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.