Dysregulation of anergy-related factors involved in regulatory T cells defects in Systemic Lupus Erythematosus patients: Rapamycin and Vitamin D efficacy in restoring regulatory T cells.

AIM: Systemic Lupus Erythematosus (SLE) patients display dysfunctions in T cell activation and anergy. Therefore the aims of our study were to explore the expression of anergy-related factors in CD4+ T cells in relationship with regulatory T cells (Tregs) frequency in SLE patients and to identify strategies to redress these defects. METHOD: Casitas B-cell lymphoma b (Cbl-b) and 'gene related to anergy in lymphocytes' (GRAIL) proteins were analyzed in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (HD) by immunoblotting. cbl-b, grail, growth response factors (egr)2 and egr3 messenger RNAs (mRNAs) were evaluated by real-time polymerase chain reaction in SLE and HD PBMCs and CD4+ T cells. Phenotypic and functional characterization of CD4+ T cells was performed by flow cytometry. Tregs expansion protocol consisted in culturing CD4+ T cells for 14 or 21 days of experimental activation with anti-CD3 and anti-CD28 monoclonal antibodies, human recombinant interleukin (hrIL)-2, in the absence or presence of rapamycin (Rapa) or 1,25-(OH)2D3 (vitamin D: VitD). RESULTS: SLE PBMCs expressed low levels of Cbl-b and GRAIL proteins. Both SLE PBMCs and CD4+ T cells expressed low levels of egr2/3 mRNAs. SLE patients had a reduced number of Tregs with impaired suppressive activity. An association between egr2 mRNA level in CD4+ T cells and Tregs percentage was identified. Experimental activation of CD4+ T cells in the presence of hrIL-2 and Rapa or VitD induced the expansion of SLE Tregs. However, on long-term, only Rapa exposure of SLE CD4+ T cells yielded high numbers of Tregs with sustained suppressive activity. CONCLUSION: Our results suggest a new strategy to correct defects in CD4+ T cell tolerance mechanisms that may prove beneficial in SLE.

Peripheral blood lymphocytes analysis detects CD100/SEMA4D alteration in systemic sclerosis patients.

It was suggested that the immune system plays an important role at least in the amplification of the main elements in systemic sclerosis (SSc), an autoimmune disease with an incompletely elucidated pathogenesis. Elucidation of the mechanisms involved in the interaction between T and B cells, major players of the immune system, could contribute to a better understanding of some of clinical and pathological manifestations of SSc. Recently, abnormalities in Semaphorin 4D (Sema4D/CD100) or CD72, two contrareceptors involved in T and B cells cooperation, were associated with autoimmunity. Therefore, we investigated CD100 and CD72 expression level on T and B cells in attempting to establish their role in SSc pathogenesis. The results revealed augmented percentages of CD100(high) T and B cells, significantly increased expression of CD100 on CD4(+) T cells and frequently detectable levels of soluble CD100 in SSc patient sera compared to healthy donors. In SSc, CD100 dysregulations were associated with anti-Scl70 antibodies production, disease type, thickening of skin, disease duration, or with active inflammation processes. In consequence, dysregulations in CD100 expression and release could play a role in SSc development and/or maintenance.

Role of cellular immunity in systemic sclerosis pathogenesis: update on CD4+T cells population studies.

Immunologic abnormalities observed in Systemic Sclerosis (SSc) patients consist of chronic mononuclear cell infiltration of affected tissues, dysregulation of lymphokine and growth factor production, and autoantibodies production. Expansion of CD4+T cells within the tissue seems to involve their activation that precedes this process. Therefore, CD4+T cells activation, as an early immune event, appears to be an important process in the development and maintaining of SSc. In SSc the disturbance of peripheral tolerance mechanisms could be also responsible for CD4+T cells activation. Consequently, we reevaluated CD4+T cells positive for CD25, GITR, CTLA-4, CD45RO, or Foxp3 in SSc patients, by comparison with healthy donors (HDs), and in correlation with clinical features of the disease. Our results reargued for activation of peripheral blood CD4+T cells in SSc patients. Thus, increased percentages of CD25+ and GITR+ CD4+T cells were found in SSc patients by comparison with HDs. Direct correlation between the percentage of GITR+CD4+T cells and disease activity recommended these cells as a good candidate for disease progression. In SSc patients, the negative regulators of T cells activation are also affected. Thus, CTLA-4+ and Foxp3+ CD4+T cell percentages were significantly reduced in SSc patients when compared to HDs. Indirect correlation between the percentage of CD152+CD4+T cells and autoantibodies (aScl70) presence or disease type highlighted the role of these cells in the disturbance of peripheral tolerance. The absence of the direct correlation between CD152+CD4+T cells and CD45RO+CD4+T cells, correlation observed only in HDs, raised the hypothesis that in SSc patients, memory T cells can be easily activated, and by consequence, they can enter within affected tissues. These data reconfirm the activation state of SSc CD4+T cells and point out some abnormalities in peripheral tolerance mechanisms that can contribute to SSc pathogeny.

