Alirio J. Melendez

IL-33 Exacerbates Autoantibody-Induced Arthritis

Rheumatoid arthritis pathogenesis comprises dysregulation in both innate and adaptive immunity. There is therefore intense interest in the factors that integrate these immunologic pathways in rheumatoid arthritis. In this paper, we report that IL-33, a novel member of the IL-1 family, can exacerbate anti–glucose-6-phosphate isomerase autoantibody-induced arthritis (AIA). Mice lacking ST2 (ST2−/−), the IL-33 receptor α-chain, developed attenuated AIA and reduced expression of articular proinflammatory cytokines. Conversely, treatment of wild-type mice with rIL-33 significantly exacerbated AIA and markedly enhanced proinflammatory cytokine production. However, IL-33 failed to increase the severity of the disease in mast cell-deficient or ST2−/− mice. Furthermore, mast cells from wild-type, but not ST2−/−, mice restored the ability of ST2−/− recipients to mount an IL-33–mediated exacerbation of AIA. IL-33 also enhanced autoantibody-mediated mast cell degranulation in vitro and in synovial tissue in vivo. Together these results demonstrate that IL-33 can enhance autoantibody-mediated articular inflammation via promoting mast cell degranulation and proinflammatory cytokine production. Because IL-33 is derived predominantly from synovial fibroblasts, this finding provides a novel mechanism whereby a host tissue-derived cytokine can regulate effector adaptive immune response via enhancing innate cellular activation in inflammatory arthritis.

A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium

BACKGROUND Leukocytes express Fc gamma receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody-directed cellular cytotoxicity. The nature of the myeloid response to Fc gamma receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability. RESULTS We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fc gamma receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor Fc gamma RI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the gamma chain, an Fc gamma RI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of Fc gamma RI resulted in the Fc gamma RIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated. CONCLUSIONS The switch in Fc gamma RI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by Fc gamma RI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.

C‐reactive protein‐mediated phagocytosis and phospholipase D signalling through the high‐affinity receptor for immunoglobulin G (FcγRI)

C‐reactive protein (CRP) is the prototypic acute‐phase protein in man which performs innate immune functions. CRP‐mediated phagocytosis may be indirect, through activation of complement and complement receptors, or direct, through receptors for the Fc portion of immunoglobulin G (IgG; FcγRs) or even a putative CRP‐specific receptor. No strong evidence has been shown to indicate which receptors may be responsible for phagocytosis or signalling responses. Using BIAcore technology, we confirm that CRP binds directly to the extracellular portion of FcγRI with a threefold higher affinity than IgG (KD = 0·81 × 10−9 m). Binding is Ca2+ dependent and is inhibited by IgG1 but not by phosphorylcholine (PC). CRP opsonization (using CRP concentrations within the normal human serum range) of PC‐conjugated sheep erythrocytes increased phagocytosis of these particles by COS‐7 cells transfected with FcγRI‐II chimaera or FcγRI/γ‐chain. Interferon‐γ‐treated U937 cells, which signal through FcγRI to activate phospholipase D (PLD) in response to cross‐linked IgG, were also activated by CRP without any requirement for further cross‐linking. These studies indicate that CRP is capable of binding to and cross‐linking FcγRI thereby resulting in PLD activation and increased phagocytosis. Uptake by FcγRI has been reported to promote various acquired immune responses suggesting that CRP could act in a similar way.

Differentiation‐dependent switch in protein kinase C isoenzyme activation by FcγRI, the human high‐affinity receptor for immunoglobulin G

Aggregation of receptors for the constant region (Fc) of immunoglobulin G on myeloid cells results in endocytosis or phagocytosis and cellular activation. Previous work has shown, using the cell line U937, that the high‐affinity immunoglobulin G receptor, FcγRI, activates alternate intracellular signalling pathways depending on the cell differentiation state, which results in a marked change in the nature of calcium transients within the cell. Here, we show that protein kinase C (PKC) is activated in both interferon‐γ (IFN‐γ) ‐primed and dibutyryl cyclic AMP (dbcAMP) ‐differentiated cells but that the nature of the particular isoenzymes recruited differs. Thus, in IFN‐γ‐primed U937 cells, FcγRI aggregation results in an increase of PKC activity which is essentially calcium independent resulting from the translocation to the membrane of the novel PKCs, δ and ε, together with the atypical PKC ζ. However, in cells differentiated to a more macrophage phenotype, all PKC enzyme activity after receptor aggregation is calcium dependent. Consistent with this finding, the isoenzymes translocated to the nuclear‐free membrane fraction are the conventional PKCs α, β and γ; results consistent with our previous finding that FcγRI couples to phospholipase C in such dbcAMP‐differentiated cells. Thus, the nature of PKC isoenzyme activated following FcγRI aggregation is defined by differentiation. The calcium dependence of the PKC isoenzyme is consistent with the duration of calcium transients previously reported in the two differentiation states.

Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics

Antisense oligonucleotides (As-ODNs) are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt), 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.

Phospholipase D1 Mediates TNFα-Induced Inflammation in a Murine Model of TNFα-Induced Peritonitis

Background Tumor Necrosis Factor alpha (TNFα) is a pleiotropic cytokine extensively studied for its role in the pathogenesis of a variety of disease conditions, including in inflammatory diseases. We have recently shown that, in vitro, that TNFα utilizes PLD1 to mediate the activation of NFκB and ERK1/2 in human monocytes. The aim of this study was to investigate the role(s) played by phospholipase D1 (PLD1) in TNFα-mediated inflammatory responses in vivo. Methodology/Findings Studies were performed in vivo using a mouse model of TNFα-induced peritonitis. The role of PLD1 was investigated by functional genomics, utilizing a specific siRNA to silence the expression of PLD1. Administration of the siRNA against PLD1 significantly reduced PLD1 levels in vivo. TNFα triggers a rapid pyrogenic response, but the in vivo silencing of PLD1 protects mice from the TNFα-induced rise in temperature. Similarly TNFα caused an increase in the serum levels of IL-6, MIP-1α and MIP-1β: this increase in cytokine/chemokine levels was inhibited in mice where PLD1 had been silenced. We then induced acute peritonitis with TNFα. Intraperitoneal injection of TNFα triggered a rapid increase in vascular permeability, and the influx of neutrophils and monocytes into the peritoneal cavity. By contrast, in mice where PLD1 had been silenced, the TNFα-triggered increase in vascular permeability and phagocyte influx was substantially reduced. Furthermore, we also show that the TNFα-mediated upregulation of the cell adhesion molecules VCAM and ICAM1, in the vascular endothelium, were dependent on PLD1. Conclusions These novel data demonstrate a critical role for PLD1 in TNFα-induced inflammation in vivo and warrant further investigation. Indeed, our results suggest PLD1 as a novel target for treating inflammatory diseases, where TNFα play key roles: these include diseases ranging from sepsis to respiratory and autoimmune diseases; all diseases with considerable unmet medical need.

FcγRI activation of phospholipase Cγ1 and protein kinase C in dibutyryl cAMP‐differentiated U937 cells is dependent solely on the tyrosine‐kinase activated form of phosphatidylinositol‐3‐kinase

The human high affinity receptor for immunoglobulin G, FcγRI, in dibutyryl cyclic AMP (dbcAMP)‐differentiated U937 cells, is coupled to the activation of phospholipase C (PLC) and the conventional protein kinase C (PKC) isoforms, α, β, and γ. Here we demonstrate that aggregation of FcγRI activates the tyrosine‐kinase regulated form of phosphatidylinositol‐3‐kinase (PI‐3‐kinase) and that an increase of phosphatidylinositol trisphosphate (PIP3) is essential for the activation and translocation of PLCγ1 in these cells. In addition, activation of the PKC isoforms was ablated by specific inhibitors of PI3‐kinase or by overexpression of a dominant negative p85 subunit of PI3‐kinase. The findings reported here demonstrate that PLCγ1 and PKC activation by FcγRI are downstream of PI3‐kinase, and that in contrast to cytokine primed cells, only the tyrosine‐kinase activated isoform of PI3‐kinase is coupled to FcγRI in dbcAMP‐differentiated cells.

Allergy therapy: The therapeutic potential of targeting sphingosine kinase signalling in mast cells

Mast cell activation is a central event in allergic diseases, and investigating the signalling pathways triggered during mast cell activation may lead to the discovery of novel therapeutic targets. Mast cells can be activated by a multitude of stimuli including antibodies/antigen, cytokines/chemokines and neuropeptides, resulting in a variety of responses including the immediate release of potent inflammatory mediators. Moreover, recent data suggest that mast cell‐mediated responses are also influenced by the differential sphingolipids/sphingosine to sphingosine‐1‐phosphate ratio. The importance of sphingolipids as potent biological mediators of both intracellular and extracellular responses is being increasingly recognized and accepted; it is now appreciated that activation of mast cells, via the high‐affinity IgE‐receptor (FcεRI) leads to the activation of sphingosine kinases (SphK), resulting in increased formation of sphingosine‐1‐phosphate. Furthermore, FcεRI activates SphK‐dependent calcium mobilization in mast cells, leading to degranulation, cytokine, and eicosanoid production, and chemotaxis. In the past two years a critical role for SphK in allergic responses in vivo has emerged. In this review, I focus on the current understanding of the role of sphingosine kinases during mast cell signalling in vitro and their role during hypersensitivity responses in vivo, and discuss the potential of these enzymes as novel therapeutic targets to treat allergic diseases.