CD138 Expression Promotes Accumulation and Activation of T Cells in Autoimmune MRL/lpr Mice

CD138+ T cells that accumulated in Fas-deficiency lupus mice, had been identified as autoreactive T cells in SLE which significantly promoted autoantibody secretion. In present study, we found CD138 expression in T cells played a key role in the progression of SLE in MRL/lpr mice. Our results indicated CD138+ T cells apoptosis was in Fas dependent way. However, CD138 expression of T cells in MRL/lpr mice could significantly prevent T cells apoptosis, contribute to accumulation of T cells and DN T cells and simultaneously promote T cells activation. Importantly, CD138 expression in DN T cells significantly increased FasL expression of DN T cells enhancing the cytotoxity of DN T cells. Phorbol 12-myristate 13-acetate and Ionomycin (PI) stimulation could significantly prevent CD138+ T cells accumulation by strikingly inducing their specific apoptosis. Moreover, PI stimulation significantly activated CD138+ T cells with increased CD69 expression in them. Importantly, our results showed CD69 expression in CD138+ T cells could significantly increase the apoptosis level of them. That indicated PI stimulation could induce specific apoptosis of CD138+ T cells via increasing CD69 expression in CD138+ T cells. In addition, our results showed CD138- T cells in MRL/lpr mice had significant defects in activation. However, to activate T cells could significantly prevent CD138 expression in CD3+ T cells of MRL/lpr mice. Our results suggested CD138 expression in CD3+ T cells of MRL/lpr mice was probably caused by the failure of activation in autoreactive T cells before self-antigens exposure to immune system.

Naja atra Cardiotoxin 1 Induces the FasL/Fas Death Pathway in Human Leukemia Cells

This study aimed to investigate the mechanistic pathway of Naja atra (Taiwan cobra) cardiotoxin 1 (CTX1)–induced death of leukemia cell lines U937 and HL-60. CTX1 increased cytoplasmic Ca2+ and reactive oxygen species (ROS) production, leading to the death of U937 cells. It was found that Ca2+-induced NOX4 upregulation promoted ROS-mediated p38 MAPK phosphorylation, which consequently induced c-Jun and ATF-2 phosphorylation. Using siRNA knockdown, activated c-Jun and ATF-2 were demonstrated to regulate the expression of Fas and FasL, respectively. Suppression of Ca2+-mediated NOX4 expression or ROS-mediated p38 MAPK activation increased the survival of U937 cells exposed to CTX1. FADD depletion abolished CTX1-induced cell death, caspase-8 activation, and t-Bid production, supporting the correlation between the Fas death pathway and CTX1-mediated cytotoxicity. Among the tested N. atra CTX isotoxins, only CTX1 induced Fas and FasL expression. Chemical modification studies revealed that intact Met residues were essential for the activity of CTX1 to upregulate Fas and FasL expression. Taken together, the data in this study indicate that CTX1 induces c-Jun-mediated Fas and ATF-2-mediated FasL transcription by the Ca2+/NOX4/ROS/p38 MAPK axis, thereby activating the Fas death pathway in U937 cells. Furthermore, CTX1 activates Fas/FasL death signaling in the leukemia cell line HL-60.

IFN-β Acts on Monocytes to Ameliorate CNS Autoimmunity by Inhibiting Proinflammatory Cross-Talk Between Monocytes and Th Cells

IFN-β has been the treatment for multiple sclerosis (MS) for almost three decades, but understanding the mechanisms underlying its beneficial effects remains incomplete. We have shown that MS patients have increased numbers of GM-CSF+ Th cells in circulation, and that IFN-β therapy reduces their numbers. GM-CSF expression by myelin-specific Th cells is essential for the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. These findings suggested that IFN-β therapy may function via suppression of GM-CSF production by Th cells. In the current study, we elucidated a feedback loop between monocytes and Th cells that amplifies autoimmune neuroinflammation, and found that IFN-β therapy ameliorates central nervous system (CNS) autoimmunity by inhibiting this proinflammatory loop. IFN-β suppressed GM-CSF production in Th cells indirectly by acting on monocytes, and IFN-β signaling in monocytes was required for EAE suppression. IFN-β increased IL-10 expression by monocytes, and IL-10 was required for the suppressive effects of IFN-β. IFN-β treatment suppressed IL-1β expression by monocytes in the CNS of mice with EAE. GM-CSF from Th cells induced IL-1β production by monocytes, and, in a positive feedback loop, IL-1β augmented GM-CSF production by Th cells. In addition to GM-CSF, TNF and FASL expression by Th cells was also necessary for IL-1β production by monocyte. IFN-β inhibited GM-CSF, TNF, and FASL expression by Th cells to suppress IL-1β secretion by monocytes. Overall, our study describes a positive feedback loop involving several Th cell- and monocyte-derived molecules, and IFN-β actions on monocytes disrupting this proinflammatory loop.

