Reduced tumor necrosis factor signaling in primary human fibroblasts containing a tumor necrosis factor receptor superfamily 1A mutant

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ObjectiveTumor necrosis factor receptor–associated periodic syndrome (TRAPS) is an autoinflammatory syndrome associated with mutations in the gene that encodes tumor necrosis factor receptor superfamily 1A (TNFRSF1A). The purpose of this study was to
  Reduced Tumor Necrosis Factor Signaling in Primary HumanFibroblasts Containing a Tumor Necrosis Factor ReceptorSuperfamily 1A Mutant Stefan Siebert 1 , Nick Amos 1 , Ceri A. Fielding 1 , Eddie C. Y. Wang 1 , Ivona Aksentijevich 2 , Bryan D. Williams 1 , and Paul Brennan 1 1 Stefan Siebert, MBBCh, MRCP, Nick Amos, MSc, Ceri A. Fielding, PhD, Eddie C. Y. Wang,PhD, Bryan D. Williams, FRCP, FRCPath, Paul Brennan, PhD: Wales College of Medicine,Cardiff University, Cardiff, UK 2 Ivona Aksentijevich, MD: National Institute of Arthritis andMusculoskeletal and Skin Diseases, NIH, Bethesda, Maryland Abstract Objective— Tumor necrosis factor receptor–associated periodic syndrome (TRAPS) is anautoinflammatory syndrome associated with mutations in the gene that encodes tumor necrosisfactor receptor superfamily 1A (TNFRSF1A). The purpose of this study was to describe a novelTNFRSF1A mutation (C43S) in a patient with TRAPS and to examine the effects of thisTNFRSF1A mutation on tumor necrosis factor α  (TNF α )–induced signaling in a patient-derivedprimary dermal fibroblast line. Methods— TNFRSF1A shedding from neutrophils was measured by flow cytometry andenzyme-linked immunosorbent assay (ELISA). Primary dermal fibroblast lines were establishedfrom the patient with the C43S TRAPS mutation and from healthy volunteers. Activation of NF- κ  B and activator protein 1 (AP-1) was evaluated by electrophoretic mobility shift assays. Cytokineproduction was measured by ELISA. Cell viability was measured by alamar blue assay. Apoptosiswas measured by caspase 3 assay in the fibroblasts and by annexin V assay in peripheral bloodmononuclear cells. Results— Activation-induced shedding of the TNFRSF1A from neutrophils was not altered bythe C43S TRAPS mutation. TNF α -induced activation of NF- κ  B and AP-1 was decreased in theprimary dermal fibroblasts with the C43S TNFRSF1A mutation. Nevertheless, the C43S TRAPSfibroblasts were capable of producing interleukin-6 (IL-6) and IL-8 in response to TNF α .However, TNF α -induced cell death and apoptosis were significantly decreased in the samplesfrom the patient with the C43S TRAPS mutation. Conclusion— The C43S TNFRSF1A mutation results in decreased TNF α -induced nuclearsignaling and apoptosis. Our data suggest a new hypothesis, in that the C43S TRAPS mutationmay cause the inflammatory phenotype by increasing resistance to TNF α -induced apoptosis.Tumor necrosis factor receptor–associated periodic syndrome (TRAPS; MIM no. 142680) isan autosomal-dominant inherited autoinflammatory syndrome characterized by recurrentfevers and abdominal pain associated with cutaneous, muscle, and joint inflammation. It isassociated with mutations in the gene that encodes tumor necrosis factor receptor © 2005, American College of RheumatologyAddress correspondence and reprint request to Stefan Siebert, MBBCh, MRCP, Section of Infection and Immunity, Henry WellcomeBuilding, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XX, UK. Europe PMC Funders Group Author Manuscript Arthritis Rheum  . Author manuscript; available in PMC 2010 March 19. Published in final edited form as: Arthritis Rheum  . 2005 April ; 52(4): 1287–1292. doi:10.1002/art.20955.  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s   superfamily 1A (TNFRSF1A) (1). At least 40 TNFRSF1A mutations associated withTRAPS have been reported on the INFEVERS (Internet periodic fevers) Web site (http:// (2). Initial studies suggested that the TRAPS mutations impairactivation-induced shedding of TNFRSF1A (1,3), although this is not the case for allmutations (4,5) and may be dependent on cell type (6). There are no published studies ontumor necrosis factor α  (TNF α )–induced activation of transcription factors or apoptosis incells derived from patients with TRAPS. The mechanisms by which these TNFRSF1A pointmutations result in the inflammatory phenotype remain unclear.TNF α  exerts its proinflammatory effects through 2 receptors, namely, TNFRSF1A (TNFRIp55) and TNFRSF1B (TNFRII p75). TNFRSF1A is widely expressed and appears to be themajor receptor for soluble TNF α -induced signaling (7). Activation of these receptorsrecruits adapter proteins to the intracellular domain of the receptor and activates downstreamsignaling cascades (8). This causes the activation of NF- κ  B and activator protein 1 (AP-1),which regulate the transcription of a variety of