CD40 (Phospho-Thr254) ELISA Kit

Cigarette smoke is a major effector of chronic obstructive pulmonary disease (COPD), and Th17 cells and dendritic cells (DCs) involve in the pathogenesis of COPD. Previous studies have demonstrated the anti-inflammatory effects of macrolides. However, the effects of macrolides on the cigarette smoke extract- (CSE-) induced immune response are unclear. Accordingly, in this study, we evaluated the effects of erythromycin (EM) on CSE-exposed DCs polarizing naive CD4(+) T cells into Th17 cells. DCs were generated from bone marrow-derived mononuclear cells isolated from male BALB/c mice and divided into five groups: control DC group, CSE-exposed DC group, CD40-antibody-blocked CSE-exposed DC group, and EM-treated CSE-exposed DC group. The function of polarizing CD4(+) T cells into Th17 cells induced by all four groups of DCs was assayed based on the mixed lymphocyte reaction (MLR) of naive CD4(+) T cells. CD40 expression in DCs in the CSE-exposed group increased significantly compared with that in the control group (P<0.05). The Th17 cells in the CSE-exposed DC/MLR group increased significantly compared with those in the control DC/MLR group (P<0.05). Moreover, Th17 cells in the CD40-blocked CSE-exposed DC/MLR group and EM-treated CSE-exposed DC/MLR group were reduced compared with those in the CSE-exposed DC/MLR group (P<0.05). Thus, these findings suggested that EM suppressed the CSE-exposed DC-mediated polarization of CD4(+) T cells into Th17 cells and that this effect may be mediated through inhibition of the CD40/CD40L pathway.

Dendritic cells (DC) are important Antigen presenting cells (APC) of the human immune system. Dendritic cells can stimulate the B and T cells. Due to their potent ability to present antigens to immune cells, they can be used as an adjuvant for antigen specific T cell response. This increases the interest of isolation and generation of dendritic cells. The Bone marrow cells are the major source of systemic DCs. The lineage committed DC progenitors of monocytes from bone marrow are differentiated to matured DC by culturing in medium containing Granulocyte/macrophage-colony stimulating factor (GM-CSF) and stimulators such as TNF (Tumor necrosis factor). Therefore, the monocytic DC progenitors express CD40 (Cluster of differentiation 40) co-stimulatory protein expressed on their surface of Dendritic cells, upon engagement with CD40Ligand (CD40L) type II glycoprotein primarily expressed on activated T-Cells upon regulate the expression of GM-CSF, an important key regulator of granulopoiesis. Therefore, this study is aimed at investigating the role of CD40L mimetic molecule, N-benzhydryl benzamide (NBB), in the development of DCs from bone marrow cells. Bone marrow cells from mouse bone marrow were isolated and treated with medium containing GM-CSF and NBB. Three days after the treatment of bone marrow cells with medium containing GM-CSF and NBB, more cells developed morphology similar to matured DC, when compared with control containing GM-CSF. Cell density assay were performed using basic procedure.