Mark D. Turner

Proinflammatory Action of the Antiinflammatory Drug Infliximab in Tumor Necrosis Factor Receptor-Associated Periodic Syndrome

OBJECTIVE Tumor necrosis factor receptor (TNFR)-associated periodic syndrome (TRAPS) is an autosomal-dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene. Unlike other autoinflammatory diseases in which anti-TNF therapy is largely a successful treatment option, therapy with the anti-TNF drug infliximab is often ineffective in patients with TRAPS. Moreover, in certain cases, infliximab actually triggers severe episodes of inflammation. The aim of this study was to elucidate the mechanisms underlying such a reaction. METHODS Peripheral blood mononuclear cells (PBMCs) were obtained from patients with TRAPS. Both caspase 3 activity and NF-kappaB subunit activity were determined by enzyme-linked immunosorbent assay. Cytokine secretion was assessed using a specific customized human multiplex bead immunoassay kit. RESULTS Unlike findings in controls, cells from a family of 9 patients, all of whom carried the T50M mutation in TNFRSF1A, failed to respond to infliximab through proapoptotic induction of caspase 3 activity. Instead, we observed enhanced antiapoptotic c-Rel subunit activity, accompanied by a significant increase in secretion of the proinflammatory cytokines interleukin- 1beta (IL-1beta), IL-1 receptor, IL-6, IL-8, and IL-12. CONCLUSION Altered extracellular conformation of TNFRI, resulting from the T50M mutation in TNFRSF1A, results in failure of PBMCs to induce an apoptotic response to infliximab. We hypothesize that failure to shed infliximab-bound TNF/TNFRI from the cell surface of cells from patients with the T50M mutation triggers c-Rel activation, and that this leads to a marked increase in cytokine secretion and an increased proinflammatory response. In light of these findings, we strongly advise caution when prescribing infliximab as anti-TNF therapy to patients with TRAPS.

Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells

Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic β cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in β cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in β cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.

Abnormal tumor necrosis factor receptor I cell surface expression and NF‐κB activation in tumor necrosis factor receptor–associated periodic syndrome

OBJECTIVE Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal-dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene. The cellular mechanisms by which mutations in this gene trigger inflammation are currently unclear. Because NF-kappaB is the major intracellular signaling component inducing secretion of proinflammatory cytokines, we sought to determine whether differences in the clinical phenotype of patients with TRAPS may be attributable to variable effects of TNFRSF1A mutations on TNFRI expression, localization, or NF-kappaB activity. METHODS Peripheral blood mononuclear cells were obtained from patients (following informed consent), and cellular nuclear and cytosolic fractions were generated by subcellular fractionation. Localization of IkappaBalpha and NF-kappaB was determined by Western blotting of the resultant fractions. NF-kappaB subunit activity was determined by enzyme-linked immunosorbent assay analysis and confirmed by electrophoretic mobility shift assay. Subcellular localization of TNFRI was determined by immunofluorescence confocal microscopy or by immunoblotting following affinity isolation of plasma membrane by subcellular fractionation. RESULTS Cells from patients with the fully penetrant C73R mutation had marked activation of the proinflammatory p65 subunit of NF-kappaB. In contrast, cells from patients with the low-penetrant R92Q mutation displayed high levels of DNA binding by the p50 subunit, an interaction previously linked to repression of inflammation. Interestingly, although cells from patients with the C73R mutation have no TNFRI shedding defect, there was nonetheless an unusually high concentration of functional TNFRI at the plasma membrane. CONCLUSION High levels of TNFRI at the cell surface in patients with the C73R mutation hypersensitizes cells to stimulation by TNF, leading to increased NF-kappaB p65 subunit activation and an exaggerated proinflammatory response.

Calpain-10: from genome search to function

Calpain‐10 (CAPN10) is the first diabetes gene to be identified through a genome scan. Many investigators, but not all, have subsequently found associations between CAPN10 polymorphism and type 2 diabetes (T2D) as well as insulin action, insulin secretion, aspects of adipocyte biology and microvascular function. However, this has not always been with the same single nucleotide polymorphism (SNP) or haplotype or the same phenotype, suggesting that there might be more than one disease‐associated CAPN10 variant and that these might vary between ethnic groups and the phenotype under study. Our understanding of calpain‐10 physiological action has also been greatly augmented by our knowledge of the calpain family domain structure and function, and the relationship between calpain‐10 and other calpains is discussed here. Both genetic and functional data indicates that calpain‐10 has an important role in insulin resistance and intermediate phenotypes, including those associated with the adipocyte. In this regard, emerging evidence would suggest that calpain‐10 facilitates GLUT4 translocation and acts in reorganization of the cytoskeleton. Calpain‐10 is also an important molecule in the β‐cell. It is likely to be a determinant of fuel sensing and insulin exocytosis, with actions at the mitochondria and plasma membrane respectively. We postulate that the multiple actions of calpain‐10 may relate to its different protein isoforms. In conclusion, the discovery of calpain‐10 by a genetic approach has identified it as a molecule of importance to insulin signaling and secretion that may have relevance to the future development of novel therapeutic targets for the treatment of T2D. Copyright