genes, including interleukin-6 (IL-6) andIL-8. Many of these TNF α -induced molecules (such as NF- κ  B, IL-6, and IL-8) are elevatedin inflammatory conditions, such as rheumatoid arthritis (9), making them good candidatesfor investigation in TRAPS. In addition, the intracellular domain of TNFRSF1A contains adeath domain motif, which is involved in TNF-induced apoptosis via activation of a caspasecascade (8).We describe a novel TNFRSF1A mutation (C43S) in a patient with TRAPS that does notimpair activation-induced shedding of TNFRSF1A. We generated a primary dermalfibroblast line from this patient that showed decreased TNF α -induced NF- κ  B and AP-1activation relative to that in normal controls. However, TNF α  was able to induce IL-6 andIL-8 to levels similar to those in the controls. TNF α -induced cell death and apoptosis weremarkedly decreased in the fibroblasts with the C43S TRAPS mutation compared with thecontrol fibroblasts. In addition, we observed decreased TNF α -induced apoptosis in thepatient’s peripheral blood mononuclear cells (PBMCs). We hypothesize that this reducedapoptosis in response to TNF α  may be a factor in the inflammatory phenotype of thispatient. MATERIALS AND METHODS Generation of primary dermal fibroblast cell line Primary skin fibroblast lines were established using an adapted method (10) after ethicalpermission and informed consent were granted. Lignocaine (1%) was injected intradermallyto anesthetize and raise the biopsy area. A small biopsy sample (~2mm 3 ) was obtained, cutinto 8 small fragments, placed into 35-mm surface-modified tissue culture dishes (PrimariaEasy Grip; Becton Dickinson, Mountain View, CA), and covered with a glass coverslip.Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing20% (volume/volume) fetal calf serum (FCS), 100 units/ml penicillin, 100  μ g/mlstreptomycin, and 2 m M   glutamine. The medium was changed at weekly intervals. Whencells were confluent, they were transferred to tissue culture flasks and maintained in DMEMcontaining 10% v/v FCS, 100 units/ml penicillin, 100  μ g/ml streptomycin, and 2 m M  glutamine. Early-passage fibroblasts were stored in liquid nitrogen. Generation of cell extracts Early-passage fibroblasts (passages 5–10) were seeded overnight at 0.3 × 10 6  cells per 60-mm tissue culture dish. They were stimulated with TNF α  (Calbiochem, La Jolla, CA).Stimulation was terminated by removal of DMEM and addition of ice-cold phosphatebuffered saline. Cells were harvested using a cell scraper. All buffers were supplemented Siebert et al.Page 2 Arthritis Rheum  . Author manuscript; available in PMC 2010 March 19.  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s   with a protease inhibitor (phenylmethylsulfonyl fluoride) and phosphatase inhibitor cocktails1 and 2 (Sigma-Aldrich, St. Louis, MO). Nuclear extracts were generated andelectrophoretic mobility shift assays (EMSAs) were performed as previously described (11). Annexin V assay in PBMCs PBMCs were isolated from fresh whole blood using a Ficoll gradient. PBMCs (2 × 10 6 )were placed in 2 ml RPMI 1640 with 10% FCS and were left untreated or were stimulatedwith TNF α  and cycloheximide (CHX; 50  μ g/ml) either alone or in combination. Apoptosiswas assayed using the TACS annexin V–fluorescein isothiocyanate kit (R&D Systems,Minneapolis, MN) according to the manufacturer’s instructions, after which fluorescencewas measured by flow cytometry. RESULTS Identification of a novel TNFRSF1A mutation (C43S) The index patient was a 50-year-old woman of Welsh srcin with recurrent attacks of fever,pharyngitis, and arthritis accompanied by a migrating skin rash and myalgia. Her first attack occurred at the age of 18 months, with attacks typically lasting 1–2 weeks. The patient’sdeceased father had similar recurrent episodic fevers. The patient’s clinical picture wasconsistent with the reported TRAPS phenotype (1). Mean ± SEM plasma levels of solubleTNFRSF1A were 3,839 ± 131 pg/ml, which is consistent with levels reported in TRAPSpatients with renal impairment (5). DNA was extracted from the patient’s blood, andpolymerase chain reaction (PCR) amplifications were performed, with subsequentsequencing of the PCR products. We identified a novel TNFRSF1A mutation (nucleotide215 G → C, exon 3) resulting in the substitution of serine for cysteine at residue 43 (C43S).This amino acid substitution disrupts the disulfide bond at this position in the firstextracellular domain of TNFRSF1A. This substitution was not observed in any of the 734control chromosomes screened by genomic sequencing. Effect of the C43S mutation on shedding of TNFRSF1A To investigate whether TNFRSF1A shedding was affected by the C43S mutation, wemeasured levels of TNFRSF1A on the surface of the patient’s neutrophils by flowcytometry. Following phorbol myristate