Targeted NF-?B Inhibition Of Asthmatic Serum-mediated Human Monocyte-derived Dendritic Cell Differentiation In A Transendothelial Trafficking Model

As professional antigen-presenting cells (APCs), dendritic cells(DCs) are not only considered to be crucial for initiating allergic airway inflammatory immune responses, but also thought to be intimately involved in the establishment and maintenance of peripheral tolerance. Various lines of evidence indicate that an inappropriate Th2-biased immune response to allergens is central to the pathogenesis of atopic disorders, however, an emerging concept is that allergic asthma in humans essentially represents a failure of immune tolerance to Ags that are common in the environment. The subtypes,the relative maturation state of DC and the local microenvironment are essential in delivering different stimulatory signals to T cell. Immature DC has been shown to have therapeutic value in models of allograft rejection and autoimmunity. Few reports regarding the ability of DC subsets to sucssessfuly prevent or treat asthma are available. The development, maturation, and function of DC are dependent on NF-?B. Since DC are rare (<1% of circulating blood mononuclearcells), we usually generate DC from monocytes under conditioned cytokine settings in vitro, such as interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). However, such high cytokine concentrations are pretty different from those in vivo. The available experimental DC development in vitro can not reflect the kinetics of monocyte-derived DC differentiation under physiologic conditions. Interestingly, it established that normal human monocyte developed into DC in the transendothelial trafficking model and serum from patients with systemic lupus erythematosus (SLE), rendering differentiation of normal monocytes into mature DC. Thus, we hypothesized that normal human monocyte could develope into DC in the transendothelial trafficking model and asthmatic serum would affect the phenotype and function of DC differentiated in the model. First, we constructed the transendothelial trafficking model. Subsequently, monocytes transfected with an adenoviral vector encoding novel truncated I?B?(203–1003 bp) were applied onto the monolayer of human umbilical vein endothelial cells (HUVECs) in the presence of asthmatic serum for 48 h. We determine whether AdI?B?M transfer could influence the differentiation and function of DC.Part?Asthmatic serum facilitaes human monocyte-derived dendritic cell differentiation in a model of transendothelial traffickingObjective To investigate the effects of asthmatic serum on the phenotype and function of human monocyte-derived DC in a model of transendothelial trafficking.Methods Normal healthy human monocytes were culured with normal serum or asthmatic serum respectively in a model of transendothelial trafficking for 48 h. The fresly isolated monocytes and monocytes cultured without HUVEC were used as controls. The phenotype, the ability to stimulate the proliferateion of allogeneic T cells, cytokine production of DC were determined by photomicrography, flow cytometry, mixed leukocyte reaction and ELISA respectivly. Asthmatic serum-induced NF-?B activation in DC was detected by Western-blot. Results After culture with normal human serum or asthmatic serum in the presence of HUVEC for 48 h, the reverse-transmigrated cells displayed characteristics of DC. CD14 was mostly lost whereas CD80, CD86, CD83 and HLA-DR were all up-regulated. These cells were large, with extended dendritic processes. Compared with normal serum-stimulated cells, the expression of CD80 and CD83 on asthmatic serum-stimulated DC were significantly increased(P<0.05, P<0.01). In contrast, there were no differences in the expression of CD86 and HLA-DR, and the proliferateion of allogeneic T cells between the cells of two groups( all P >0.05).The production of IL-12 p70 and IL-10 in supernatants were significantly increased in asthmatic serum-stimulated cells(P<0.05, P<0.01).In addition, asthmatic serum induced the nuclear translocation of NF-?B p65 in DC. Conclusion These data suggest that some monocytes become DC like cells in a model of transendothelial trafficking and asthmatic serum may induce the maturation of DC through selectively determining the phenotype and function of monocyte-derived DC.Part?Targeted NF-?B inhibition of asthmatic serum-mediated human monocyte-derived dendritic cell differentiation in a transendothelial trafficking modelObjective To evaluate whether adenoviral gene transfer of a novel mutated I?B?(AdI?B?M) would influence the differentiation and function of DC in an asthmatic serum-transendothelial trafficking model. Methods Monocytes, untransferred or transferred with AdI?B?M or AdLacZ, were applied onto the monolayer of human umbilical vein endothelial cells (HUVEC) in the presence and absence of asthmatic serum for 48 h. The expression of I?B?M gene was detected by Western-blot. The phenotype, cytokine production, the ability to stimulate and polarize allogeneic T cells of the reverse-transmigrated cells were determined by photomicrography, flow cytometry, ELISA, mixed leukocyte reaction and Western-blot, respectivly. Confocal microscopic analysis and electrophoretic mobility shift assay were used to detect NF-?B activity in reverse-transmigrated cells. Results I?B?M was detected in the reverse-transmigrated cells differentiated from AdI?B?M-transferred monocytes. AdLacZ or AdI?B?M-transferred monocytes also differentiated into DC in the transendothelial trafficking model because they displayed elaborate cellular processes consistent with DC. There were no differences in the expression of CD14. Moreover, AdI?B?M inhibited the expression of costimulatory molecules, decreased the IL-12 p70 production in DC, and reduced the capacity of DC to simulate allogeneic T cell proliferation accompanied by less Th1 and Th2 polarization. Conclusion These data suggest that monocytes transferred with I?B?M still differentiated into DC like cells in a model of transendothelial trafficking and AdI?B?M might pharmacologically modify the phenotype and function of DC which initiate commitment of naive Th cells toward Th1 or Th2 subsets.Part?Induction of CD4+CD25+ T regulatory cells in asthmatic patient by dendritic cells overexpressing I?B?MObjective To investigate the effect of I?B?M-overexpressed DC on the induction of CD4+CD25+ T regulatory cells in asthmatic patients in vitro. Methods?The CD4+ T cells of normal and asthmatic patients were isolated, and the percentage of CD4+CD25+ T regulatory cells in CD4+ T cells was calculated by flow cytometry. The asthmatic CD4+ T cells were cocultured with the same number of asthmatic and normal CD4+CD25+ T cells separately. The proliferation of CD4+ T cells was measured by MTT.?Monocytes, untransferred or transferred with AdI?B?M or AdLacZ, were applied onto the monolayer of human umbilical vein endothelial cells (HUVEC) in the presence and absence of asthmatic serum for 48 h. The asthmatic CD4+CD25- T cells were isolated and cocultured with DC for 72 h, the percentage of CD4+CD25+ T regulatory cells among the CD4+ T cells was analysed with flow cytometry. The levels of IL-10 and TGF-?1 in supernant of the medium were measured by ELISA. Results?The percentage of CD4+CD25+ T regulatory cells in blood were decreased in the asthmatic group. However, there was no significant difference in the effects of the same amount of asthmatic and normal CD4+CD25+ T regulatory cells on the proliferation of CD4+ T cells.?Compared with the untransferred and AdLacZ-transferred group, the DC overexpressing I?B?M could significantly induce the CD4+CD25-T cells to differentiate into CD4+CD25+T cells, increase IL-10 level in the supernatant, without influencing TGF-?1. Conclusions?The decreased percentage of CD4+CD25+ T regulatory cells in blood may contribute to the up-regulated Th2 response in asthma.?DC derived from I?B?M-transferred monocyte could induce the differentiation of the CD4+CD25+ T regulatory cells and increase the production of inhibitory cytokine IL-10 in asthmatic patient in vitro.