Klaus Tenbrock

Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock

To identify new components that regulate the inflammatory cascade during sepsis, we characterized the functions of myeloid-related protein-8 (Mrp8, S100A8) and myeloid-related protein-14 (Mrp14, S100A9), two abundant cytoplasmic proteins of phagocytes. We now demonstrate that mice lacking Mrp8-Mrp14 complexes are protected from endotoxin-induced lethal shock and Escherichia coli–induced abdominal sepsis. Both proteins are released during activation of phagocytes, and Mrp8-Mrp14 complexes amplify the endotoxin-triggered inflammatory responses of phagocytes. Mrp8 is the active component that induces intracellular translocation of myeloid differentiation primary response protein 88 and activation of interleukin-1 receptor–associated kinase-1 and nuclear factor-κB, resulting in elevated expression of tumor necrosis factor-α (TNF-α). Using phagocytes expressing a nonfunctional Toll-like receptor 4 (TLR4), HEK293 cells transfected with TLR4, CD14 and MD2, and by surface plasmon resonance studies in vitro, we demonstrate that Mrp8 specifically interacts with the TLR4-MD2 complex, thus representing an endogenous ligand of TLR4. Therefore Mrp8-Mrp14 complexes are new inflammatory components that amplify phagocyte activation during sepsis upstream of TNFα–dependent effects.

Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV

Systemic lupus erythematosus (SLE) T cells express high levels of cAMP response element modulator (CREM) that binds to the IL-2 promoter and represses the transcription of the IL-2 gene. This study was designed to identify pathways that lead to increased binding of CREM to the IL-2 promoter in SLE T cells. Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) was found to be increased in the nucleus of SLE T cells and to be involved in the overexpression of CREM and its binding to the IL-2 promoter. Treatment of normal T cells with SLE serum resulted in increased expression of CREM protein, increased binding of CREM to the IL-2 promoter, and decreased IL-2 promoter activity and IL-2 production. This process was abolished when a dominant inactive form of CaMKIV was expressed in normal T cells. The effect of SLE serum resided within the IgG fraction and was specifically attributed to anti-TCR/CD3 autoantibodies. This study identifies CaMKIV as being responsible for the increased expression of CREM and the decreased production of IL-2 in SLE T cells and demonstrates that anti-TCR/CD3 antibodies present in SLE sera can account for the increased expression of CREM and the suppression of IL-2 production.

Glucocorticoids induce an activated, anti-inflammatory monocyte subset in mice that resembles myeloid-derived suppressor cells

Glucocorticoids (GC) are still the most widely used immunosuppressive agents in clinical medicine. Surprisingly, little is known about the mechanisms of GC action on monocytes, although these cells exert pro‐ and anti‐inflammatory effects. We have shown recently that GC induce a specific monocyte phenotype with anti‐inflammatory properties in humans. We now investigated whether this also applies for the murine system and how this subset would relate to recently defined murine subtypes. After treatment with dexamethasone for 48 h, monocytes up‐regulated scavenger receptor CD163 and Gr‐1, down‐regulated CX3CR1, and shared with human GC‐treated monocytes functional features such as low adhesiveness but high migratory capacity. They specifically up‐regulated anti‐inflammatory IL‐10, but not TGF‐β, and in contrast to their human counterparts, they down‐regulated IL‐6. Although GC‐induced monocytes down‐regulated CX3CR1, a distinctive marker for classical/proinflammatory human and murine monocytes (CX3CR1loCCR2+Ly6Chi), they differed from this physiologically occurring subset, as they remained Ly6Cmed and unactivated (CD62 ligand++). In addition to their immunosuppressive effects, they were CD11b+Gr‐1+ and expressed the IL‐4Rα chain (CD124), a recently described, signature molecule of tumor‐induced myeloid‐derived suppressor cells (MDSC). We therefore generated murine MDSC in B16 melanoma‐bearing mice and indeed found parallel up‐regulation of CD11b+Gr‐1+ and CD124 on GC‐induced monocytes and MDSC. These data allow us to speculate that the GC‐induced subtype shares with inflammatory monocytes the ability to migrate quickly into inflamed tissue, where they, however, exert anti‐inflammatory effects and that similarities between GC‐induced monocytes and MDSC may be involved in progression of some tumors observed in patients chronically treated with GC.