acetate (PMA) treatment, membrane TNFRSF1Adecreased to levels comparable with those of healthy controls (Figure 1A). To establishwhether this decrease in surface TNFRSF1A was a result of internalization or cleavage of the receptor, levels of soluble TNFRSF1A were measured in the supernatant of the cells.Soluble TNFRSF1A levels increased in response to PMA, indicating that the loss of surfaceTNFRSF1A was a result of receptor shedding (Figure 1B). Activation-induced cleavage wastherefore not impaired by the C43S mutation, and could not account for the inflammatoryphenotype in this patient. Generation of a primary TRAPS fibroblast line To facilitate the study of the C43S TNFRSF1A mutation, we generated a primary dermalfibroblast cell line from a skin biopsy sample taken from the patient. The skin is anaccessible site for obtaining fibroblasts and is also the site of the prototypical rash thatcharacterizes TRAPS. The sample was taken from an area of normal skin during anasymptomatic period. Similar dermal fibroblast lines were generated from age- and sex-matched healthy volunteers and used as controls. Siebert et al.Page 3 Arthritis Rheum  . Author manuscript; available in PMC 2010 March 19.  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s   Role of the C43S TNFRSF1A mutation in NF- κ  B and AP-1 activation and induction of IL-6or IL-8 TNFRSF1A nuclear signaling was investigated using the dermal fibroblast lines. Cells werestimulated with 10 ng/ml of TNF α  for 1 hour, after which nuclear extracts were generated.NF- κ  B activation was determined by EMSA using radiolabeled DNA corresponding to aspecific NF- κ  B site. All the fibroblast lines were able to activate NF- κ  B in response toTNF α . However, the activation was consistently less in fibroblasts from the patient withTRAPS compared with fibroblasts from healthy controls (Figure 2A). There was noevidence of constitutive activation of NF- κ  B in the TRAPS fibroblasts. In response to IL-1stimulation, the TRAPS fibroblasts activated NF- κ  B to levels similar to those of healthycontrols (data not shown). Supershift assays with antibodies to NF- κ  B subunits indicatedthat the complexes are very similar, with both the TRAPS and control fibroblasts activatingan NF- κ  B complex containing p50 and RelA subunits (Figure 2B).To determine whether the NF- κ  B activation in C43S TRAPS fibroblasts was more sensitiveto TNF α  or more prolonged than in healthy controls, TNF α  dose-response and time-courseexperiments were performed. TNF α -induced NF- κ  B activation was consistently lower inthe TRAPS fibroblasts at all doses of TNF α  tested (Figure 2C). In addition, the duration of TNF α -induced NF- κ  B activation was significantly shorter in the C43S TRAPS fibroblastscompared with the control fibroblasts (Figure 2D). There was also no increase in NF- κ  Bactivation at any later time points up to 24 hours (results not shown). The C43S fibroblastsalso resulted in less activation of AP-1 compared with the normal fibroblasts across a rangeof TNF α  doses and time points (results not shown). The above results were consistent forboth control fibroblast lines.To determine whether the C43S TRAPS mutation altered TNF α -induced production of IL-6and IL-8, the levels of these cytokines in the culture supernatants of the primary dermalfibroblasts were measured by enzyme-linked immunosorbent assay. While absolute valuesvaried with the passage number, the induction of IL-6 (Figure 2E) and IL-8 (Figure 2F) inresponse to TNF α  in the C43S TRAPS fibroblast line was not statistically different fromthat observed in the controls. Baseline levels of the cytokines were also similar in thefibroblast lines. Therefore, in spite of the reduced NF- κ  B and AP-1 activation, the C43STRAPS fibroblast line was able to produce IL-6 and IL-8 in response to TNF α . Reduction of TNF α  -induced apoptosis in dermal fibroblasts and PBMCs due to C43STRAPS mutation Another important TNFRSF1A-mediated effect is the induction of apoptosis. TNF α  alsoinduces antiapoptotic genes, and therefore the apoptotic response to TNF α  is usuallydependent on the inhibition of protein synthesis (12). Fibroblasts were stimulated withTNF α  (10 ng/ml) either alone or in the presence of CHX (13). Three assays for cell survivalwere used. First, cells were examined by light microscopy (BX41 microscope; Olympus,Lake Success, NY) after 24 and 48 hours. Dramatic differences were observed at both timepoints, with more TRAPS-derived fibroblasts than wild-type cells surviving the combinationof TNF α  and CHX (Figure 3A). The differences observed with light microscopy werequantified using an alamar blue assay (14). This nontoxic dye is chemically reduced by theinnate metabolic activity of cells, which allows quantification of cell viability byfluorometry. Figure 3B shows the effect of a range of doses of TNF α  on the survival of C43S TRAPS fibroblasts and a control fibroblast line. TRAPS-derived fibroblasts weremarkedly less sensitive to TNF α -induced cell death. Statistically significant ( P   < 0.05)differences in cell viability were observed at doses of TNF α  >1 ng/ml. Caspase 3 activity, ameasure of apoptosis, was also assayed. TNF α  and CHX induced significantly less caspase3 activity in the TRAPS fibroblasts at all time points measured (Figure 3C). Siebert et al.Page 4 Arthritis Rheum  . Author manuscript; available in PMC 2010 March 19.  