Emerging functions of the calpain superfamily of cysteine proteases in neuroendocrine secretory pathways

The first calpain protease was discovered over 40 years ago now, yet despite the vast amount of literature that has subsequently emerged detailing their involvement in the pathophysiology of a variety of human diseases, it is only in the last decade that calpain‐mediated actions along the secretory pathway have begun to emerge. However, the number of secretory pathway substrates identified and their diversity of function continues to grow. This review summarizes our current knowledge of calpain‐mediated mechanisms of action that are pertinent to synaptic vesicle assembly and budding, cytoskeletal organization, endosomal recycling, and exocytotic membrane fusion.

Tumour necrosis factor receptor trafficking dysfunction opens the TRAPS door to pro-inflammatory cytokine secretion

Cytokines are secreted from macrophages and other cells of the immune system in response to pathogens. Additionally, in autoinflammatory diseases cytokine secretion occurs in the absence of pathogenic stimuli. In the case of TRAPS [TNFR (tumour necrosis factor receptor)-associated periodic syndrome], inflammatory episodes result from mutations in the TNFRSF1A gene that encodes TNFR1. This work remains controversial, however, with at least three distinct separate mechanisms of receptor dysfunction having been proposed. Central to these hypotheses are the NF-κB (nuclear factor κB) and MAPK (mitogen-activated protein kinase) families of transcriptional activators that are able to up-regulate expression of a number of genes, including pro-inflammatory cytokines. The present review examines each proposed mechanism of TNFR1 dysfunction, and addresses how these processes might ultimately impact upon cytokine secretion and disease pathophysiology.

High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts

Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic beta-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28mM glucose resulted in approximately 30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin 1A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion.

Differential cytokine secretion results from p65 and c-Rel NF-κB subunit signaling in peripheral blood mononuclear cells of TNF receptor-associated periodic syndrome patients

Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene which encodes the tumor necrosis factor (TNF) receptor, TNFR1. We investigated the effect of three high penetrance and three low penetrance TNFRSF1A mutations upon NF-κB transcription factor family subunit activity, and the resulting impact upon secretion of 25 different cytokines. Whilst certain mutations resulted in elevated NF-κB p65 subunit activity, others instead resulted in elevated c-Rel subunit activity. Interestingly, high p65 activity was associated with elevated IL-8 secretion, whereas high c-Rel activity increased IL-1β and IL-12 secretion. In conclusion, while all six TNFRSF1A mutations showed enhanced NF-κB activity, different mutations stimulated distinct NF-κB family subunit activities, and this in turn resulted in the generation of unique cytokine secretory profiles.

Lessons from anti-TNF biologics: infliximab failure in a TRAPS family with the T50M mutation in TNFRSF1A.

Tumour necrosis factor (TNF) receptor-associated syndrome (TRAPS) is a chronic inherited autoinflammatory disorder. Typical features of TRAPS include recurrent fever, myalgia, rashes, and joint and abdominal pains. At the molecular level, TRAPS is associated with autosomal dominant mutations in the gene encoding the 55 kDa TNF receptor (TNFRSF1A). TRAPS affords a unique opportunity to study the biology of TNF in humans, as it is the only human disease currently known to be caused by mutations in the TNFR1 receptor. Although the inflammatory attacks of TRAPS generally fit with the notion of TNF as an inflammatory cytokine, there remain a number of questions to be answered. In particular, why do only certain patients present with cachexia, why do some patients develop systemic amyloidosis and not others, and why is erosive arthritis not seen in TRAPS although it is observed in TNF transgenic mice [20]? Perhaps some of these outcomes are related to the specific mutations seen in TRAPS, whereas others may be the result of still-undefined environmental or genetic factors. With the identification of new TRAPS mutations, it is likely that additional pathogenetic mechanisms will be identified.

Pseudoislets as primary islet replacements for research : report on a symposium at King's College London, London UK

Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying β-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 β-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed ‘pseudoislets’ largely recapitulates the function of primary islet β-cells. The Diabetes Research Group at King’s College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on “Pseudoislets as primary islet replacements for research”, which was funded by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or β-cell lines but who do not currently use pseudoislets. This short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research.