Rewiring the T-cell: signaling defects and novel prospects for the treatment of SLE

Abstract Activation of T cells from patients with systemic lupus erythematosus (SLE) leads to increased signaling responses, detected by increased calcium and protein tyrosine phosphorylation patterns. This overexcitability occurs in spite of decreased levels of T-cell receptor ζ chain. The replacement of the ζ chain by the Fc receptor (FcR) γ chain and the formation of signaling molecule aggregates on the surface of T cells are considered to be responsible for the observed signaling phenotype. Decreased production of the ζ-chain promoter binding form of the transcription factor Elf-1 is responsible for the decreased transcription of the ζ chain gene. In addition, transcription of the interleukin-2 (IL-2) gene is decreased because of the presence of the transcriptional repressor cyclic adenine mono-phosphate (cAMP) response element modulator. Replenishment of the ζ chain and elimination of the repressor by antisense approaches leads to increased expression of IL-2, suggesting that gene therapy approaches might represent tangible modalities in the treatment of human SLE.

Inflammatory Cytokines in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown origin affecting virtually all organ systems. Beyond genetic and environmental factors, cytokine imbalances contribute to immune dysfunction, trigger inflammation, and induce organ damage. The key cytokine that is involved in SLE pathogenesis is interferon alpha. Interferon secretion is induced by immune complexes and leads to upregulation of several inflammatory proteins, which account for the so-called IFN signature that can be found in the majority of SLE PBMCs. Additionally IL-6 and IFN-y as well as T-cell-derived cytokines like IL-17, IL-21, and IL-2 are dysregulated in SLE. The latter induce a T-cell phenotype that is characterized by enhanced B-cell help and enhanced secretion of proinflammatory cytokines but reduced induction of suppressive T cells and activation-induced cell death. This paper will focus on these cytokines and highlights pathophysiological approaches and therapeutic potential.

Antisense Cyclic Adenosine 5′-Monophosphate Response Element Modulator Up-Regulates IL-2 in T Cells from Patients with Systemic Lupus Erythematosus

The cAMP response element modulator (CREM) has been shown to bind specifically to the −180 site of the IL-2 promoter in vitro. CREM protein is increased in T cells of patients with systemic lupus erythematosus (SLE), and it has been considered responsible for the decreased production of IL-2. In this work we show that transcriptional up-regulation is responsible for the increased CREM protein levels and that CREM binds to the IL-2 promoter in live SLE T cells. Suppression of the expression of CREM mRNA and protein by an antisense CREM plasmid, which was force expressed in SLE T cells by electroporation, resulted in decreased CREM protein binding to the IL-2 promoter and increased expression of IL-2 mRNA and protein. Our data demonstrate that antisense constructs can be used to effectively eliminate the expression of a transcriptional repressor. This approach can be used therapeutically in conditions where increased production of IL-2 is desired.

cAMP-responsive element modulator (CREM)α protein induces interleukin 17A expression and mediates epigenetic alterations at the interleukin-17A gene locus in patients with systemic lupus erythematosus.

Background: Expression levels of both IL-17A and transcription factor CREMα are increased in T cells from SLE patients. Results: In primary human T cells, CREMα binds to the proximal IL17A promoter and induces IL-17A expression by transcriptional activation and epigenetic modifications. Conclusion: CREMα promotes IL-17A expression. Significance: Suppression of CREMα expression should mitigate IL-17A-driven inflammatory responses. IL-17A is a proinflammatory cytokine that is produced by specialized T helper cells and contributes to the development of several autoimmune diseases such as systemic lupus erythematosus (SLE). Transcription factor cAMP-responsive element modulator (CREM)α displays increased expression levels in T cells from SLE patients and has been described to account for aberrant T cell function in SLE pathogenesis. In this report, we provide evidence that CREMα physically binds to a cAMP-responsive element, CRE (−111/−104), within the proximal human IL17A promoter and increases its activity. Chromatin immunoprecipitation assays reveal that activated naïve CD4+ T cells as well as T cells from SLE patients display increased CREMα binding to this site compared with T cells from healthy controls. The histone H3 modification pattern at the CRE site (−111/−104) and neighboring conserved noncoding sequences within the human IL17A gene locus suggests an accessible chromatin structure (H3K27 hypomethylation/H3K18 hyperacetylation) in activated naïve CD4+ T cells and SLE T cells. H3K27 hypomethylation is accompanied by decreased cytosine phosphate guanosine (CpG)-DNA methylation in these regions in SLE T cells. Decreased recruitment of histone deacetylase (HDAC)1 and DNA methyltransferase (DNMT)3a to the CRE site (−111/−104) probably accounts for the observed epigenetic alterations. Reporter studies confirmed that DNA methylation of the IL17A promoter indeed abrogates its inducibility. Our findings demonstrate an extended role for CREMα in the immunopathogenesis of SLE because it contributes to increased expression of IL-17A.

