B-lymphocytes from Malignant Hyperthermia-susceptible Patients Have an Increased Sensitivity to Skeletal Muscle Ryanodine Receptor Activators*

Malignant hyperthemia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1β after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1β from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1β production.

(1-4). The clinical signs of an impending MH reaction are highly variable and are caused by a hypermetabolic state with muscle rigidity, metabolic acidosis, rhabdomyolysis, tachycardia, and/or an increase in body temperature (5). In some individuals MH reactions appear to be triggered by physical exercise or emotional stress. The latter observation has led to the suggestion that MH, heat stroke, and exercise-induced rhabdomyolysis might have a common denominator (2,6,7). The underlying causes of MH are abnormalities in the skeletal muscle calcium metabolism (8,9) and molecular genetic studies have mapped the primary locus of MH to chromosome 19q, the gene encoding the ryanodine receptor calcium release channel (RYR1) (2,4,10). Approximately 50% of MH families have mutations in the RYR1 gene, and mutations have been reported in other loci (for recent reviews, see Refs. 11 and 12).
The ryanodine receptors are large tetrameric oligomers that function as intracellular calcium release channels. Three different isoforms have been identified at the molecular level: type 1 (RYR1), which is preferentially expressed in skeletal muscle; type 2, which is in the heart and cerebellum; and type 3, which is in the central nervous system as well as in a variety of other tissues (13)(14)(15)(16). RYR1 can be pharmacologically activated by a number of compounds, among which are caffeine, halothane, thymol, 4-chloro-m-cresol, E218, bastadin, polylysine, and calcium (17)(18)(19)(20)(21). Activation causes the channel to open and thus to a transient calcium flow from the sarcoplasmic reticulum, leading to an increase in the calcium concentration of the myoplasm.
In B-lymphocytes Ca 2ϩ signaling has been implicated in various physiological responses such as cell proliferation, gene expression, and antibody secretion (22). In a recent report, Sei et al. (23) have presented evidence supporting the existence of RYR1 in B-lymphocytes. Thus, in this cell type, changes in the [Ca 2ϩ ] i may be under the control of both the inositol trisphosphate receptor and the RYR1. B-lymphocytes are capable of responding to and producing several cytokines among which IL-1, IL-6, and tumor necrosis factor (24), although what controls cytokine release is not fully understood. IL-1 is a mediator of the host inflammatory response in innate immunity and stimulates other cell types such as macrophages and endothelial cells to synthesize and secrete other cytokines. When released in large quantities, IL-1 causes fever, i.e. it is an endogenous pyrogen and can induce the synthesis of acute phase plasma proteins and initiate metabolic wasting (25). Because of the different clinical symptoms during an MH episode, we were interested in establishing whether there is a link between IL-1 release and malignant hyperthermia. The results of the pres-* This work was supported in part by Swiss National Foundation Grant 3200-063959.00, Telethon Italy Grant 1259 (to F. Z.), a grant from the Ministero Università e Ricerca Scientifica e Tecnologica ex 40%, and by the Department of Anesthesia, Basel Kantonsspital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ ent study raise the possibility that some of the symptoms seen during an MH episode may be the result of IL-1 production.

EXPERIMENTAL PROCEDURES
Materials fura-2/AM, 1,2-bis (2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM), thapsigargin, and peroxidase-conjugated anti-mouse IgG were from Sigma; cDNA synthesis system kit, DNA isolation kit from mammalian blood, Taq polymerase, and BM chemiluminescence kit were from Roche Molecular Biochemicals. RNA isolation kit was from Biotecx Laboratories (Houston, TX). DNA-modifying enzymes were from New England Biolabs. Human IL-1␤ and IL-6 ELISA kits were from CLB (Amsterdam, The Netherlands). Cyclosporin A was from Novartis (Basel, Switzerland). Nitrocellulose and Ficoll-Hypaque were from Amersham Biosciences, Inc. Caffeine was from Merck (Darmstadt, Germany); 4-chloro-m-cresol was from Fluka Chemicals (Buchs, Switzerland). Tissue culture media and reagents were from Life Technologies, Inc. All other chemicals were reagent or of the highest available grade.

