Defective apoptosis of peripheral-blood lymphocytes in hyper-IgD and periodic fever syndrome

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 × 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 NFκB. 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.⁷  However, available data on apoptosis measured in patient cells is limited. Two studies in Turkish patients with FMF show normal apoptosis of peripheral-blood leukocytes in symptom-free patients but increased apoptosis of neutrophils and monocytes during inflammatory attacks⁸  or elevated serum soluble FAS concentrations.⁹  A third study showed decreased TNF-induced neutrophil apoptosis in patients with TRAPS.¹⁰  We included 2 symptom-free patients with FMF who showed no defect in lymphocyte apoptosis. We did not find an apoptosis defect in 7 patients with TRAPS. Preliminary studies from our laboratory suggest that the apoptotic potential of lymphocytes in TRAPS is not affected by an attack (data not shown). Conceptually it would be interesting to measure apoptosis in patients with HIDS during inflammatory attacks. However the profound lymphopenia that occurs early during attacks so far has hampered these measurements. At recovery from an attack, we measured restored apoptotic activity in 2 patients (59.8% early apoptosis after 24-hour stimulation with anisomycin) compared with controls. This might mean that apoptotic pathways are activated at that stage to end the attack. An explanation for the difference between HIDS and the other syndromes found in our study may be the cell type studied. Reasoning from the clinical phenotype, we chose to investigate apoptosis of lymphocytes in HIDS. Lymphadenopathy, lymphocytic infiltrates in skin biopsies, and many B lymphocytes containing IgD in the cytoplasm in bone marrow can be observed in HIDS,^11,12  and this may be related to the lymphocyte apoptosis defect. In the other periodic fever syndromes, different cell types are more prominent and point to cell-specific defects. FMF is characterized by infiltrates that are almost exclusively composed of neutrophils, whereas the urticarial response in certain cryopyrin-associated periodic syndromes could point to a role of mast cells. This hypothesis of cell-type specificity is supported by the study by D'Osualdo et al,¹⁰  showing abnormal neutrophil apoptosis in 8 patients with TRAPS. In this study, one patient with HIDS was measured and found to have normal neutrophil apoptosis.¹⁰  In conclusion, we have shown decreased apoptosis of circulating lymphocytes ex vivo in fever-free patients with HIDS after stimulation with anisomycin. This lymphocytic defect was not seen in TRAPS or FMF. Decreased apoptosis may be central to the pathogenesis of HIDS.

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@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.).