Satoshi Jodo

Up-regulated expression of Fas antigen (CD95) by peripheral naive and memory T cell subsets in patients with systemic lupus erythematosus (SLE): a possible mechanism for lymphopenia.

Fas antigen (CD95) is a membrane‐associated molecule that mediates apoptotic cell death and may play a role in the induction and maintenance of T cell tolerance. To elucidate the involvement of Fas antigen in human autoimmune diseases, we analysed Fas antigen expression by peripheral T cells from patients with SLE and rheumatoid arthritis (RA), using three‐colour flow cytometry. Both CD4+ and CD8+ T cell from SLE patients expressed Fas antigen in a higher density than did these cell from healthy donors and from RA patients. Enhancement of Fas antigen density was noted in Fas+CD45RO+ memory T cell from SLE patients. More remarkably, a significant expression of Fas antigen was observed in CD45RO− naive T cells from SLE patients CD4+CD45RO− T cells from SLE patients co‐expressed Fas antigen and early to intermediate activation antigens such as CD25 and CD71, and late activation antigen HLA‐DR in only FashiCD4+ naive T tells. Such up‐regulation of Fas antigen expression in SLE patients seems to be clinically meaningful, because mean fluorescence intensity (MFI) of Fas antigen on CD4+ T cell subsets inversely correlates with the absolute size of CD4+ T tell subsets in peripheral blood of SLE patients. These results suggest that T cells with increased Fas antigen expression may be highly susceptible to apoptotic cell death, in vivo. A putative mechanism for lymphopenia in SLE patients is discussed.

Serum levels of soluble Fas/APO-1 (CD95) and its molecular structure in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases

There are two major forms of the Fas molecule, membranous Fas and soluble Fas (sFas). To clarify the clinical significance of sFas in autoimmune diseases, we designed a sandwich ELISA to determine serum concentrations of sFas and its molecular structure, and we then analysed the correlation between levels of sFas and laboratory findings in patients with SLE and other autoimmune diseases. The levels of serum sFas were significantly higher in SLE patients than in subjects with other autoimmune diseases and in healthy donors, and the frequency of a positive serum sFas was much greater in SLE patients with high SLE disease activity index scores than in those with low scores. In addition, sFas‐positive SLE patients showed a significant difference in various laboratory parameters from sFas‐negative SLE patients. Serial measurements of serum sFas levels in SLE patients with active disease revealed that the elevated level of sFas dramatically decreased with improvement in clinical and laboratory findings, following corticosteroid therapy. We propose that the serum level of sFas can serve as an appropriate marker for evaluating SLE disease activity. Serum sFas is heterogeneous with respect to molecular structure, thus several mechanisms are involved in the generation of sFas.

CD95 (Fas) Ligand-Expressing Vesicles Display Antibody-Mediated, FcR-Dependent Enhancement of Cytotoxicity

Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1–10 μg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1–100 ng/ml significantly enhanced cytotoxicity against the FcR+ LB27.4, M59, and LF+ targets, but not the FcR− Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR+ A20 B lymphoma but not with the FcR− A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR− Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.

Anti-CD95-induced Lethality Requires Radioresistant FcγRII+ Cells A NOVEL MECHANISM FOR FULMINANT HEPATIC FAILURE

The Jo2 anti-mouse CD95 monoclonal antibody induces lethality in mice characterized by hepatocyte death and liver hemorrhage. Mice bearing a defect in Fas expression or in the Fas-mediated apoptotic pathway are resistant to Jo2. Here we show that FcγRII knockout mice or mice with monoclonal antibody-blocked FcγRII are also resistant to Jo2. The critical FcγRII+ cells are radioresistant and could not be reconstituted with splenic cells. Death of sinusoidal lining cells and destruction of sinusoids were observed, consistent with the characteristic liver hemorrhage and the selective FcγRII expression in sinusoidal lining cells but not hepatocytes. Hemorrhage developed coincident with hepatocyte death and the sharp rise of serum alanine aminotransferase and alanine aminotransferase. Invariably, moribund mice showed severe liver hemorrhage and destruction of sinusoids. The data demonstrate a novel mechanism by which the destruction of liver sinusoids, induced by the Jo2-mediated co-engagement of Fas and FcγRII, leads to severe hemorrhage and lethal fulminant hepatitis.