The Cyclic Adenosine 5′-Monophosphate Response Element Modulator Suppresses IL-2 Production in Stimulated T Cells by a Chromatin-Dependent Mechanism

The production of IL-2 is tightly controlled by several transcription factors that bind to the IL-2 promoter. The cAMP response element modulator (CREM) is known to form complexes with CREB and bind to the −180 site of the IL-2 promoter in anergic and in systemic lupus erythematosus T cells. In this study we show that CREM is transcriptionally induced in T cells following stimulation through CD3 and CD28, binds to the IL-2 promoter in vivo, and suppresses IL-2 production. Transfection of an antisense CREM plasmid into T cells blocked the expression and binding of CREM to the IL-2 promoter and the decrease of IL-2 production, which follows the early increase after T cell stimulation with CD3 and CD28. In addition, as assessed by chromatin immunoprecipitation experiments, antisense CREM prevented the binding of protein 300 and cAMP response element binding protein and promoted the acetylation of histones. Antisense CREM also enhanced the accessibility of the IL-2 promoter to endonucleases and prevented the condensation of chromatin in vivo. Our data suggest that upon T cell activation, CREM gradually replaces phosphorylated CREB at the −180 site of the IL-2 promoter. CREM, in turn, binds protein 300 and cAMP response element binding protein, but CREM is unable to activate its histone acetyltransferase activity, which results in condensation of chromatin and down-regulation of IL-2 production.

Defective Production of Functional 98-kDa Form of Elf-1 Is Responsible for the Decreased Expression of TCR ζ-Chain in Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE), the prototypic autoimmune disease, is characterized by defective expression of TCR ζ-chain. Elf-1 (E-74-like factor) is a member of the Ets (E-26-specific) family and is crucial for the basal transcription of TCR ζ-chain in Jurkat cells. We previously demonstrated that Elf-1 exists in the cytoplasm mainly as 80-kDa form and after phosphorylation and O-glycosylation it moves to the nucleus as a 98-kDa which binds DNA. We now demonstrate that Elf-1 is crucial for the transactivation of TCR ζ-chain promoter in normal and SLE T cells. Defective expression of TCR ζ-chain in SLE T cells is associated with two distinct molecular defects in the generation of the 98-kDa DNA binding Elf-1 form. In the first, the levels of the 98-kDa form were either decreased or absent. In the second, the apparent levels of the nuclear Elf-1 form were normal but included only two of the three bands into which the nuclear Elf-1 form separated in isoelectric focusing gels. Because both the transcription and the translation processes of Elf-1 gene are normal in SLE T cells, our data demonstrate that abnormal posttranslational mechanisms of the Elf-1 protein result in defective expression of functional Elf-1, and consequently, the transcriptional defect of TCR ζ-chain in patients of SLE.

Regulatory T cells in systemic lupus erythematosus

Systemic lupus erythematosus (SLE), an autoimmune disease, develops when immunologic self‐tolerance fails. Treg cells are a subset of CD4+ T cells that maintain self‐tolerance by suppressing autoreactive lymphocytes. Defects in Treg cells are therefore considered to be an aspect of SLE pathogenesis. Nevertheless, reports on the numbers and function of Treg cells in SLE are contradictory and the definitive role of Treg cells in SLE remains unclear. In this review, we summarize findings from murine models and ex vivo experiments, which provide insights into the mechanisms that result in the breakdown of tolerance. We also include recent findings about Treg‐cell subsets and their markers in human SLE. The identification of unique markers to identify bona fide Treg cells, as well as therapies to reconstitute the balance between Treg cells and autoreactive T cells in SLE, are the future challenges for SLE research.