RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-β-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-β stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-β-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-β-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.     

RGC-32 and diseases: the first 20 years

The response gene to complement (RGC)-32 acts as a cell cycle regulator and mediator of TGF-β effects. However, recent studies have revealed other functions for RGC-32 in diverse processes such as cellular migration, differentiation, and fibrosis. In addition to its induction by complement activation and the C5b-9 terminal complement complex, RGC-32 expression is also stimulated by growth factors, hormones, and cytokines. RGC-32 is induced by TGF-β through Smad3 and RhoA signaling and plays an important role in cell differentiation. In particular, RGC-32 is essential for the differentiation of Th17 cells. RGC-32^−/− mice display an attenuated experimental autoimmune encephalomyelitis phenotype that is accompanied by decreased central nervous system inflammation and reductions in IL-17- and GM-CSF-producing CD4⁺ T cells. Accumulating evidence has drawn attention to the deregulated expression of RGC-32 in human cancers, atherogenesis, metabolic disorders, and autoimmune disease. Furthermore, RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases. A better understanding of the mechanism(s) by which RGC-32 contributes to the pathogenesis of all these diseases will provide new insights into its therapeutic potential.

     

The complement system as a biomarker of disease activity and response to treatment in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system. The complement system has an established role in the pathogenesis of MS, and evidence suggests that its components can be used as biomarkers of disease-state activity and response to treatment in MS. Plasma C4a levels have been found to be significantly elevated in patients with active relapsing-remitting MS (RRMS), as compared to both controls and patients with stable RRMS. C3 levels are also significantly elevated in the cerebrospinal fluid (CSF) of patients with RRMS, and C3 levels are correlated with clinical disability. Furthermore, increased levels of factor H can predict the transition from relapsing to progressive disease, since factor H levels have been found to increase progressively with disease progression over a 2-year period in patients transitioning from RRMS to secondary progressive (SP) MS. In addition, elevations in C3 are seen in primary progressive (PP) MS. Complement components can also differentiate RRMS from neuromyelitis optica. Response gene to complement (RGC)-32, a novel molecule induced by complement activation, is a possible biomarker of relapse and response to glatiramer acetate (GA) therapy, since RGC-32 mRNA expression is significantly decreased during relapse and increased in responders to GA treatment. The predictive accuracy of RGC-32 as a potential biomarker (by ROC analysis) is 90% for detecting relapses and 85% for detecting a response to GA treatment. Thus, complement components can serve as biomarkers of disease activity to differentiate MS subtypes and to measure response to therapy.     

Memory and naïve B-cell subsets in patients with multiple sclerosis

Memory and naïve B cells are considered to play distinct roles in immune regulation. However, the roles of memory and naïve B-cell subsets in multiple sclerosis (MS) have not yet been elucidated. In this study, we examined whether memory and naïve B-cell subsets differ between patients with MS and healthy subjects and whether interferon beta (IFNβ)-1b can affect these subsets in patients with MS. We also studied these subsets in relapsing and remitting stages of MS. Subjects included 31 patients with relapsing–remitting MS in the remitting stage, of which 15 were treated with IFNβ-1b and 16 were not treated, and 22 healthy control subjects. For 11 of the 16 untreated patients, blood samples were also obtained in the relapsing stage. Expression of CD5, CD80, CD86, CCR5, CXCR3, CD11a, and CD49d in memory and naïve B cells in blood samples was examined by flow cytometry. The percentages of CD86⁺ cells and CCR5⁺ cells in the naïve B-cell subset were significantly higher in untreated patients than in control subjects or IFNβ-treated patients. In patients with MS, the percentages of CD86⁺ cells and CCR5⁺ cells in the naïve B-cell subset and the percentage of CD5⁺ cells in the memory B-cell subset were significantly greater in the remitting stage than in the relapsing stage. These results indicate that memory and naïve B-cell subsets, especially CD86⁺ naïve B cells, CCR5⁺ naïve B cells, and CD5⁺ memory B cells, might be useful in the study of the pathogenesis of and therapy for MS.     

Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells

Proliferation of vascular endothelial cells (EC) and smooth muscle cells (SMC) is a critical event in angiogenesis and atherosclerosis. We previously showed that the C5b-9 assembly during complement activation induces cell cycle in human aortic EC (AEC) and SMC. C5b-9 can induce the expression of Response Gene to Complement (RGC)-32 and over expression of this gene leads to cell cycle activation. Therefore, the present study was carried out to test the requirement of endogenous RGC-32 for the cell cycle activation induced by C5b-9 by knocking-down its expression using siRNA. We identified two RGC-32 siRNAs that can markedly reduce the expression of RGC-32 mRNA in AEC. RGC-32 silencing in these cells abolished DNA synthesis induced by C5b-9 and serum growth factors, indicating the requirement of RGC-32 activity for S-phase entry. RGC-32 siRNA knockdown also significantly reduced the C5b-9 induced CDC2 activation and Akt phosphorylation. CDC2 does not play a role in G1/S transition in HeLa cells stably overexpressing RGC-32. RGC-32 was found to physically associate with Akt and was phosphorylated by Akt in vitro. Mutation of RGC-32 protein at Ser 45 and Ser 47 prevented Akt mediated phosphorylation. In addition, RGC-32 was found to regulate the release of growth factors from AEC. All these data together suggest that cell cycle induction by C5b-9 in AEC is RGC-32 dependent and this is in part through regulation of Akt and growth factor release.     

Astrocytic Fas ligand expression is required to induce T‐cell apoptosis and recovery from experimental autoimmune encephalomyelitis

In T‐cell‐mediated autoimmune diseases of the CNS, apoptosis of Fas⁺ T cells by FasL contributes to resolution of disease. However, the apoptosis‐inducing cell population still remains to be identified. To address the role of astrocytic FasL in the regulation of T‐cell apoptosis in experimental autoimmune encephalomyelitis, we immunized C57BL/6 glial fibrillary acid protein (GFAP)‐Cre FasL^fl/fl mice selectively lacking FasL in astrocytes with MOG_35–55 peptide. GFAP‐Cre FasL^fl/fl mice were unable to resolve EAE and suffered from persisting demyelination and paralysis, while FasL^fl/fl control mice recovered. In contrast to FasL^fl/fl mice, GFAP‐Cre FasL^fl/fl mice failed to induce apoptosis of Fas⁺ activated CD4⁺ T cells and to increase numbers of Foxp3⁺ Treg cells beyond day 15 post immunization, the time point of maximal clinical disease in control mice. The persistence of activated and GM‐CSF‐producing CD4⁺ T cells in GFAP‐Cre FasL^fl/fl mice also resulted in an increased IL‐17, IFN‐γ, TNF, and GM‐CSF mRNA expression in the CNS. In vitro, FasL⁺ but not FasL^? astrocytes induced caspase‐3 expression and apoptosis of activated T cells. In conclusion, FasL expression of astrocytes plays an important role in the control and elimination of autoimmune T cells from the CNS, thereby determining recovery from EAE.     

Overexpression of RGC-32 in colon cancer and other tumors

Tumors often exhibit deregulation of the cell cycle and overexpression of cyclins and cyclin-dependent kinases (CDKs). Response gene to complement (RGC)-32 is a substrate and regulator of CDC2 and its overexpression induces cell cycle activation. We investigated RGC-32 mRNA and protein expression in tumors with special emphasis in colon carcinoma. By using an expression array technique we found that 19% of tumor tissues showed increased RGC-32 mRNA expression over the levels of corresponding normal tissues. On the other hand, an increased RGC-32 protein was found in 70% of colon adenocarcinoma samples tested. In colon carcinomas, two major patterns of RGC-32 immunoreactivity were seen: staining of malignant epithelial cells only in some tumors and RGC-32 reactivity of both malignant epithelia as well as cells in the interstitium in others. Colonic epithelium obtained from normal individuals was consistently negative for RGC-32 protein. Overexpression of RGC-32 protein was found in other tumors including prostate, bladder, breast, lung, and other digestive tract tumors. RGC-32 expression was present in the same malignant epithelial cells that also expressed the proliferation marker Ki-67. Our data suggest that RGC-32 overexpression might be part of the deregulation of the cell cycle that is required for the growth of tumor cells.     

