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.

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.

Human autologous mixed lymphocyte reaction as an in vitro model for autoreactivity to apoptotic antigens.

Recent studies have indicated that cells undergoing apoptosis are the source of autoantigens which drive autoimmune responses in systemic lupus erythematosus (SLE). It has been recognized for many years that in vitro stimulation of T cells with irradiated major histocompatibility complex (MHC) class II-bearing autologous cells results in T-cell proliferation with immunological specificity and memory, namely the autologous mixed lymphocyte reaction (AMLR). The nature of the major stimulants in the AMLR is still unclear. We investigated whether apoptotic fragments from irradiated cells act as antigenic stimulators for AMLR or nucleohistone-primed T cells. T-cell proliferation in the primary AMLR was significantly suppressed by the presence of a caspase inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD.fmk), indicating that apoptotic antigens released from irradiated autologous feeder cells act as stimulators of AMLR T cells. This inhibitory effect of Z-VAD was not caused by toxic effects, because the T-cell response to the mitogen phytohaemagglutinin (PHA) was not inhibited by Z-VAD. A nucleohistone preparation was shown to contain antigens that are important in the AMLR, as culture with nucleohistone (but not with thyroglobulin or hen-egg lysozyme) primed T cells to respond with secondary kinetics in a subsequent AMLR that was also suppressed by Z-VAD. Our data provide evidence that the AMLR constitutes a model for the evaluation of cellular and molecular mechanisms that may be relevant to the pathogenesis of SLE and similar autoimmune diseases.