Differential gene expression analysis identifies murine Cacnb3 as strongly upregulated in distinct dendritic cell populations upon stimulation.

Langerhans cells (LCs) represent the dendritic cell (DC) population in the epidermis. Among the set of genes induced in primary mouse LCs in response to stimulation, both isoforms of the voltage-dependent Ca²(+) channel (VDCC) regulatory subunit Cacnb3 as well as the DC maturation marker Fscn1 were upregulated most strongly. Comparable results were obtained for a recently described myeloid DC line (SP37A3). Other antigen presenting cell populations, namely, bone marrow-derived DCs, macrophages and primary B cells, showed no stimulation-associated upregulation of Cacnb3 expression. Pharmacological inhibition of Ca²(+) channel activity during the stimulation of SP37A3 cells enhanced their T cell stimulatory capacity, while selective inhibition of L-type VDCC had no effect. Both Cacnb3 isoforms, similar to Fscn1, required JNK and p38 kinase activity for stimulation-associated upregulation, and this process was inhibited by ERK and PI(3)K. The putative promoter region of Cacnb3 isoform 2, which we found to be less ubiquitously expressed than Cacnb3 isoform 1, exerted reporter activity in LC-like cell lines. Our findings suggest that Cacnb3 exerts its function in distinct activated DC populations. Further analysis of the regulatory region(s) facilitating stimulation-induced upregulation of Cacnb3 expression in these DC subsets will help to gain better insight into DC subset specific gene regulation.

A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid.

The phenotype and function of murine dendritic cells (DCs) are primarily studied using bone-marrow-derived DCs (BM-DCs), but may be hampered by the heterogeneous phenotype of BM-DCs due to their differential state of maturation. Here we characterize a newly established murine DC line (SP37A3) of myeloid origin. During maintainance in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF, SP37A3 cells resemble immature DCs characterized by low expression of major histocompatibility complex (MHC) II and costimulatory molecules and low T-cell stimulatory capacity. Upon stimulation, SP37A3 cells acquire a mature phenotype and activate naive T cells as potently as BM-DCs. Similar to BM-DCs, SP37A3 cells activated in the presence of dexamethasone-induced regulatory T cells, which were anergic upon restimulation and suppressed proliferation of naive T cells. This tolerogenic state was reflected by lower expression levels of costimulatory molecules and proinflammatory cytokines compared with mature cells, as well as up-regulated expression of FcgammaRIIB and interleukin-1RA (IL-1RA). SP37A3 cells were responsive to dexamethasone even when applied at later time points during activation, suggesting functional plasticity. Thus, DC line SP37A3 represents a suitable model to study functions of immature and mature as well as tolerogenic myeloid DCs, circumventing restrictions associated with the use of primary DCs and BM-DCs.

Antiangiogenic combination tumor therapy blocking alpha(v)-integrins and VEGF-receptor-2 increases therapeutic effects in vivo.

Anti-angiogenesis is a promising strategy for cancer therapy currently evaluated in clinical trials. The aim of the study was to investigate the effects of an antiangiogenic combination therapy inhibiting alpha(v)-integrins by a c(yclic)RGD-peptide (EMD270179) and blocking VEGFR-2 by SU5416 on tumor angiogenesis and progression in vivo. Experiments were performed in dorsal skinfold chamber preparations of Syrian golden hamsters (60 +/- 5 g) bearing A-Mel-3 tumors. From day 3-10 after tumor-cell implantation, animals (n = 6 per group) were treated by monotherapies using the cRGD-peptide (114 mg/kg/day; i.p.), the VEGFR-2 antagonist (6 mg/kg/day; i.p.) or by the combination of both monotherapies. A control group received only the solvent DMSO. Using intravital microscopy parameters of intratumoral microcirculation were analyzed on day 5, 7 and 10. In separate experiments subcutaneous tumor growth and metastasis formation was evaluated starting therapy on day 0. Functional vessel density was significantly reduced by the combination therapy compared to that by all other groups on day 10. Although intratumoral red blood cell velocity and vessel diameters were less affected, blood flow in vessel segments and the microcirculatory perfusion index were lower after combined therapy compared to controls. In addition, we observed a significantly stronger inhibition of subcutaneous tumor growth and metastasis formation using the combination therapy. These data clearly support the concept of antiangiogenic combination therapy and demonstrate that it may especially be effective when scheduled as an early or prophylactic treatment regimen, thus avoiding angiogenesis-dependent tumor and metastasis initiation or tumor recurrence.