Quantification and molecular characterization of regulatory T cells in connective tissue diseases.

The aim of our study was to investigate and characterize regulatory T cells (Treg) in peripheral blood of patients with connective tissue diseases (Systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, poly- and dermatomyositis) as compared with blood from healthy controls. Treg cells were quantified and phenotypically characterized by flow cytometry while the expression level of Foxp3 mRNA was evaluated by real time PCR. A reduced percentage of peripheral blood Treg cells was found in patients than in controls, irrespective of the type of connective tissue disease. Treg cells, especially those expressing one of the phenotypical markers, seemed to differ not only between patients and healthy controls but also among types of diseases. Additionally, the presence of autoantibodies as well as disease activity appeared to be correlated with particular Treg cell populations, especially those expressing one of the examined phenotypical markers. Correlations with therapy suggested that glucocorticoids plus antimalarial or other immunosuppressor drugs diminished the percentage of Treg cells, especially of those with memory phenotype. These findings indicated dysregulations at the level of Treg cells and suggested an involvement of these cells in the pathology of connective tissue diseases. Moreover, our data are in agreement with the suggestion that Treg cells could be therapeutic targets for some autoimmune diseases.

PI3K/Akt signaling in peripheral T lymphocytes from systemic lupus erythematosus patients.

PI3K/Akt/mTOR signaling pathway plays an important role in cellular proliferation and growth signaling. It was demonstrated that murine models presenting activated PI3K/Akt/mTOR signaling pathway in lymphocytes develop features of systemic autoimmunity, linking this pathway to autoimmune diseases. Therefore, the goal of our study was to analyze this signaling axis in Systemic Lupus Erythematosus (SLE), the prototype of systemic autoimmune diseases, focusing on Akt and p70S6k, two components of this pathway. Our results demonstrated that both expression and phosphorylation levels of Akt are more increased in SLE than in healthy donors (HDs) CD4+ T cells suggesting an up-regulation of PI3K and mTOR activities. This result was also suggested when p70S6k, one of mTOR substrate, was evaluated. Indeed, in SLE CD4+ T cells an enhancement of p70S6k activity, in direct correlation with its expression level, was found. Since p27kip1, an inhibitor of cell cycle progression, is one of the Akt substrates, we analyzed its expression level in relationship with cell cycle progression and apoptosis. The results demonstrated that p27kip1 expression level was significantly decreased in SLE than in HDs CD4+ T cells. In SLE p27kip1 level was inversely correlated with the percentage of peripheral lymphocytes in apoptosis and in S phase of the cell cycle. Therefore, the increased activity of PI3K/Akt/mTOR signaling pathway and, as a result, the drop of p27kip1 levels observed in CD4+ T cells isolated from SLE patients might explain the accumulation of SLE lymphocytes in S and G2/M cell cycle phases where they undergo apoptosis.

Roles of CD147 on T lymphocytes activation and MMP-9 secretion in systemic lupus erythematosus.

