Novel markers of inflammation identified in tumor necrosis factor receptor-associated periodic syndrome (TRAPS) by transcriptomic analysis of effects of TRAPS-associated tumor necrosis factor receptor type I mutations in an endothelial cell line.

OBJECTIVE: To analyze the effects of tumor necrosis factor receptor-associated periodic syndrome (TRAPS)-associated mutant tumor necrosis factor receptor type I (TNFRI) expression in a cell type directly relevant to the inflammation in TRAPS, and to identify novel markers associated with mutant TNFRI expression. METHODS: Transcriptome analysis on 30,000 human genes was performed on SK-Hep-1 human endothelial cells transfected with either wild-type (WT) or TRAPS-associated mutant TNFRI. Quantitative reverse transcriptase-polymerase chain reaction and protein expression levels measured by enzyme-linked immunosorbent assay verified transcriptional changes for selected genes both in supernatants from cells expressing mutant TNFRI and in patient plasma. RESULTS: Cells expressing mutant TNFRI showed up-regulation of multiple proinflammatory genes relative to WT transfectants, including genes for pentraxin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, CCL2, and CCL5, which were also expressed as proteins. In addition, the expression of most of these markers was increased in the plasma and peripheral blood mononuclear cells from TRAPS patients relative to those from healthy controls. The cysteine mutations (C33Y and C52F), which are associated with a more severe clinical phenotype, induced more genes than the low-penetrance mutation R92Q, which is associated with a milder phenotype. The expression of most genes was induced by a death domain (DD)-dependent mechanism, since they were not induced by expression of TNFRI mutants with an inactivated DD. CONCLUSION: TRAPS-associated TNFRI mutants induce the expression of multiple genes encoding inflammatory molecules, cellular receptors, transcription factors, and regulators of apoptosis in endothelial cells that require the cytoplasmic signaling properties of the receptor. Different mutants have specific expression profiles, indicating mutation-specific effects. The expression of some of these markers was also elevated in samples from TRAPS patients.

Mutant tumor necrosis factor receptor associated with tumor necrosis factor receptor-associated periodic syndrome is altered antigenically and is retained within patients' leukocytes.

OBJECTIVE: To investigate the effect of mutations in tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) in TNFR-associated periodic syndrome (TRAPS) on the binding of anti-TNFRSF1A monoclonal antibodies (mAb), and to investigate the subcellular distribution of mutant versus wild-type (WT) TNFRSF1A in patients with TRAPS. METHODS: HEK 293 cells transfected with WT and/or mutant TNFRSF1A were used to investigate the interaction of anti-TNFRSF1A mAb with the WT and mutant proteins. Monoclonal antibodies that differentially bound to C33Y TNFRSF1A were used to investigate the distribution of WT and mutant TNFRSF1A in TRAPS patients with the C33Y mutation. RESULTS: We identified a mAb whose binding to TNFRSF1A was completely abolished by the C33Y or C52F TRAPS-associated mutations, whereas other mutations (T50M, C88Y, R92Q) had lesser effects on the binding of this mAb. A different mAb was found to bind efficiently to all of the mutant forms of TNFRSF1A examined as well as to the WT receptor. Exploitation of the differential binding properties of these mAb indicated that mutant (as distinct from WT) TNFRSF1A showed abnormal intracellular retention in the neutrophils of TRAPS patients with the C33Y mutation, with little if any expression of mutant TNFRSF1A on the cell surface or as soluble receptor in plasma. CONCLUSION: TRAPS-associated mutant TNFRSF1A has an antigenically altered structure and shows abnormal retention in the leukocytes of patients with TRAPS, which is consistent with previous findings from in vitro and transgenic model systems. This is consistent with a misfolded protein response contributing to the pathophysiology of TRAPS.

Modeling of tumor necrosis factor receptor superfamily 1A mutants associated with tumor necrosis factor receptor-associated periodic syndrome indicates misfolding consistent with abnormal function.

OBJECTIVE: To investigate the effect of mutations in the tumor necrosis factor receptor superfamily 1A (TNFRSF1A) gene on the conformation and behavior of the TNFRSF1A protein. Mutations in TNFRSF1A cause the autosomal-dominant, autoinflammatory TNFR-associated periodic syndrome (TRAPS). METHODS: The expression of recombinant TNFRSF1A was compared in SK-HEp-1 endothelial cells and HEK 293 epithelial cells stably transfected with full-length R347A or Deltasig constructs of wild-type or TRAPS-associated mutant TNFRSF1A. TNF binding was assessed in HEK 293 cell lines expressing R347A wild-type or mutant TNFRSF1A. Homology modeling of the 3-dimensional structure of the ectodomains of wild-type and mutant TNFRSF1A was performed. RESULTS: TRAPS-associated mutant and wild-type TNFRSF1A behaved differently and had different localization properties within the cell, as a direct result of mutations in the ectodomains of TNFRSF1A. From a structural perspective, mutants with a predicted structure similar to that of the wild-type protein (e.g., R92Q) behaved similarly to wild-type TNFRSF1A, whereas forms of TNFRSF1A with mutations predicted to drastically destabilize the protein structure (e.g., cysteine mutations) showed defects in cell surface expression and TNF binding. CONCLUSION: The results obtained from the in vitro experiments, in combination with the modeled structures, indicate that the phenotype and clinical differences between different TRAPS-associated mutants of TNFRSF1A result from different conformations of the TNFRSF1A ectodomains.

Shedding of mutant tumor necrosis factor receptor superfamily 1A associated with tumor necrosis factor receptor-associated periodic syndrome: differences between cell types.

OBJECTIVE: To investigate the effect of mutations in tumor necrosis factor receptor superfamily 1A (TNFRSF1A) on the ability of the receptors to be cleaved from the cell surface upon stimulation. The mutations we studied are associated with clinically distinct forms of TNF receptor-associated periodic syndrome (TRAPS). We also investigated different cell types within the same form of TRAPS. METHODS: The shedding of TNFRSF1A in response to stimulation with phorbol myristate acetate was assessed in leukocytes and dermal fibroblasts from patients with C33Y TRAPS, and in HEK 293 cell lines stably transfected with constructs containing wild-type TNFRSF1A and/or TNFRSF1A mutants identified in TRAPS patients. RESULTS: The shedding of TNFRSF1A differed between cell types within the same form of TRAPS. In particular, dermal fibroblasts, but not leukocytes, from C33Y TRAPS patients demonstrated reduced shedding of TNFRSF1A. Shedding of both wild-type and mutant TNFRSF1A from the transfected HEK 293 cells showed minor differences, but was in all cases induced to a substantial extent. CONCLUSION: Differences in TNFRSF1A shedding are not purely a function of the TNFRSF1A structure, but are also influenced by other features of genetic makeup and/or cellular differentiation. It is unlikely that a defect in TNFRSF1A shedding per se can fully explain the clinical features that are common to TRAPS patients with different TNFRSF1A mutations.