New regulatory CD19(+)CD25(+) B-cell subset in clinically isolated syndrome and multiple sclerosis relapse. Changes after glucocorticoids.

In multiple sclerosis (MS), the immune damage to the central nervous system results from the net balance between self-reactive and immunoregulatory cells, among other factors. We identified novel perforin-expressing regulatory B-cells (BReg) in patients with clinically isolated syndrome, significantly enriched within the cerebrospinal fluid when compared to peripheral blood, of memory B cell phenotype (CD19(+)CD25(+), CD19(+)CD25(+)FoxP3(+) and CD19(+)FoxP3(+), p=0.007, p=0.06 and p=0.03, respectively). These BReg subsets were also higher in relapsing-remitting MS during relapse symptoms than in non-clinically active MS patients. Suppressive effects by CD19(+)CD25(+hi) BReg on CD4(+) T cell proliferation seem to be mediated at least in part by perforin/granzyme pathway. To our knowledge, this is the first report that shows cytolytic perforin/granzyme granule storage in B cells; the interesting point is its involvement on BReg cell immunosuppressive mechanisms, similarly to that in TReg cells. Our data may extend the understanding of pathophysiological processes in MS immunoregulation.

Perforin expression by CD4+ regulatory T cells increases at multiple sclerosis relapse: sex differences.

Multiple sclerosis (MS) represents the leading cause of neurological deficit among young adults, affecting women more frequently than men. In MS, the extent of central nervous system lesions is determined by the net balance between self-reactive and regulatory T-cells at any given time, among other factors, as well as by the effect of inflammatory response. Here, we studied both CD4+ and CD8+ T(Reg) in parallel in blood and CSF during MS relapse. A recruitment of both regulatory CD4+ and CD8+ T cells (T(Reg)) within the cerebrospinal fluid (CSF) takes place during MS relapse. Not previously described, the presence of CD4+ T(Reg) in CSF was higher in women than in men, which could account for the sexual dimorphism in the incidence of MS. A direct correlation between plasma oestradiol (E2) and IL-2 levels was observed, in line with a putative circuit of E2 and perforin expression by CD4+ T(Reg) playing a role in MS. Also, serum IFN-alpha was higher in females, with direct correlation with serum E2 levels. This is the first study to analyze perforin expression by CD4+ T(Reg) in MS, which was greatly enhanced in CSF, what points out a relevant role of this molecule in the suppressive effects of the CD4+ T(Reg) in MS, and contributes to the understanding of MS pathophysiology.

Long-term decrease in VLA-4 expression and functional impairment of dendritic cells during natalizumab therapy in patients with multiple sclerosis.

Myeloid and plasmacytoid dendritic cells (mDCs, pDCs) are central to the initiation and the regulation of immune processes in multiple sclerosis (MS). Natalizumab (NTZ) is a humanized monoclonal antibody approved for the treatment of MS that acts by blocking expression of VLA-4 integrins on the surface of leukocytes. We determined the proportions of circulating DC subsets and analyzed expression of VLA-4 expression in 6 relapsing-remitting MS patients treated with NTZ for 1 year. VLA-4 expression levels on pDCs and mDCs decreased significantly during follow-up. In vitro coculture of peripheral blood mononuclear cells and pDCs, with different doses of NTZ in healthy controls (HC) and MS patients showed dose-dependent down-regulation of VLA-4 expression levels in both MS patients and HC, and reduced functional ability to stimulate antigen-specific T-lymphocyte responses. The biological impact of NTZ may in part be attributable to inhibition of transmigration of circulating DCs into the central nervous system, but also to functional impairment of interactions between T cells and DC.

Sex-hormone receptors pattern on regulatory T-cells: clinical implications for multiple sclerosis.

