopolysaccharide receptor (CD14) is released via two different mechanisms from human monocytes and CD14 transfectants

The receptor for lipopolysaccharide LPS (CD14) exists in a membrane-associated (mCD14) and a soluble form (sCD14). Previous studies indicate that monocytes produce sCD14 by limited proteolysis of the membrane-bound receptor. In this study we demonstrate that human monocytes also produce sCD14 by a protease-independent mechanism. To investigate the molecular nature of this second pathway we studied sCD14 formation in the monocytic cell line Mono Mac 6 (MM6) and in CD14 transfectants. Both MM6 and the CD14 transfectants constitutively produce sCD14 by a protease-independent mechanism. Structural analysis of sCD14 produced by the CD14 transfectants reconfirmed the presence of the COOH terminus predicted from the cDNA. Since glycosylphosphatidyl-inositol anchor attachment is associated with the removal of a hydrophobic C-terminal signal peptide, our finding demonstrates that the transfectants secrete sCD14 which escaped this posttranslational modification. Identical results obtained for sCD14 derived from peritoneal dialysis fluid of a patient with kidney dysfunction show the in vivo relevance of this pathway for sCD14 production.     

Apoptotic cells selectively suppress the Th1 cytokine interferon gamma in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2

Apoptotic cells are readily recognized and engulfed by phagocytes and usually do not induce inflammation or tissue damage. Furthermore, they can actively suppress a pro-inflammatory response in phagocytes: In the presence of apoptotic cells, activated monocytes/macrophages produce more of the anti-inflammatory and immunoregulatory cytokines IL-10 and TGF-beta, but less of the pro-inflammatory cytokines TNFalpha, IL-1beta and IL-12. This immunoregulatory effect is most likely mediated by several receptors on monocytes/macrophages including the thrombospondin receptor (CD36). In addition to the modulation of cytokine secretion, apoptotic cell material inhibited the expression of MHC class II molecules on the surface of monocytes/macrophages. Decreased MHC II expression appeared to be mediated predominantly by increased IL-10 secretion in a para-/autocrine manner. Here, we show that the functional modulation of antigen-presenting monocytes/macrophages by apoptotic cells also influences T cell activation and function. When human peripheral blood mononuclear cells were stimulated with recall antigens in the presence of apoptotic cells, interferon gamma (IFN gamma) secretion was markedly suppressed, whereas secretion of the Th2 cytokine IL-4 was not significantly altered. Hence, apoptotic cells shift the T cell cytokine secretion pattern towards a Th2-like response. This Th2 shift can largely be prevented by neutralizing IL-10, indicating an important role of this cytokine for modulating T cell cytokine secretion patterns.     

CD31 (PECAM-1) is a differentiation antigen lost during human CD4 T-cell maturation into Th1 or Th2 effector cells.

The CD31 antigen (PECAM-1) has been reported to be a stable marker for a human CD4 T-cell subpopulation unable to produce interleukin-4 (IL-4). We show here that CD31 expression is not stable inasmuch as CD4 T-cell lines and clones derived from cell-sorted neonatal CD31+ cells lose CD31 upon repetitive cycles of stimulation and IL-2 expansion. Moreover, various cytokines (IL-1 alpha, IL-4, IL-6, transforming growth factor-beta) fail to reinduce CD31 on CD31- clones. Whereas all CD31+ CD4 T cells rapidly express high levels of the CD45RO antigen and down-regulate the L-selectin antigen after priming, CD31 disappears more slowly because only part of the cells lose CD31 expression upon each cycle of stimulation. Loss of CD31 reflects a functional maturation of CD45RO+ cells since, in a system which favours the development of Th2 effectors, IL-4 is produced by CD31- but not CD31+ effector T cells, whereas interferon-gamma is produced by both types of cells. However, CD31 is not a Th1 marker since it is not expressed on several Th1 antigen-specific clones. We conclude that CD31 is a maturation marker expressed on the great majority of naive CD45RO- CD4 T cells and on a subset of CD45RO+ CD4 T cells that are at an intermediate stage of maturation.     

Endogenous IL-4 and IFN-gamma are essential for expression of Th2, but not Th1 cytokine message during the early differentiation of human CD4+ T helper cells

CD4+ T cells can be divided into several distinct effector subpopulations, including Th1 and Th2. Human Th1 cells are essential for the establishment of cellular immune responses, whereas Th2 cells for immunoglobulin E synthesize by B cells and immunoregulation. This study determines the involvement of exogenously and endogenously produced T cell-derived cytokines during early differentiation of naive CD4+ T cells into Th1 and Th2 cells. Cytokine gene expression of purified experienced and naive CD4+T cells in the presence or absence of Th-directing cytokines and neutralizing anti-cytokine antibodies, was determined at early (20 and 40 h) time points, after in vitro activation. These studies demonstrated that: (1) endogenously produced, T cell-derived cytokines (interferon [IFN]-gamma and interleukin [IL]-4), play an important role in the regulation of early gene expression of Th2, but not Th1 type cytokines; (2) Th1-related cytokines, IFN-gamma, and IL-2, are preferentially expressed in cultures directed toward Th1, as compared with Th2; and (3) IL-4 and IFN-gamma showed early message expression in both differentiating populations, indicating a mixed profile of Th1 and Th2 cytokine production in early human Th cell development. These findings point to the critical role for endogenously produced cytokines in the early differentiation of human Th1 or Th2 cells.     

