Importantly, BMSC rescued B cells from corticosteroid-induced apoptosis, indeed only 30% of B cells were dying in BMSC-B cell co-cultures. proteins. First, mesenchymal stromal cell morphology, cytoskeleton assembly, cell cycle, survival and cytokine production were evaluated. Then, these cells were co-cultured with either T or B lymphocytes and we analyzed: 1) the inhibition of T-cell proliferation to mitogenic stimuli; 2) B-cell survival. Results Fluvastatin altered the assembly of actin microfilaments, inactivated RhoA guanosin triphosphate binding protein, inhibited the S-phase of the cell cycle, induced apoptosis in a small fraction of cells but preserved cytokine production. Preincubation of mesenchymal stromal cells with fluvastatin, or manumycin A, down-regulated the expression of adhesion molecules, reduced cell-to-cell interactions and prevented the inhibition exerted by these stromal cells on CD3/T-cell receptor-induced lymphocyte proliferation. Mevalonic acid could revert morphological, phenotypic and functional effects of fluvastatin. Finally, fluvastatin significantly reduced the mesenchymal stromal cells-mediated rescue of B cells in the presence of dexamethasone, although it did not function in the absence of corticosteroids. Conclusions Fluvastatin-mediated effects on bone marrow mesenchymal stromal cells were conceivably due to the inhibition of isoprenylation of small guanosin triphosphate binding proteins, occurring for the lack of mevalonate. Altogether these findings suggest that drugs acting on the mevalonate biosynthetic pathway can regulate mesenchymal stromal cell-induced T-cell suppression and B-lymphocyte survival. and and and values are shown when statistically significant. (C) BMSC were cultured with DMSO as in panel A or with manumycin A and analyzed for the expression of the indicated surface molecules with specific mAbs followed by Alexafluor647-conjugate anti-isotype specific GAM. Results are expressed as Log far red fluorescence intensity in a.u. and are Rabbit polyclonal to THIC representative of 6 Carboxypeptidase G2 (CPG2) Inhibitor independent experiments. (D) Results of each molecule analyzed in panel C are depicted as the meanSD of 6 independent experiments. Results are shown as MFI in a.u. and they were analyzed by one-tail Students t-test at 95% confidence. The values are shown when statistically significant. Fluvastatin impairs the S-phase of the cell cycle in BMSC but it does not induce apoptosis nor affect secretion of constitutive cytokines Due to its action on cytoskeleton and RhoA GTP-binding proteins, we analyzed the effect of fluvastatin on cell cycle progression of BMSC, on BMSC survival and on constitutive release of cytokines by BMSC. Indeed, other GTP-binding protein, such as Ras and Raf, involved in the regulation of cell proliferation can be affected by fluvastatin as previously reported;25,27 in addition, secretion of cytokines may occur via granule exocytosis depending on cytoskeleton activity.25,27,31 We found that fluvastatin strongly reduced or even abrogated the S phase of the cell cycle, partially affected G0/G1 phase but not G2/M phase after 48 h of treatment (and and values are shown when statistically significant. Fluvastatin inhibits BMSC immunosuppressive effect on T-cell proliferation BMSC can inhibit T-cell proliferation thus exerting a potent immunosuppressive effect.5C15 Thus, Carboxypeptidase G2 (CPG2) Inhibitor we analyzed whether this effect was affected by fluvastatin. To this aim, BMSC were treated for 48 h with fluvastatin, washed and used for co-culture experiments with CFSE-labeled PBMC at the 1:5 BMSC:PBMC ratio and proliferation of T cells was analyzed by staining cell cultures with anti-CD3 mAb to gate T cells: reduction of CFSE content along time was proportional to cell proliferation. As shown in Figure 2A, evident clumps of PBMC were detectable in the presence of PHA. These clumps were not evident in PBMC cultured with PHA and BMSC. In PBMC co-cultures with fluvastatin pre-treated BMSC, lymphocyte cell clumps were evident again and this effect was abrogated when BMSC were incubated with fluvastatin and L-mevalonate (Figure 2A). As shown in Figure 2B, BMSC strongly inhibited T-cell proliferation induced Carboxypeptidase G2 (CPG2) Inhibitor by the polyclonal mitogen PHA or through the engagement of the CD3/TCR complex. This effect was almost completely abolished by 48 h pre-incubation of BMSC with fluvastatin 10 M (Figure 2B, D and E). This inhibiting effect was not detected when BMSC were pre-incubated with 1 M fluvastatin for 48 h (2D and E). Interestingly, BMSC treated with fluvastatin for 48 h did not affect the inhibition of T-cell proliferation (Figure 2F) detected in PBMC-BMSC TW cultures. This suggests that fluvastatin does not alter the efficiency of putative inhibiting factors produced when BMSC and PBMC were not in contact. Results concerning conditioned medium from co-cultures of fluvastatin-treated BMSC and PBMC.
Importantly, BMSC rescued B cells from corticosteroid-induced apoptosis, indeed only 30% of B cells were dying in BMSC-B cell co-cultures
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