Tumor necrosis factor receptor–associated periodic syndrome (TRAPS; MIM 142680), which is characterized by recurrent fevers and certain inflammatory symptoms, is associated with autosomal-dominant mutations in the gene encoding the 55-kd tumor necrosis factor receptor (TNFRSF1A; also known as TNFR1, CD120a, and p55 TNFR). More than 30 different mutations have been reported (1–15).
The syndrome presented in the prototypical TRAPS family was originally termed familial Hibernian fever (FHF) (16, 17). Individuals with FHF experience febrile attacks, abdominal pain, localized myalgia, large, painful, erythematous skin lesions, conjunctivitis, periorbital edema, and inguinal hernias (17, 18). The associated mutation results in the substitution of cysteine by tyrosine at position 33 (C33Y) of the TNFRSF1A first cysteine-rich domain (CRD1) (1).
Both TNFRSF1A and the 75-kd TNF receptor (TNFRSF1B; also known as TNFR2, CD120b, and p75 TNFR) exist in soluble forms that are produced by cleavage by metalloproteinases upon cell activation (19). In common with patients bearing other TNFRSF1A mutations, the affected members of the C33Y prototype TRAPS family exhibit low levels of serum TNFRSF1A that rise to only just within the normal range during febrile attacks, unlike patients with, for example, rheumatoid arthritis, in whom soluble TNFRSF1A (sTNFRSF1A) can reach 10 times normal levels (1, 2, 20). Consistent with these in vivo observations, peripheral blood leukocytes from TRAPS family members with a C52F mutation were found to express higher than normal levels of TNFRSF1A and to exhibit little, if any, shedding of their surface TNFRSF1A when stimulated with phorbol myristate acetate (PMA), whereas TNFRSF1B did not share these abnormalities (1). This TNFRSF1A shedding defect led to the hypothesis that there might not be enough sTNFRSF1A to neutralize the free TNF, thus resulting in the continuous inflammatory stimulus that is notable in TRAPS (1). In addition, low levels of sTNFRSF1A are seen in all families with TRAPS, regardless of the mutation present (1). However, not all the TRAPS-related TNFRSF1A mutations result in defective receptor shedding by leukocytes (4, 14), suggesting that this may not be the sole cause of the low levels of sTNFRSF1A, and that other pathophysiologic mechanisms may also be involved in generating the disease phenotype.
In order to further elucidate the possible association of TNFRSF1A shedding with the pathophysiology of TRAPS, we investigated the shedding of surface TNFRSF1A from leukocytes and dermal fibroblasts from TRAPS patients with the C33Y mutation and from normal controls. Dermal fibroblasts were considered relevant to study because the patients demonstrate skin lesions, and fibroblasts are a potentially important source of inflammatory cytokines. Furthermore, in order to compare the shedding of TNFRSF1A with different mutations under conditions where all other potential genetic and cellular variables are constant, we produced HEK 293 cloned cell lines that stably expressed either wild-type (WT) and/or a single mutant recombinant TNFRSF1A in the absence of TNFRSF1B. Mutants with the following single–amino acid substitutions were produced as truncated forms, representing the extracellular and transmembrane regions but lacking the cytoplasmic signaling domain (Δ-sig): C33Y, T50M, C52F, C88Y, and R92Q. C33Y, T50M, and C52F are mutations within the CRD1 of TNFRSF1A; C88Y and R92Q are mutations in CRD2. We used these truncated forms of WT and mutant TNFRSF1A to assess receptor shedding in response to stimulation, because these do not affect cell viability when expressed, whereas expression of the full-length transfected receptor can induce apoptosis (ref. 21 and Todd I, et al: unpublished observations). Each of the single–amino acid substitutions used to generate the mutant cell lines has been previously described (1, 4).
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- MATERIALS AND METHODS
Not all TRAPS-related TNFRSF1A mutations result in reduced receptor shedding by leukocytes (4, 14), but there is evidence that serum TNFRSF1A is low in patients with TRAPS (1, 20). However, no previous studies have investigated other types of cells from TRAPS patients. In addition, no previous study has assessed the shedding from homozygous cells expressing high levels of only TRAPS-related mutant TNFRSF1A. Here we demonstrated that the defective receptor shedding varies not only between mutations, but also between cell types bearing the same mutation, and is not purely a function of the mutations in TNFRSF1A.
Despite an earlier finding that the C52F mutation was associated with reduced shedding from leukocytes (1), we have shown here that leukocytes from C33Y TRAPS patients do not show any defect in PMA-induced TNFRSF1A shedding. However, dermal fibroblasts from the same C33Y TRAPS patients had a marked reduction in TNFRSF1A shedding. This difference between cell types indicates that factors in addition to the mutations in TNFRSF1A influence receptor shedding. It also suggests that there is a difference between the two cell types in their roles in the inflammatory response seen in these patients. The observed difference is not dependent on the dose of PMA used (10 ng/ml with fibroblasts and 100 ng/ml with leukocytes), since treating leukocytes with 10 ng/ml also failed to distinguish TNFRSF1A shedding between C33Y TRAPS patients and controls (data not shown). The method used to induce TNFRSF1A shedding from the leukocytes was identical to that used for the other TRAPS families, such as patients with C52F TRAPS, in which a reduction in the shedding of TNFRSF1A was observed.