Blood levels of transforming growth factor-beta 1 (TGF-β1) are elevated in both relapsing remitting and chronic progressive multiple sclerosis (MS) patients and are further augmented by treatment with interferon-beta 1b (IFN-β1b)

The serum levels of TGF-β1, measured by solid-phase ELISA, were determined to be significantly augmented in patients with both relapsing remitting (RR) and secondary chronic progressive (CP) MS compared with sex- and age-matched healthy controls. Moreover, in RR MS patients, the blood levels of the cytokine were further augmented either during relapses or, in a rapid but reversible fashion, by s.c. injection with 8 million International Units (MIU) IFN-β1b. Because TGF-β1 possesses multiple anti-inflammatory activities, we hypothesize that the increase in its circulating levels in RR and CP MS patients might represent an endogenous anti-inflammatory mechanism aimed at counteracting ongoing immunoinflammatory events, and that IFN-β may further potentiate this natural defensive apparatus.     

Role of response gene to complement 32 in diseases

The role of response gene to complement (RGC)-32 as a cell cycle regulator has been attributed to its ability to activate cdc2 kinases and to induce S-phase entry and mitosis. However, recent studies revealed novel functions for RGC-32 in diverse processes such as cellular differentiation, inflammation, and fibrosis. Besides responding to C5b-9 stimulation, RGC-32 expression is also induced by growth factors, hormones, and cytokines. Transforming growth factor β activates RGC-32 through Smad and RhoA signaling, thus initiating smooth muscle cell differentiation. Accumulating evidence has drawn attention to the deregulated expression of RGC-32 in human malignancies, hyper-immunoglobulin E syndrome, and fibrosis. RCG-32 expression is up-regulated in cutaneous T cell lymphoma and colon, ovarian, and breast cancer, but down-regulated in invasive prostate cancer, multiple myeloma, and drug-resistant glioblastoma. A better understanding of the mechanism by which RGC-32 contributes to the pathogenesis of these diseases will provide new insights into its therapeutic potential. In this review we provide an overview of this field and discuss the most recent research on RGC-32.     

MAPT gene rs1052553 variant is not associated with the risk for multiple sclerosis

Background/Objectives

Some experimental data suggest a possible role of tau protein in the pathogenesis of multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis. The aim of this study was to investigate a possible influence of the SNP rs1052553 in the MAPT gene in the risk for relapsing bout onset (relapsing–remitting and secondary progressive) MS.

Methods

We analyzed the allelic and genotype frequency of MAPT rs1052553, which has been associated with some neurodegenerative diseases, in 259 patients with relapsing bout onset MS and 291 healthy controls, using TaqMan Assays.

Results

MAPT rs1052553 allelic and genotype frequencies did not differ significantly between relapsing bout onset MS patients and controls, and were unrelated with the age of onset of MS or gender.

Conclusions

These results suggest that MAPT rs1052553 polymorphism is not related with the risk for relapsing bout onset MS.     

CD32 expression and signaling is down-regulated by transforming growth factor-β1 on human monocytes

CD32 (FcγRII) is the most abundantly distributed class of IgG Fc receptors in the human body. In this study, we analyzed the effect of transforming growth factor (TGF)-β1, a cytokine with strong immunosuppressive function, on the expression and function of CD32 on freshly isolated peripheral blood monocytes and three human monocytic cell lines, U937, THP-1 and Mono mac-6. We found that TGF-β1 down-regulates CD32 expression on monocytes and all monocytic cell lines in a dose- and time-dependent fashion. A mean down-regulation of CD32 expression on THP-1 cells of 54 ± 3.2% after 24 h was found at a concentration of 1 ng/ml TGF-β1. At the mRNA level, TGF-β1 induced a twofold down-regulation of CD32. Cross-linking of CD32 induced an increase in the concentration of intracellular Ca²⁺, which was reduced by 50% by TGF-β1, suggesting a decreased downstream signaling mediated by the receptor.     