The cellular and molecular mechanisms involved in many abnormalities described in Systemic Lupus Erythematosus (SLE) are still unclear. Some of these abnormalities referred to the hyperactivation of T lymphocytes and the enhanced secretion of MMP-9 by peripheral blood mononuclear cells (PBMCs). Therefore, in this paper we investigated the potential role of CD147 molecule in these abnormalities. Our results demonstrated that CD147 molecule is overexpressed on CD3+T lymphocytes from SLE patients when compared with CD3+T lymphocytes from healthy donors. Monoclonal anti-CD147 antibodies, MEM-M6/1 clone, were able to inhibit protein tyrosine phosphorylation only in CD3 x CD28 costimulated T lymphocytes from SLE patients. However, this monoclonal antibody was unable to inhibit the enhanced activity of MMP-9 secreted by SLE PBMCs.

MMP-9 and MMP-2 gelatinases and TIMP-1 and TIMP-2 inhibitors in breast cancer: correlations with prognostic factors.

The goal of our study was to analyse the prognostic values for some matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in breast cancer. We evaluated the activity and the expression levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 in malignant versus benign fresh breast tumor extracts. For this purpose, gelatinzymography, immunoblotting and ELISA were used to analyse the activity and expression of MMPs and TIMPs. We found that MMP-9 expression level and activity are increased in malignant tumors. In addition, MMP-9/TIMP-1 and MMP-2/TIMP-2 ratio values obtained by us were significantly different in malignant tumors compared to benign tumors. We suggest that the abnormal MMP-9/TIMP-1 balance plays a role in the configuration of breast invasive carcinoma of no special type and also in tumor growth, while altered MMP-2/TIMP-2 ratio value could be associated with lymph node invasion and used as a prognostic marker in correlation with Nottingham Prognostic Index. Finally, we showed that in malignant tumors high expression of estrogen receptors is associated with enhanced activity of MMP-2 and increased bcl- 2 levels, while high expression of progesterone receptors is correlated with low TIMP-1 protein levels.

Serum levels of matrix metalloproteinases MMP-2 and MMP-9 and their tissue natural inhibitors in breast tumors.

In this study, the levels of matrix metalloproteinases MMP-2 and MMP-9 were simultaneously analyzed with the levels of their tissue natural inhibitors TIMP-1 and TIMP-2 in sera of patients with breast tumors. At the same time, the activity of these two matrix metalloproteinases was evaluated. The decrease of TIMP-2 level in sera from patients with breast cancer as well as an imbalance between MMP-2 and TIMP-2 in neoplasic processes were found. The serum levels of MMP-2, MMP-9 and TIMP-1 were comparable between the patients with breast cancer and benign tumors. These experimental studied parameters were found to correlate with some of clinicopathological disease variables (TNM or pTNM staging system, tumor size and node invasion) suggesting their potential value for diagnosis and prognosis of breast cancer. Matrix metalloproteinases or their natural inhibitors and tumor markers (CA15.3 and CEA) not correlated between but, each of them correlated with another clinicopathological disease variable, suggesting their usefulness in the evaluation.

Matrix metalloproteinase-9 and its natural inhibitor TIMP-1 expressed or secreted by peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

Matrix metalloproteinase-9 (MMP-9) was involved in inflammation and immune system dysfunctions. Besides immunologic abnormalities, systemic lupus erythematosus (SLE) also presents chronic inflammatory components. Therefore, a role of MMP-9 in SLE pathology might be supposed. To verify this hypothesis, SLE patients and healthy donors were compared for the MMP-9 and MMP-9 mRNA levels in peripheral blood mononuclear cells (PBMCs), the spontaneous secretion of MMP-9 and TIMP-1 and the MMP-9 activity. Thus, we found that fresh PBMCs from SLE patients expressed a significantly higher activity of MMP-9 and spontaneously released higher levels of MMP-9, as compared to healthy donors, while the secreted TIMP-1 level was the same for both groups. When the patients were sub-grouped based on disease status, the most increased pro-MMP-9 activity inside the PBMCs was identified for relapse SLE sub-group. A similar observation for SLE patients with positive serum fibrinogen was found. Following culture, the PBMCs from remission SLE patients secreted significantly higher MMP-9 level, than the PBMCs from relapse SLE patients. PBMCs from relapse SLE patients secreted the highest levels of TIMP-1, although this difference was not statistically significant. Taken together, these observations suggested the multiple roles of MMP-9 and TIMP-1 in progress of inflammation and tissue damage and/or in repair, depending on clinical stages of SLE.