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Blood, 15 March 2007, Vol. 109, No. 6, pp. 2416-2418. Prepublished online as a Blood First Edition Paper on November 30, 2006; DOI 10.1182/blood-2005-10-039578.
HEMATOPOIESIS Defective apoptosis of peripheral-blood lymphocytes in hyper-IgD and periodic fever syndrome1 Department of General Internal Medicine, 2 Department of Haematology, and 3 Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Hereditary periodic fever syndromes are characterized by incapacitating attacks of fever and generalized inflammation. While the mutated genes for the major syndromes in this group are known, the pathogenesis remains unclear. The aim of this study was to investigate apoptosis in patients with periodic fever as a possible pathogenic factor. We measured anisomycin-induced apoptosis with annexin-V flow cytometry and caspase-3/7 activity in peripheral-blood lymphocytes from symptom-free patients with hyper-IgD and periodic fever syndrome (HIDS; n = 10), TNF-receptor–associated periodic syndrome (TRAPS; n = 7), and familial Mediterranean fever (FMF; n = 2). HIDS lymphocytes showed a decreased percentage of apoptosis during remission by both methods compared with controls (17.8% vs 55.4%), whereas no difference was observed in TRAPS or FMF lymphocytes. This defective apoptosis of lymphocytes may be a central pathogenic mechanism in HIDS, since dysfunction of one of the inhibitory mechanisms to curtail the immunologic response could cause an unbridled generalized inflammation after a trivial stimulus.
Hereditary periodic fever syndromes, also known as autoinflammatory disorders, are characterized by incapacitating attacks of fever and generalized inflammation. Patients present with a long history of recurrent episodes with spiking fever, abdominal discomfort, diarrhea, vomiting, chest pain, or arthralgia. The most prevalent examples of this group of syndromes are familial Mediterranean fever (FMF), TNF-receptor associated periodic syndrome (TRAPS), and hyper-IgD and periodic fever syndrome (HIDS).1 Although the genetic defects are known, the mechanisms by which these syndromes cause inflammatory attacks are largely unclear.1,2 Factors known to precipitate an attack are infection, trauma, vaccination, and both physical and psychological stress. It is hypothesized that the normal immunologic response to relatively trivial insults is not stopped in time, but, instead, leads on to an overwhelming and generalized inflammatory attack. This may be caused by a lack of inhibitory signals to down-regulate the inflammation, or an inability to respond to such signals. Apoptosis plays an important role in down-regulation of the inflammatory response, for example, by reducing the lifespan of activated lymphocytes. We hypothesized that a defect in apoptosis regulation is the cause of the exaggerated inflammatory response in periodic fever patients. This was investigated in peripheral-blood lymphocytes of patients with periodic fever syndromes.
Ten patients with HIDS (2 female, age range 18-44 years), 7 patients with TRAPS (6 female, age range 20-66 years), and 2 patients with FMF (both male, ages 24 and 36 years) were included in this study. All patients showed the relevant pathogenic mutations (Table 1)Nineteen healthy unrelated persons served as controls (13 females, age range 20-51 years). The study was approved by the local ethics committee, and written informed consent was obtained from all participants. Patients and healthy volunteers signed informed consent forms in accordance with the Declaration of Helsinki. Peripheral venous blood from patients in a symptom-free interval and from controls was collected simultaneously and processed in pairs. Peripheral-blood mononuclear cells (PBMCs) were isolated using Ficoll gradient separation (Ficoll-Paque Plus, Amersham Biosciences, Amersham, United Kingdom). Cells were resuspended at a concentration of 5.0 x 106/mL in RPMI 1640 medium (Dutch modification; Invitrogen, Paisley, United Kingdom) and incubated at 37°C. The protease inhibitor anisomycin (Sigma-Aldrich, St Louis, MO) was added in a concentration of 20 µg/mL to induce apoptosis. At different times (0, 2, 5, and 24 hours) the reaction was stopped. At 0, 5, and 24 hours cells were stained with annexin-V–FITC and PI (Apoptest-FITC; VPS Diagnostics, Hoeven, The Netherlands) and analyzed by flow cytometry (Coulter XL; Beckman Coulter, Fullerton, CA) The lymphocyte population was selected based on forward and side scatter and staining with labeled antibodies to CD14, CD3, and CD19.3 At 2 hours cells were permeabilized and the profluorescent caspase-3/7 substrate was added (Apo-One Homogenous Caspase-3/7 Assay; Promega, Madison, WI). Fluorescence was measured using a fluorometer (POLARstar Galaxy; BMG Labtech, Offenburg, Germany) at excitation 485 nm and emission 520 nm. Results shown are corrected for background emission. Results were analyzed using the unpaired t test by GraphPad Prism, version 4.00 (GraphPad Software, San Diego, CA).
