dc.description.abstract | Dendritic cells (DCs) are antigen presenting cells important in both innate and adaptive immune systems. DCs in peripheral tissues are capable of capturing antigens and migrating to local lymphoid tissues where the DCs become mature and enable to activate antigen-specific T cells. Elevation of intracellular cAMP concentration suppresses various inflammatory responses in almost all inflammatory cell types, including DCs. However, the information on how cAMP signaling regulates the differentiation, maturation and function of DCs is limited. Type 4 phosphodieasterases (PDE4s), enzymes that degrade cAMP with high affinity, are the predominant PDE isozymes expressed in most inflammatory cells, and are critical in regulation of various inflammatory responses. To determine whether PDE4, thereby the cAMP signaling, regulates DC differentiation and maturation, in this study we stimulated mouse bone marrow cells with GM-CSF in the presence of cAMP-elevating agents to assess their effects on DC differentiation. Flow cytometry analyses revealed that 81.7 ± 1.1 % cells harvested on day 8 of culture developed into immature DCs (imDCs), demonstrated by the expression of CD11c on these cells. This process was inhibited by the PDE4 inhibitor Rolipram, the combination of Rolipram and the PDE3 inhibitor cilostazol, dibutyryl cAMP (dbcAMP), and the non-selective PDE inhibitor IBMX at both the CD11+ population (12.26 ± 1.3 %, 35.5 ± 1.6 %, 18.3 ± 1.4 %, and 16.8 ± 2.4 %, respectirely) and the level of CD11c expression (i.e. CD11c mean fluorescence intensity; 21.8 ± 2 %, 35 ± 2.9 %, 58.8 ± 0.3 % and 41.4 ± 2.21 %, respectirely). However, Cilostazol alone lacked these effects. The experiments using PDE4 null bone marrow cells further demonstrated that PDE4B and possibly PDE4A, but not PDE4D, mediated the Rolipram effect on the differentiation of imDC population. During LPS induced DC maturation, the CD11c+ population and the mean fluorescence intensity (MFI) of CD11c+ were significantly decreased (p<0.05), whereas the CD11c+CD86+ population (mature DCs) was increased by approximately 3.2 folds. This increase however was not accompanied by an increase in MFI of CD86. Rolipram also showed an inhibition (21.4 ± 2.36 %) in the LPS-induced CD11c+CD86+ population when the total gated cells were analyzed, but this decrease was not obtained when the CD11c+ population was analyzed. Further study on PDE4 null DCs suggested that the Rolipram effect on the CD11+CD86+ population was probably mediated by inhibiton of both PDE4A and PDE4B. Similar to LPS, our results showed that dbcAMP or Rolipram alone also induced DC maturation albeit at less extent. In a functional study using ovalbumin (OVA) – primed spleen CD4+ T cells, we found that DC-induced T cell proliferation in response to OVA was attenuated in PDE4B-deficient DCs compared to PDE4B wild - type DCs. Taken together, these findings indicate an involvement of PDE4, particularly PDE4B, in the development and function of DCs. They also form the basis for the development of PDE4 inhibitors for the treatment of inflammatory diseases that are mediated by DCs. | en_US |