Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis

The endothelial cell monolayer of cerebral vessels and its basement membrane (BM) are ensheathed by the astrocyte endfeet, the leptomeningeal cells, and their associated parenchymal BM, all of which contribute to establishment of the blood–brain barrier (BBB). As a consequence of this unique structure, leukocyte penetration of cerebral vessels is a multistep event. In mouse experimental autoimmune encephalomyelitis (EAE), a widely used central nervous system inflammatory model, leukocytes first penetrate the endothelial cell monolayer and underlying BM using integrin β1-mediated processes, but mechanisms used to penetrate the second barrier defined by the parenchymal BM and glia limitans remain uninvestigated. We show here that macrophage-derived gelatinase (matrix metalloproteinase [MMP]-2 and MMP-9) activity is crucial for leukocyte penetration of the parenchymal BM. Dystroglycan, a transmembrane receptor that anchors astrocyte endfeet to the parenchymal BM via high affinity interactions with laminins 1 and 2, perlecan and agrin, is identified as a specific substrate of MMP-2 and MMP-9. Ablation of both MMP-2 and MMP-9 in double knockout mice confers resistance to EAE by inhibiting dystroglycan cleavage and preventing leukocyte infiltration. This is the first description of selective in situ proteolytic damage of a BBB-specific molecule at sites of leukocyte infiltration

Common Interactions between S100A4 and S100A9 Defined by a Novel Chemical Probe.

S100A4 and S100A9 proteins have been described as playing roles in the control of tumor growth and metastasis. We show here that a chemical probe, oxyclozanide (OX), selected for inhibiting the interaction between S100A9 and the receptor for advanced glycation end-products (RAGE) interacts with both S100A9 and S100A4. Furthermore, we show that S100A9 and S100A4 interact with RAGE and TLR4; interactions that can be inhibited by OX. Hence, S100A4 and S100A9 display similar functional elements despite their primary sequence diversity. This was further confirmed by showing that S100A4 and S100A9 dimerize both in vitro and in vivo. All of these interactions required levels of Zn(++) that are found in the extracellular space but not intracellularly. Interestingly, S100A4 and S100A9 are expressed by distinct CD11b(+) subpopulations both in healthy animals and in animals with either inflammatory disease or tumor burden. The functions of S100A9 and S100A4 described in this paper, including heterodimerization, may therefore reflect S100A9 and S100A4 that are released into the extra-cellular milieu

Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9. Methods: Flow cytometry, confocal microscopy and real-time PCR of flow cytometry-sorted cells were used to measure cell surface S100A8/A9, intracellular S100A8/A9 and mRNA levels of S100A8 and S100A9, respectively. Results: Cell surface S100A8/A9 was detected on all leukocyte subpopulations investigated except for T cells. By confocal microscopy, real-time PCR and stimulation assays, we could demonstrate that pDCs, monocytes and PMNs could synthesize S100A8/A9. Furthermore, pDC cell surface S100A8/A9 was higher in patients with active disease as compared to patients with inactive disease. Upon immune complex stimulation, pDCs up-regulated the cell surface S100A8/A9. SLE patients had also increased serum levels of S100A8/A9. Conclusions: Patients with SLE had increased cell surface S100A8/A9, which could be important in amplification and persistence of inflammation. Importantly, pDCs were able to synthesize S100A8/A9 proteins and up-regulate the cell surface expression upon immune complex-stimulation. Thus, S100A8/A9 may be a potent target for treatment of inflammatory diseases such as SLE