Bioactivities of Fas Ligand-Expressing Retroviral Particles

Culture supernatants from retroviral packaging cells carrying the human Fas ligand (FasL) gene killed both human (Jurkat) and mouse (LB27.4) targets within 5 h of incubation. Cytotoxicity was found both in a fraction ≥500 kDa and a fraction between 50 and 500 kDa. Following ultracentrifugation, the activity in the ≥500-kDa fraction was concentrated in the pellet (FasL vector preparation (VP)), which was also infective when added to NIH-3T3 cells. Both Polybrene and poly-l-lysine significantly enhanced the cytotoxicity of FasL VP but not anti-Fas mAb, soluble FasL (sFasL), and cell-associated FasL. In the presence of Polybrene, FasL VP killed targets that are resistant to anti-Fas mAb and sFasL. The infectivity but not FasL cytotoxicity of FasL VP was sensitive to irradiation and heat shock. By contrast, cytotoxicity of FasL VP could be enhanced or inhibited depending on the doses of anti-FasL mAb. Interestingly, the infectivity of FasL VP was specifically enhanced by anti-FasL mAb, suggesting that a nonviral gene product could be used to regulate the behavior of the retroviral vector. Thus, in addition to expressing potent FasL cytotoxicity, the FasL VP exhibits unique properties heretofore not attributed to anti-Fas mAb, sFasL, and cell-associated FasL. Our study raises the possibility of using the retroviral gene-packaging technology to make powerful, versatile, and regulatable bioactive vesicles expressing a predetermined function of the protein encoded by the target gene.

Novel Negative Regulator of Expression in Fas Ligand (CD178) Cytoplasmic Tail: Evidence for Translational Regulation and against Fas Ligand Retention in Secretory Lysosomes

Fas ligand ((FasL) CD178), a type II transmembrane protein, induces apoptosis of cells expressing the Fas receptor. It possesses a unique cytoplasmic tail (FasLCyt) of 80 aa. As a type II transmembrane protein, the early synthesis of FasLCyt could affect FasL translation by impacting FasL endoplasmic reticulum translocation and/or endoplasmic reticulum retention. Previous studies suggest that the proline-rich domain (aa 43–70) in FasLCyt (FasLPRD) inhibits FasL membrane expression by retaining FasL in the secretory lysosomes. This report shows that deletion of aa 2–33 of FasLCyt dramatically increased total FasL levels and FasL cell surface expression. This negative regulator of FasL expression is dominant despite the presence of FasLPRD. In addition, retention of proline-rich domain-containing FasL in the cytoplasm was not observed. Moreover, we demonstrated that FasLCyt regulates FasL expression by controlling the rate of de novo synthesis of FasL. Our study demonstrated a novel negative regulator of FasL expression in the FasLCyt region and its mechanism of action.

Cutting Edge: Fas Ligand (CD178) Cytoplasmic Tail Is a Positive Regulator of Fas Ligand-Mediated Cytotoxicity

The cytotoxic function of CD178 (Fas ligand (FasL)) is critical to the maintenance of peripheral tolerance and immune-mediated tissue pathology. The active site of FasL resides at the FasL extracellular region (FasLExt) and it functions through binding/cross-linking Fas receptor on target cells. In this study, we report that FasLExt-mediated cytotoxicity is regulated by the FasL cytoplasmic tail (FasLCyt). Deleting the N-terminal 2–70 aa (Δ70) or N-terminal 2–33 aa (Δ33) reduced the cytotoxic strength as much as 30- to 100-fold. By contrast, change in the cytotoxic strength was not observed with FasL deleted of the proline-rich domains (45–74 aa, ΔPRD) in the FasLCyt. Our study identifies a novel function of FasLCyt and demonstrates that FasL2–33, a sequence unique to FasL, is critically required for the optimal expression of FasLExt-mediated cytotoxicity.