In Vitro Effects of Sodium Benzoate on Th1/Th2 Deviation in Patients with Multiple Sclerosis

Interleukin 4 (IL-4) can improve the clinical manifestations in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Sodium benzoate (NaB) deviates the cytokine profile to Th2 (or IL-4 producing) cells in EAE and thus might be effective in the treatment of MS. Therefore, in this study the effect of different concentrations of NaB on the percentage and mRNA levels of IL-4 and interferon gamma (IFN-γ)-producing peripheral blood mononuclear cells (PBMCs) of 20 Relapsing-remitting multiple sclerosis (RR-MS) patients and eight healthy controls was evaluated in the presence of mitogen (phytohemagglutinin, PHA) or specific antigen (myelin basic protein, MBP). Our results showed that in the patient’s group the percentage of CD4⁺IL-4⁺ cells was significantly increased in the presence of all concentrations of NaB when PBMCs were stimulated by MBP (p = 0.001) or PHA (p < 0.03). The same results were obtained for normal donors in the highest concentration of NaB, 1000 µg/ml (p = 0.02). Moreover, in the patient’s group the percentage of CD4⁺IFN-γ⁺ cells was decreased significantly when the PBMCs were stimulated by PHA and NaB (p < 0.004) or by MBP and 1000 µg/ml of NaB (p < 0.03). The effect of NaB on IL-4 and IFN-γ production was also documented at the mRNA levels. In conclusion, our data suggest that NaB is able to induce IL-4 production by human PBMCs and therefore might be a useful candidate for conjunctive therapy in RR-MS.     

TLS interaction with NMDA R1 splice variant in retinal ganglion cell line RGC-5

Translocated in liposarcoma (TLS or FUS) is a multifunctional protein component of the heterogenous ribonuclear complex involved in the splicing of pre-mRNA and the export of fully processed mRNA from the nucleus to the cytoplasm. As we determined that TLS was substantially expressed in the adult retina, we investigated the functions of TLS in a rat retinal ganglion cell (RGC) line RGC-5. TLS was found to be associated with N-methyl-d-aspartate (NMDA) receptor 1 (NR1) and myosinVa (MyoVa) in a calcium-dependent manner. We demonstrated that TLS-associated NR1 could be one of the NR1 alternative splice variants, NR1-4, which was predominantly expressed in RGC-5. The degree of colocalization between TLS and NR1 was significantly decreased by depolarization of RGC-5 cells, indicating that the depolarization-induced Ca²⁺-influx triggered a redistribution of NR1 from the TLS–protein complex. These results suggested that TLS might be involved in a calcium-dependent trafficking of specific NR1 splice variants in RGCs.     

Glucocorticoid treatment reduces T-bet and pSTAT1 expression in mononuclear cells from relapsing remitting multiple sclerosis patients

High dose glucocorticoid (GC) treatment has been demonstrated to have a short-term beneficial effect on functional recovery in relapsing multiple sclerosis (MS) patients but the exact mechanism of action of GCs in MS is unclear. We found that high dose intravenous GCs strongly reduced T-bet and pSTAT1 expression in CD4+, CD8+, CD14+ circulating cells in RRMS patients in relapse. pSTAT1and T-bet reduction was associated with the decline of IFNgamma production by PBMCs. A significant increase of AV-positive CD4+ and CD8+ T cells was detectable after GC treatment without any variation in the percentage of annexin V-positive monocytes. By in vitro analysis, patients during relapse, either before or after GC treatment, exhibited a lower proportion of apoptotic lymphocytes than remitting patients and controls. Our study suggests that GCs can modulate T-bet and STAT1 expression and that IFNgamma signalling inhibition contributes to anti-inflammatory action of GCs in the treatment of relapses of MS patients.     

Transforming growth factor-β1 induces antigen-specific unresponsiveness in naive t cells

Transforming growth factor-β1 (TGF-β1) is a cytokine with complex immunomodulatory effects including the ability to inhibit the onset or seventy of autoimmune disease. This study was designed to test the possibility that one mechanism by which TGF-β1 exerts its immunosuppressive effects is by inducing antigen (Ag)-specific unresponsiveness in CD4⁺ cells. TGF-β1 was shown here to inhibit the Ag-specific proliferation of naive CD4⁺ cells from T cell receptor (TCR) transgenic mice. More importantly, the naive CD4⁺ cells exposed to TGF-β1 and Ag, but not to TGF-β1 alone, in primary cultures were unable to proliferate or secrete IL-2 in response to a subsequent Ag challenge following removal of TGF-β1 from the cultures. Anti-CD28 mAb partially blocked the Ag-specific inactivation induced by TGF-β1 in naive CD4⁺ cells. The inhibitory effects of TGF-β1 on CD4⁺ cells are not mediated by alterations in APC costimulation since TGF-β1 did not inhibit the Ag-induced expression of MHC class II molecules, CD80 or CD86 on splenic APC. Taken together, the results suggest that the immunosuppressive activities of TGF-β1 encompass direct induction of Ag-specific unresponsiveness in naive CD4⁺ cells.     

