Inflammation is modulated by Cav3.2 expressed in macrophages and lymphocytes T CD4+
The presence of Cav3.2 channels in BMDM and CD4+ T cells led us to investigate the impact of these channels on the activation of both types of cells. To demonstrate the role of Cav3.2 channels in BMDM, in vitro studies were performed on BMDM culture of Cav3.2 KO and WT mice. The analysis of BMDM morphology, performed by phalloidin-immunofluorescence labelling of actin, revealed swollen morphology in WT BMDM in presence of LPS (cells area without LPS = 283 ± 15 µm²; with LPS = 643 ± 22 µm²). Cav3.2 KO BMDM had the same basal morphology as WT BMDM but no morphological change was observed after stimulation by LPS (cell area without LPS = 307 ± 12 µm²; with LPS = 260 ± 10 µm²; Figures 6A and 6B left panel ). In addition, Cav3.2 KO BMDM produced a smaller amount of IL-6 than WT BMDM in presence of LPS (Figure 6B right panel ). A reduction of TNF-α production was also observed in LPS-stimulated Cav3.2 KO BMDM (WT: 1,400.56 ± 193.08 pg/ml vs KO Cav3.2: 462.22 ± 196.79 pg/ml, p = 0.0045, Mann Whitney test) as compared with levels of unstimulated BMDM (WT: 65.67 ± 21.19 pg/ml vs KO Cav3.2: 19.86 ± 19.39 pg/ml). As calcium is a major factor in macrophage activation (Zhou et al., 2006), we thought it would be of interest to investigate variations of intracellular calcium concentration after LPS stimulation. The results showed that 30.2 ± 5.6% of WT BMDM cells and only 6.7 ± 2.1% of Cav3.2 KO BMDM cells induced elevation of intracellular calcium in response to LPS stimulation (Figure 6C ).
An assessment of the immune cell phenotype was performed by using flow cytometry analysis of total spleen cells from WT and Cav3.2 KO mice. The expression of CD86, which is required for T cells activation, was used to measure the activation status of antigen-presenting cells (APC) (Hellman and Eriksson, 2007). Median fluorescence intensity in macrophages, CD11b+dendritic cells and inflammatory monocytes was significantly lower in spleen cells from Cav3.2 KO mice than in those of the WT mice (Figure 6D ) whereas the absolute number of these APC were unchanged (data not shown). These results indicate, at the basal level, a defect in APC activation that could alter T-cells activation and thus the immune response. Moreover, we analyzed the proliferation capacities of WT and Cav3.2 KO CD4+ T cells activated ex vivo by CD3/CD28. T-cell proliferation, assessed at day 4, was significantly lower in Cav3.2 KO mice than in WT mice (54.6% as compared with 91.3% divided cells, respectively;Figure 6E ). These findings led to conclude that the absence of Cav3.2 channels in T cells impaired or delayed their proliferation, which could partly account for the reduced edema size observed in the Cav3.2 KO mice.