Cellular mechanisms underlying sexual dimorphism in the immune response remain largely unknown. Concerning the interactions among the nervous, endocrine and immune systems, we reported that during gestation, a period during which multiple sclerosis (MS) clearly ameliorates, there is a physiological expansion of regulatory T-lymphocytes (T(Reg)). Given that alterations in T(Reg) proportions and suppressive function are involved in MS pathophysiology, we investigated the in vitro effect of sex hormones on T(Reg). Here, we show that both E2 and progesterone (P2) enhance T(Reg) function in vitro, although only E2 further induces a T(Reg) phenotype in activated responder T-cells (CD4(+)CD25(-)) (P < 0.01). E2 receptor beta (ERß) percentages and mean fluorescence intensity (MFI) on T(Reg) were lower in MS patients than in controls (P < 0.05), in parallel with lower E2 plasma levels (P < 0.05). Importantly, percentages and MFI of ERß were higher in T(Reg) than in T-responder cells (P < 0.0001) both in MS patients and controls. We show a unique differential pattern of higher ER and PR levels in T(Reg), which may be relevant for the in vivo responsiveness of these cells to sex hormones and hence to MS physiopathology.

Estradiol-dependent perforin expression by human regulatory T-cells.

BACKGROUND: CD4+CD25+FoxP3+ regulatory T-cells (nT(Reg)) have been shown to suppress immune responses to autoantigens and to other diverse antigens, this suppression is mainly mediated by a cell contact-dependent mechanism not yet fully defined. It has been reported that both human natural and induced T(Reg) exert cytotoxic activity against autologous target cells, which suggests that the perforin/granzyme pathway may be a relevant candidate mechanism for the suppressive function of T(Reg). Previous reports have shown that oestradiol (E2) modulates T(Reg) percentages and function. METHODS: We have evaluated in pregnant and non-pregnant subjects perforin intracellular expression in CD4+CD25+FoxP3+ regulatory T-cells by flow cytometry in whole blood, ex-vivo purified nT(Reg) and ex-vivo purified nT(Reg) after TCR and E2 stimulation. The expression of cellular degranulation markers was also phenotypically determined. RESULTS: We show that E2 expands T(Reg), enhances in vitro T(Reg) function and induces a T(Reg) phenotype in activated responder (CD4+CD25) T-cells, further increasing the expression of perforin on T(Reg) than in vitro T-cell receptor activation alone. We found surface lysosomal-associated membrane glycoproteins (LAMP)-1 and LAMP-2 expression by T(Reg), which is a sign of cell degranulation and therefore of cytotoxicity exerted by these cells. CONCLUSION: Our data demonstrates the presence of functional T(Reg) cytotoxic properties in biological systems and support the concept that E2 enhances the number and function of T(Reg) suggesting the potential interaction between E2 and immunoregulatory mechanisms.

Leptin increases proliferation of human steosarcoma cells through activation of PI(3)-K and MAPK pathways.

BACKGROUND: Serum leptin levels are strongly and directly related to fat body mass (FBM). Bone mineral density (BMD) increases with FBM, and obesity has a protective effect against osteoporosis. We have previously demonstrated that leptin therapy has a significant effect in preventing ovariectomy-induced bone loss in rats and leptin also exerts direct osteogenic effects in vitro. To obtain a better understanding of the physiology and pharmacology of leptin in bone metabolism, we evaluated the leptin-induced signal transduction pathways and proliferative response in the human osteosarcoma cell line Saos-2. MATERIAL/METHODS: Saos-2 cell lines were used. Leptin receptor common form (OB-Ra) and long form (OB-Rb) were detected by RT-PCR and immunocytochemistry. PI(3)-K activity was immunoprecipitated using antibodies directed against tyrosine-phosphorylated proteins and IRS-1. The activated form of p42/p44 MAPK was investigated in cytosolic extracts of confluent Saos-2 in response to leptin. RESULTS: In this study, we tested the hypothesis that leptin might be a mediator linking obesity and bone cell proliferation. We found that Saos-2 cells expressed OB-Ra and OB-Rb. Leptin (10 nmol/L - 2 umol/L) caused a significant increase in the activation of PI (3)-K that was accompanied by an increase in cell proliferation dose dependently based on the [3H]-thymidine incorporation. The specific PI (3)-K inhibitors LY294002 and wortmannin blocked leptin-induced cell proliferation. Interestingly, leptin activated MAPK and the specific MAPK-inhibitor PD98059 blocked DNA synthesis induced by leptin. CONCLUSIONS: Our data support the hypothesis that leptin may increase bone mass by stimulating osteoblast proliferation through activation of the P1 (3)-K and MAPK signaling pathways.