After 24 hours of incubation with anisomycin, lymphocytes from 10 patients with HIDS showed a significantly smaller percentage of apoptosis than healthy controls, as shown by annexin-V staining (17.8% vs 55.4%, P < .001; Figure 1B,E). This difference was not observed in unstimulated cells (Figure 1A). There were no differences in the percentages of apoptosis in lymphocytes from patients with TRAPS (n = 7) and FMF (n = 2) compared with those of healthy donors, whether stimulated with anisomycin or unstimulated (Figure 1C-D). At all times, PI binding of cells was less than 0.5%, indicating that the amount of necrotic or late apoptotic cells was negligible. A caspase-3/7 activity assay confirmed the difference in apoptosis found with annexin-V staining in patients with HIDS compared with controls (9060 vs 15 971, P = .026; Figure 1F).
Decreased apoptosis in stimulated lymphocytes in HIDS may well be central to the pathogenesis of the inflammatory attacks: dysfunction of one of the inhibitory mechanisms to curtail the immunologic response can explain the unbridled generalized inflammation seen after a trivial stimulus. The causative mutations in HIDS are located in the gene encoding mevalonate kinase (MVK), an enzyme in the isoprenoid metabolism, the end-products of which include cholesterol, protein isoprenylation (including the prenylated Ras/Rho proteins), dolichol, and ubiquinone.1 The exact pathogenesis of HIDS is unclear. However, several of the isoprenoid end-products have been linked with apoptosis.4 A link between the isoprenoid pathway and apoptosis was also suggested by Nagashima et al,5 who showed that inhibition of this pathway by statins induced apoptosis in rheumatoid arthritis synoviocytes. This might offer an explanation for the beneficial effect of statins seen in patients with HIDS.6
Other periodic fever syndromes are caused by mutations in molecules that contain a death domain, death-effector domain, and/or CARD, involved in the protein-protein interactions required for apoptosis or signaling through NF
Contribution: E.J.B., J.P.H.D., W.H., and A.S. designed research; E.J.B. and J.C.H.H. performed research; E.J.B., W.H., and A.S. analyzed data; E.J.B., J.W.M.M, A.S., and W.H. wrote the paper; and all authors checked the final version of the manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Evelien J. Bodar, Department of General Internal Medicine 463, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands; e-mail: e.bodar{at}aig.umcn.nl.
This work was supported by a program grant from the Netherlands Organization for Health Research and Development (ZonMW, no. 912-03-024) and a Vidi grant from the Netherlands Organization for Health Research and Development (ZonMw, no. 016.066.082) (J.P.H.D.).
Submitted October 26, 2005; accepted August 16, 2006.
Prepublished online as Blood First Edition Paper, November 30, 2006
DOI: 10.1182/blood-2005-10-039578
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
B. These include the TNF-receptor type 1 (TNFRSF1A) in TRAPS, pyrin in FMF, cryopyrin in cryopyrin-associated periodic syndrome (CAPS), and NOD2/CARD15 in Blau syndrome. A central role for an apoptosis defect in these syndromes has therefore been hypothesized and such a defect has been found in vitro; that is, decreased apoptosis of macrophages from pyrin-mutant mice.