CD4+ Th2 cells are directly regulated by IL-10 during allergic airway inflammation

Interleukin-10 (IL-10) is an important regulatory cytokine required to control allergy and asthma. IL-10-mediated regulation of T cell-mediated responses was previously thought to occur indirectly via antigen-presenting cells. However, IL-10 can act directly on regulatory T cells and T helper type 17 (Th17) cells. In the context of allergy, it is therefore unclear whether IL-10 can directly regulate T helper type 2 (Th2) cells and whether this is an important regulatory axis during allergic responses. We sought to determine whether IL-10 signaling in CD4⁺ Th2 cells was an important mechanism of immune regulation during airway allergy. We demonstrate that IL-10 directly limits Th2 cell differentiation and survival in vitro and in vivo. Ablation of IL-10 signaling in Th2 cells led to enhanced Th2 cell survival and exacerbated pulmonary inflammation in a murine model of house dust mite allergy. Mechanistically, IL-10R signaling regulated the expression of several genes in Th2 cells, including granzyme B. Indeed, IL-10 increased granzyme B expression in Th2 cells and led to increased Th2 cell death, identifying an IL-10-regulated granzyme B axis in Th2 cells controlling Th2 cell survival. This study provides clear evidence that IL-10 exerts direct effects on Th2 cells, regulating the survival of Th2 cells and severity of Th2-mediated allergic airway inflammation.     

三种不同方法体外诱导CD4+T细胞向Th17细胞分化与增殖的效率

背景：研究认为Th17细胞参与了许多炎症性疾病的发生,但其体外诱导分化效率尚不明确。 目的：比较采用3种不同方法在体外诱导CD4⁺T细胞向Th17细胞分化与增殖的效率。 方法：分别采取以下3种方法体外诱导Th17细胞：①给予CD3及CD28抗体刺激,并在培养体系中加入白细胞介素6及转化生长因子β,培养3 d。②在方法①的基础上加入白细胞介素1β及肿瘤坏死因子α,培养3 d。③在方法②的基础上第3天洗去细胞因子,培养2 d;再次给予CD3及CD28抗体刺激,并在培养体系中加入白细胞介素23,培养3 d。 结果与结论：3种方法培养后CD4⁺T细胞中Th17细胞的比例分别为(8.5±2.8)%,(26.9±4.3)%,(44.3±5.5)%,三组之间差异有显著性意义(P < 0.01)。提示在培养体系中加入白细胞介素1β、肿瘤坏死因子α及白细胞介素23,更有利于增加CD4⁺T细胞向Th17细胞分化的比例。     

Enhanced T-cell activation and T-cell-dependent IL-2 production by CD83+, CD25high, CD43high human monocyte-derived dendritic cells

Although standardized protocols are widely used for the generation of monocyte-derived immunostimulatory dendritic cells (DC(ims)), the inducibility of Th1 cells by DC(ims) may considerably differ. As a measure for the quality of DC(ims) generated from an individual donor at a certain time point, CD83 is used in combination with HLA-DR and CD86 to assess DC maturation. When phenotypically analyzing DC(ims), we identified a subpopulation ( approximately 60%) of CD83+, CD86+, and HLA-DR+ DC(ims) that co-expressed CD25. DC within a given DC(ims) preparation identified by lower expression of CD83 and by selective expression of CD14, however, did not co-express CD25. In order to establish CD25 as an additional maturation marker of DC(ims), we studied the DC phenotype of these cells as well as the DC-dependent T-cell proliferation and T-cell cytokine production profile after co-incubation with sorted CD25(high) and CD25(low) subpopulations of CD83+, HLA-DR+, CD86+ DC(ims). CD25(high) DC(ims) showed significant up-regulation of the DC activation molecule CD43 and induced increased levels of IL-2 secretion in allogeneic T-cells (170.7+/-86.7pg/mL) as compared to T-cells coincubated with CD25(low) DC(ims) (86.6+/-37.6pg/mL) [p=0.0224]. This was reflected by a significantly lower T-cell stimulatory capacity of CD25(low) DC(ims) (84.0% of CD25(high) DC(ims), 1:10 ratio; p=0.014) whereas the T-cell stimulatory capacity of CD25(low) DC(ims) was much higher when compared to IL-10 induced regulatory DC (55.3% of CD25(high) DC(ims); 1:10 ratio). With regard to cancer vaccination protocols, we propose to use CD25 and CD43 as additional markers for DC quality control, assessment of maturational status, and positive selection.