A Fas/FasL mediated Feed-forward Controller of T-cell Differentiation Reconciles MIS-C and Discrepant Youth and Adult Covid-19 Severity

Fas expression is quickly upregulated on CD8+ T cells following stimulation, while FasL expression is limited to Tcm and later. A phenomenon of T cell differentiation via paracrine Fas signal has been previously described. Here, we describe such differentiation in a pool fits the Feed-forward model which can correct for disturbances in the system, as seen during in vitro T cell stimulation. This feed-forward controller exerts control via Fas/ FasL expression, and the effect is uncoupled with use of lz-FasL. Interestingly, the feed-forward model provides us with evolutionary insight as to why Fas stimulation becomes apoptotic at terminal differentiation, in order to exhibit a perfect and extinguished control and response.

Repeated Intrathecal Administration of Ropivacaine Hydrochloride Induces Apoptosis Via Up-Regulating of Fas/FasL Expression in the Rat Spinal Cord

Background: Ropivacaine hydrochloride (RH) is a local anesthetic and frequently used for perioperative anesthesia and analgesia; but it has potential neurotoxicity, especially when intrathecally used repeatedly for a long time, although the exact mechanism is unclear.Methods: In this study, the expression of Factor associated suicide receptor (Fas) and its ligand (FasL) in the spinal cord cells was detected in rats receiving repeated intrathecal injection of RH. The paw mechanical withdraw threshold (MWT) was measured before and 3 days after intrathecal cannulation and 24 h after intrathecal administration. Rats received intrathecal injection of 0.5%, 1%, 2% RH at 0.12 ml/kg or saline of equal volume alone. Intrathecal injection was done 8 times with an interval of 1.5h in 12 h. The spinal cord was collected for pathological examination; TUNEL staining was used to assess the apoptosis in the spinal cord and the expression of Fas, FasL, caspase-3, and caspase-8 was detected by qPCR and Western blotting.Results: 1% and 2% RH groups significantly increased the MWT (P<0.05) and more vacuoles or edema was observed in the spinal cord after RH treatment. The apoptosis rates and expression of Fas, FasL, caspase-3, and caspase-8 increased in the RH groups (P<0.05).Conclusions: Repeated intrathecal administration of RH may cause damage to the spinal cord and induce apoptosis in the spinal cord via up-regulating Fas/FasL expression.

Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3′-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes.

Polymyxin-Induced Cell Death of Human Macrophage-Like THP-1 and Neutrophil-Like HL-60 Cells Associated with the Activation of Apoptotic Pathways

ABSTRACT Innate immunity is crucial for the host to defend against infections, and understanding the effect of polymyxins on innate immunity is important for optimizing their clinical use. In this study, we investigated the potential toxicity of polymyxins on human macrophage-like THP-1 and neutrophil-like HL-60 cells. Differentiated THP-1 human macrophages (THP-1-dMs) and HL-60 human neutrophils (HL-60-dNs) were employed. Flow cytometry was used to measure the concentration-dependent effects (100 to 2,500 μM for THP-1-dMs and 5 to 2,500 μM for HL-60-dNs) and time-dependent effects (1,000 μM for THP-1-dMs and 300 μM for HL-60-dNs) of polymyxin B over 24 h. Effects of polymyxin B on mitochondrial activity, activation of caspase-3, caspase-8, and caspase-9, and Fas ligand (FasL) expression in both cell lines were examined using fluorescence imaging, colorimetric, and fluorometric assays. In both cell lines, polymyxin B induced concentration- and time-dependent loss of viability at 24 h with 50% effective concentration (EC50) values of 751.8 μM (95% confidence interval [CI], 692.1 to 816.6 μM; Hill slope, 3.09 to 5.64) for THP-1-dM cells and 175.4 μM (95% CI, 154.8 to 198.7 μM; Hill slope, 1.42 to 2.21) for HL-60-dN cells. A concentration-dependent loss of mitochondrial membrane potential and generation of mitochondrial superoxide was also observed. Polymyxin B-induced apoptosis was associated with concentration-dependent activation of all three tested caspases. The death receptor apoptotic pathway activation was demonstrated by a concentration-dependent increase of FasL expression. For the first time, our results reveal that polymyxin B induced concentration- and time-dependent cell death in human macrophage-like THP-1 and neutrophil-like HL-60 cells associated with mitochondrial and death receptor apoptotic pathways.