The method chosen to detect TNFRSF1A shedding from the fibroblasts differed slightly from the method used for the leukocytes. In particular, fibroblasts must adhere to the culture plate to remain viable, resulting in 100-fold fewer cells for analysis than was available for the leukocytes. A longer incubation period was therefore necessary for the sTNFRSF1A released by these cells to be within the detection range of the ELISAs used. However, further samples taken at 72 and 96 hours after PMA stimulation showed a continued release of sTNFRSF1A from the normal fibroblasts, suggesting that the time subsequent to stimulation did not limit the release of sTNFRSF1A from these cells (data not shown).
TRAPS is characterized by an augmented inflammatory response, and flares have been related to infection and trauma (17). Such inflammatory stimuli liberate proinflammatory cytokines (TNF, interleukin-1, and interleukin-6) and in TRAPS, defective negative feedback (such as reduced sTNFRSF1A production) may be unable to terminate these reactions. In the skin, there is a tightly regulated microenvironment of cytokines and growth factors produced by keratinocytes and dermal fibroblasts. Hence, fibroblasts in the skin and other tissues may also contribute to the feedback mechanisms. In this study, we have demonstrated that C33Y TRAPS dermal fibroblasts have a reduced ability to produce sTNFRSF1A. These results, together with the reduced serum levels of TNFRSF1A in these patients, suggest that fibroblasts may normally be an important source of sTNFRSF1A in the control of inflammation.
All of the HEK 293 Δ-sig cell lines expressing mutant forms of TNFRSF1A, as well as the WT, showed PMA-induced receptor shedding, although to differing extents. Since these recombinant receptors lacked the intracellular region, this finding is consistent with that of a previous study reporting that the intracellular region of TNFRSF1A is not essential for receptor shedding (25). In all cases, shedding was at least partially inhibited by the metalloprotease inhibitor TAPI-1, indicating that the mechanism of shedding of these recombinant truncated receptors is the same as the mechanism for natural TNFRSF1A (19).
Similar levels of shedding were seen in transfectants expressing high levels of mutant TNFRSF1A alone or mutant plus WT TNFRSF1A, suggesting that shedding behavior is similar in either situation. We also observed that most of the mutant forms of Δ-sig TNFRSF1A showed substantially reduced TNF binding but that, in the dual transfectants, WT TNFRSF1A still bound TNF in the presence of the mutant receptors. This is consistent with the previous report that leukocytes from TRAPS patients and healthy controls show similar affinities of binding for TNF (1).
Although minor differences in the degree of shedding from the transfectants were seen between WT and the various mutants of TNFRSF1A, all showed substantial shedding. All features of the transfected HEK 293 cell lines are very similar, apart from the mutations in the transfected TNFRSF1A. This, again, therefore indicates that potential shedding differences are not purely a function of TNFRSF1A structure, but are influenced by other genetic and/or cellular variables. This conclusion is consistent with the differences in C33Y TNFRSF1A shedding by leukocytes and fibroblasts, as discussed above. It is also consistent with the findings of Aganna et al (14), who reported that not only do TRAPS patients differ with respect to a TNFRSF1A shedding defect, but some patients with autosomal-dominant periodic fever without TNFRSF1A mutations also demonstrate reduced shedding of TNFRSF1A and have low serum levels of the soluble receptor.
It has been difficult to conceive how diverse mutations in the cysteine-rich domains in the distal regions of the TNFRSF1A ectodomain could influence shedding that is a result of cleavage proximal to the plasma membrane (26). However, it has recently been found that cleavage depends on interaction of the TNFRSF1A ectodomain with a type II integral membrane protein called aminopeptidase regulator of TNFRSF1A shedding (ARTS-1) (27). Although the precise sites of interaction between ARTS-1 and TNFRSF1A have not been defined, it is possible that TRAPS-associated mutations may reduce interactions between TNFRSF1A and ARTS-1 and therefore reduce shedding.
Another possibility that should be kept in mind is that mutations may affect the internalization of TNFRSF1A into the cytoplasm that has been reported to occur following TNF binding (28). This might, for example, prolong signaling, with inflammatory consequences.
In general, our data support the concept that a combination of functional abnormalities resulting from mutations in TNFRSF1A may contribute to TRAPS. Shedding defects are influenced not only by different mutations in TNFRSF1A, but also by other factors, including cell type. This raises the possibility that reduced shedding of TNFRSF1A may be one of several factors that determine the clinical picture of TRAPS and that other mechanisms of TNF receptor signaling and control of inflammation are also involved. The complex and variable clinical picture in TRAPS may relate to differences in several functional effects of the different mutations.