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s   Because the effects of TNF α  can vary in different cells (12), we investigated whether theC43S TNFRSF1A mutation would also result in decreased sensitivity to TNF α -inducedapoptosis in circulating inflammatory cells. PBMCs from the patient with the C43S TRAPSmutation and from healthy volunteers were isolated. PBMCs were stimulated with TNF α and CHX for either 2 or 6 hours. Cells were stained with annexin V and propidium iodide(PI) and analyzed by flow cytometry. Cells in the live gate (PI negative) were analyzed forannexin V staining. Although similar percentages of unstimulated cells were positive forannexin V, after stimulation with TNF α  and CHX, fewer PBMCs from the patient withTRAPS were annexin V positive at both time points (Figure 3D). The difference observed at6 hours was statistically significant ( P   < 0.05). The C43S TNFRSF1A mutation thereforeresulted in decreased TNF α -induced apoptosis in both fibroblasts and PBMCs. DISCUSSION This report describes a novel TNFRSF1A mutation (C43S) associated with TRAPS andcharacterizes the signaling abilities of this mutation in a primary dermal fibroblast lineestablished from the patient. TNF α  activated the transcription factors NF- κ  B and AP-1 atreduced levels in C43S TRAPS fibroblasts but was able to induce the proinflammatorycytokines IL-6 and IL-8 to levels similar to those in healthy controls. TNF α -inducedapoptosis was significantly decreased in the fibroblasts bearing the C43S TNFRSF1Amutation. This defect in TNF α -induced apoptosis was also seen in PBMCs isolated from thepatient. Thus, this study demonstrates that this TRAPS mutation results in reduced TNF α -induced nuclear signaling and apoptosis in this patient.The initial studies of TRAPS cells proposed a mechanism of impaired activation-inducedcleavage of TNFRSF1A that could cause the systemic inflammation associated with thissyndrome (1,3). However, impaired shedding of TNFRSF1A does not appear to be the casefor all TRAPS mutations (4,5). Our results support the hypothesis that TRAPS does notrequire impaired activation-induced shedding of TNFRSF1A from neutrophils.Our data show that the C43S TRAPS fibroblasts exhibit reduced NF- κ  B and AP-1 activationand decreased apoptosis in response to stimulation with TNF α . However, no significantdifference in the induction of proinflammatory cytokines was observed, findings consistentwith those of a previous study (1). This suggests a possible hypothesis to explain theinflammatory pathology: cells survive longer because of impaired apoptosis, but remaincapable of producing proinflammatory cytokines. The levels of these cytokines would beexpected to accumulate and could result in an inflammatory phenotype. Apoptosis of inflammatory cells is an important homeostatic mechanism for limiting an inflammatoryresponse once it is established (15). Our hypothesis is also compatible with the high levelsof serum amyloid A and C-reactive protein, surrogate markers for serum IL-6, noted in ourand other TRAPS patients (1). However, this hypothesis is based on results from a singlepatient, and it remains to be established whether this is also the case in other TRAPSpatients.Because both IL-6 and IL-8 are regulated by NF- κ  B, the question remains of how theinduction of these proinflammatory cytokines is normal, despite the reduced NF- κ  Bactivation. We propose 2 possible explanations. The first is that the threshold for TNF α -induced cytokine production is lower than that for apoptosis. Thus, reduced NF- κ  B stillgenerates a sufficient signal to allow induction of both IL-6 and IL-8. A second explanationis that signaling via TNFRSF1B (TNFRII) may play a role. TNFRSF1B is able to activateNF- κ  B, but does not contain a death domain (8) and, thus, could induce IL-6 and IL-8without causing apoptosis. TNF α  generally results in less activation via TNFRSF1B than Siebert et al.Page 5 Arthritis Rheum  . Author manuscript; available in PMC 2010 March 19.  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s  E  ur  o p e P M C F  un d  e r  s A u t  h  or M a n u s  c r i   p t   s 
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