T regulatory cells lacking CD25 are increased in MS during relapse

Dysregulation of inflammatory responses is considered to be a key element in autoreactive immune responses. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4⁺CD25⁺FoxP3⁺ Tregs in autoimmunity has been extensively investigated. Recently, it was shown that Tregs in systemic lupus erythematosus lacked CD25 but were biologically functional. These data warrants for further investigation of CD25^? Tregs in human autoimmunity. We analyzed relapsing–remitting multiple sclerosis (MS) patients by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3, and the IL7R (CD127). Further, the level of Tregs was compared in remitting and relapsing patients and correlated with disease duration. Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls (p < 0.05), indicating that Tregs attempt to restrain immune activity during relapse. The proportion of Tregs tended to be decreased with disease duration, while CD25⁺CD4⁺ and CD25⁺CD8⁺ effector T-cell proportions were elevated and positively correlated with overall disease duration (p < 0.05). In conclusion, while MS patients in remission have normal levels of Tregs of different phenotype, relapsing patients show an increased proportion of systemic CD25^? FoxP3⁺ Tregs. With time, the proportion of Tregs decrease while effector T cells expand.     

FasL Expression on Human Nucleus Pulposus Cells Contributes to the Immune Privilege of Intervertebral Disc by Interacting with Immunocytes

The mechanisms of immune privilege in human nucleus pulposus (NP) remain unclear. Accumulating evidence indicates that Fas ligand (FasL) might play an important role in the immune privilege of the disc. We aimed for addressing the role of FasL expression in human intervertebral disc degeneration (IDD) and immune privilege in terms of the interaction between NP cells and immunocytes via the FasL-Fas machinery. We collected NP specimens from 20 patients with IDD as degenerative group and 8 normal cadaveric donors as control. FasL expression was detected by qRT-PCR, western blotting and flow cytometry (FCM). We also collected macrophages and CD8⁺ T cells from the peripheral blood of patients with IDD for co-cultures with NP cells. And macrophages and CD8⁺ T cells were harvested for apoptosis analysis by FCM after 2 days of co-cultures. We found that FasL expression in mRNA, protein and cellular resolutions demonstrated a significant decrease in degenerative group compared with normal control (p<0.05). FCM analysis found that human NP cells with increased FasL expression resulted in significantly increased apoptosis ratio of macrophages and CD8⁺ T cells. Our study demonstrated that FasL expression tends to decrease in degenerated discs and FasL plays an important role in human disc immune privilege, which might provide a novel target for the treatment strategies for IDD.     

IFNβ-1a therapy for multiple sclerosis expands regulatory CD8 T cells and decreases memory CD8 subset: A longitudinal 1-year study

The beneficial effects of interferon β-1a (IFNβ-1a) in multiple sclerosis (MS) remain only partially understood. CD8⁺ T cells are key cells in MS pathogenesis that contribute to axonal damage in MS, whereas CD4⁺ regulatory T cells (T_Reg) and CD8⁺ regulatory/suppressor T cells (Ts) play an important role in protecting against subsequent MS activity. We analysed ex vivo changes on T_Reg and on the different subsets of CD4⁺ and CD8⁺ T lymphocytes, before IFNβ-1a (Rebif) therapy and at 3, 6, and 12 months after treatment, in 23 MS patients and in 26 healthy controls. IFNβ-1a significantly increased the proportions of CD4⁺ T_Reg and regulatory CD8⁺ T cells (Tr). Memory CD8⁺ T cells were significantly decreased after 1 year of treatment, maybe reflecting down-regulation of abnormally persistent systemic activation in MS patients. After 1 year of IFNβ-1a, a direct correlation was observed between plasmacytoid dendritic cells and effector CD8⁺ T cells.