Hypothesis: A Recurrent, Moderate Activation Fosters Systemic Autoimmunity – The Apoptotic Roles of TCR, IL-2 and Fas Ligand

Studies of several gene knockout mice suggest an interesting association of a moderate T cell response with systemic autoimmune diseases. In addition, CD95 ligand (FasL) expression in some strains of these mice is impaired. Because FasL is critically involved in regulating peripheral tolerance, there may be a link between autoimmune diseases and a moderate T cell response that cannot activate the FasL gene. Here, we propose that there are two thresholds of T cell activation. When moderately stimulated, T cells can be activated to the low (1st) threshold, which permits the induction of CD40L, IL-2, IL-4, and other components that help the immune response. The high (2nd) activation threshold can only be achieved by a strong and concurrent stimulation through TCR and IL-2R. Once the high threshold is reached, FasL is produced to induce apoptosis of the activated T and B cells. In the absence of the FasL-mediated downregulation, the activated B cells become efficient antigen-presenting cells for self-antigens and excellent responders for T cell help. Such an exacerbating condition, induced by recurrent and moderate activation, favors the development of autoreactive T cells and autoantibody production. Evidence supporting this hypothesis and some predictions that can be tested are described.

Maintenance treatment using abatacept with dose reduction after achievement of low disease activity in patients with rheumatoid arthritis (MATADOR) – A prospective, multicenter, single arm pilot clinical trial

Abstract Objectives: To preliminarily evaluate the feasibility of maintenance therapy with reduced dose of intravenous abatacept (ABT) to 250 mg/body/month after achieving remission or low disease activity (LDA). Patients and methods: RA patients treated with ABT at 13 sites were enrolled in this prospective interventional pilot study during the period between March 2013 and March 2015. Inclusion criteria were (1) age at 20 years or older, (2) under treatment with monthly intravenous ABT at approved doses, (3) DAS28-CRP lower than 2.7 at least for 6 months, (4) agreed to join this trial with written informed consent and (5) body weight under 125 kg. Enrolled patients were maintained with intravenous monthly ABT at a reduced dose of 250 mg/body (MATADOR protocol). The primary end point was the proportion of the patients continued with MATADOR protocol at week 48. MATADOR protocol was discontinued upon disease flare or other reasons such as patients’ request or severe adverse event (AE). Disease activities and structural changes were also evaluated. Results: Fifty-three patients fulfilled the entry criteria and were followed for 1-year. MATADOR protocol was continued for 1-year in 43 (81%) of the evaluated patients. Three patients experienced severe AEs. Mean DAS28-CRP and remission rate were 1.56 and 88% when ABT reduced and 1.80 and 81% at 1-year, respectively. Structural remission was achieved in 34 out of 42 evaluated patients. Conclusions: Reduced dose of intravenous ABT was proposed as a feasible choice for maintenance therapy for RA after achievement of remission/LDA, although further randomized trials would be awaited.

Elderly-Onset Varicella Pneumonia in a Patient With Rheumatoid Arthritis Treated With Tofacitinib

A 68-year-old woman with rheumatoid arthritis, who was treated with tofacitinib, a type of Janus kinase (JAK) inhibitor, was admitted for dyspnea and generalized purpuric vesicular rash, following 3 days of fever and malaise. She developed hypoxia from respiratory insufficiency. On physical examination, the systemic eruptions had an erythematous base and were present in various stages, from maculopapular to vesicular and pustular, with crusting (left). Chest radiograph demonstrated diffuse infiltrates with reticular and nodular lesions in the bilateral lung fields (right). Laboratory findings revealed high varicella-zoster virus (VZV) immunoglobulin M antibody titers and positive VZV-DNA in the serum. The patient denied previous varicella history. Therefore, we diagnosed the patient with chickenpox and pneumonia. The patient fully recovered following treatment with intravenous acyclovir. This article is protected by copyright. All rights reserved.