SMAC mimetics promote NIK-dependent inhibition of CD4+ TH17 cell differentiation

Second mitochondria-derived activator of caspase (SMAC) mimetics (SMs) are selective antagonists of the inhibitor of apoptosis proteins (IAPs), which activate noncanonical NF-κB signaling and promote tumor cell death. Through gene expression analysis, we found that treatment of CD4+ T cells with SMs during T helper 17 (TH17) cell differentiation disrupted the balance between two antagonistic transcription factor modules. Moreover, proteomics analysis revealed that SMs altered the abundance of proteins associated with cell cycle, mitochondrial activity, and the balance between canonical and noncanonical NF-κB signaling. Whereas SMs inhibited interleukin-17 (IL-17) production and ameliorated TH17 cell–driven inflammation, they stimulated IL-22 secretion. Mechanistically, SM-mediated activation of NF-κB–inducing kinase (NIK) and the transcription factors RelB and p52 directly suppressed Il17a expression and IL-17A protein production, as well as the expression of a number of other immune genes. Induction of IL-22 production correlated with the NIK-dependent reduction in cMAF protein abundance and the enhanced activity of the aryl hydrocarbon receptor. Last, SMs also increased IL-9 and IL-13 production and, under competing conditions, favored the differentiation of naïve CD4+ T cells into TH2 cells rather than TH17 cells. These results demonstrate that SMs shape the gene expression and protein profiles of TH17 cells and inhibit TH17 cell–driven autoimmunity

Specific effect of immunomodulatory quinoline-3-carboxamide ABR-215757 in GM-CSF stimulated bone marrow cell cultures: Block of initiation of proliferation of Gr-1(+) cells.

Quinoline-3-carboxamides are currently in clinical development for treatment of both autoimmune disease and cancer. Carboxamides such as ABR-215757 (5757) have shown efficacy in several in vivo mouse models of human inflammatory autoimmune disease. Some microbial infections in mice cause GM-CSF dependent accumulation of dendritic cells expressing TNFα and inducible nitric oxide synthase (iNOS; Tip-DCs) in lymphoid organs. Functionally similar DCs develop in GM-CSF stimulated bone marrow (BM) cell cultures and offered an in vitro model that allowed us to study the impact of 5757 on cellular development of relevance for in vivo inflammatory conditions. We show in here that addition of 5757 to such cultures, in a dose-dependent way increased the frequency of DCs, while it reduced the frequency of Gr-1(+) cells by inhibiting their proliferation. This effect was specific as the compound neither influenced DC development from myeloid progenitors, nor the development of granulocytes in G-CSF stimulated BM cell cultures. Importantly, we also show that 5757 treatment reduced the accumulation of Gr-1(+) cells during inflammation in vivo. We therefore propose that this compound may ameliorate autoimmune disease by blocking proliferation of Gr-1(+) cells during inflammation-induced mobilization of myeloid cells

Early TCR alpha beta expression promotes maturation of T cells expressing Fc epsilon RI gamma containing TCR/CD3 complexes

In a previous study we presented data indicating that the expanded population of CD4(-)CD8(-) (DN) alphabeta T cells in TCRalpha-chain-transgenic mice was partially if not entirely derived from gammadelta T cell lineage cells. The development of both gammadelta T cells and DN alphabeta T cells is poorly understood; therefore, we thought it would be important to identify the immediate precursors of the transgene-induced DN alphabeta T cells. We have in this report studied the early T cell development in these mice and we show that the transgenic TCRalpha-chain is expressed by precursor thymocytes already at the CD3(-)CD4(-)CD8(-) (triple negative, TN) CD44(+)CD25(-) stage of development. Both by using purified precursor populations in reconstitution experiments and by analyzing fetal thymocyte development, we demonstrated that early TN precursors expressing endogenous TCRbeta-chains matured into DN alphabeta T cells at several stages of development. The genes encoding the gamma-chain of the high affinity receptor for IgE (FcepsilonRIgamma) and the CD3zeta protein were found to be reciprocally expressed in TN thymocytes such that during development the FcepsilonRIgamma expression decreased whereas CD3zeta expression increased. Furthermore, in a fraction of the transgene-induced DN alphabeta T cells the FcepsilonRIgamma protein colocalized with the TCR/CD3 complex. These data suggest that similarly to gammadelta T cells and NKT cells, precursors expressing the TCR early in the common alphabetagammadelta developmental pathway may use the FcepsilonRIgamma protein as a signaling component of the TCR/CD3 complex

S100A9 and tumor growth.

We have investigated the role of the Ca(2+)-binding protein S100A9 on tumor growth in prostate cancer and T-cell lymphoma models. We found that the expression of, S100A9 and its interaction with Toll-like receptor 4 (TLR4) is critical for tumor growth in these settings