Methods
Mononuclear Cells and EBV-transformed Cell Lines-after informed consent whole blood was collected in EDTA-treated tubes from 4 healthy volunteers and from 4 patients who had undergone a diagnostic skeletal muscle biopsy to determine malignant hyperthermia susceptibility and were known to carry the MH-associated V2168M mutation. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. For infection with Epstein-Barr virus, mononuclear cells were exposed to supernatants of the B95.8 cell line in the presence of cyclosporin A and IL-6, according to standard procedures. Cells were cultured in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 100 units of penicillin and streptomycin.
Mutation Screening-The presence of the MH-linked V2168M RYR1 gene mutation in selected patients from four unrelated families was determined by DNA PCR amplification, followed by restriction enzyme digestion. Genomic DNA was isolated using a DNA isolation kit for mammalian blood. Total RNA was isolated using an RNA isolation kit; poly(A ϩ ) RNA was converted into cDNA using a cDNA synthesis system kit following the instructions provided by the manufacturer (Roche Molecular Biochemicals, catalog no. 1117831). Approximately 100 ng of DNA were used for each PCR amplification using a GeneAmp2400 thermocycler (PerkinElmer Life Sciences). The following primers were used to amplify genomic DNA and cDNA: forward, 5Ј-GGG CCC AAG AGG ACT TCG TGC; reverse, 5Ј-GCC CCC GAG GAC GTT GAC CAT. Amplification conditions were: 5 min, 95°C followed by 35 cycles for genomic DNA or 40 cycles for cDNA: 30-s annealing at 60°C, 45-s extension at 72°C, 30-s denaturation at 92°C, and extension for 4 min at 72°C. The presence of the mutation was detected by restriction enzyme digestion using MslI. Subcellular Membrane Fractionation and Western Blotting-Total microsomes were isolated from rabbit brain, heart, and EBV-transformed B-lymphocytes as described previously (20) and stored in liquid nitrogen. Terminal cisternae were isolated from the white muscle of New Zealand White rabbits as described by Saito et al. (26). Proteins were separated on a 6% SDS-PAGE transferred onto nitrocellulose and probed with a monoclonal antibody specific for type 1 RYR (27). Peroxidase-conjugated anti-mouse IgG was used to detect the primary antibody followed by chemiluminescence.

Intracellular Ca 2ϩ
Measurements-changes in the intracellular calcium concentration of the EBV-transformed B-lymphocyte cell lines were monitored with the fluorescent calcium indicator fura-2/AM (final concentration, 5 M) as described (20,28). Fluorescent changes (ratio 340/380 nm) were measured in a PerkinElmer Life Sciences spectrofluorometer equipped with a magnetic stirrer and thermostated at 37°C. All measurements were made in Ca 2ϩ -free Krebs-Ringer containing 0.5 mM EGTA.
Release of Cytokines-Peripheral blood mononuclear leukocytes were placed in the wells of a microtiter plate in Krebs-Ringer solution and incubated 37°C for 60 min under the specified conditions. For experiments in which the [Ca 2ϩ ] i was to be buffered, cells were pre-incubated with 50 M of the calcium chelator BAPTA/AM for 30 min in Ca 2ϩ -free Krebs-Ringer. Cells were then washed with Ca 2ϩ -free Krebs-Ringer medium and treated as indicated. After 60 min, cells were centrifuged and the amount of IL-1␤ (or IL-6) released into the supernatant was determined by using the CLB PeliKine Compact indirect ELISA kit following the manufacturer's instructions. All tests were performed in triplicate.
Data Analysis-For the two group comparisons, Student's t test was used; three or more groups were compared by one-way ANOVA. Doseresponse measurements were compared by repeated measurement ANOVA. Where ANOVA revealed a significant difference, the Fisher protect least significant difference post hoc test was performed. The overall statistical significance level was set to 5%. StatView™ from SAS Institute Inc. was used for statistical analysis.

RESULTS
The EBV-transformed B-cell lines were assessed for the presence of type 1 RYR using a monoclonal antibody we previously developed and characterized (27). Fig. 1 shows that an immunopositive high molecular weight band is present in the microsomal fraction obtained from the EBV-immortalized B-lymphocytes (lane 4). The antibody failed to react with type 2 RYR, which is present in heart microsomes (lane 2), or with any high molecular weight protein present in the microsomal fraction of rabbit brain (lane 3). The antibody reacted strongly with the RYR present in rabbit skeletal muscle terminal cisternae, a fraction that is highly enriched in this protein (lane 1).
Having determined that the B-cell lines do indeed express RYR 1, we performed RT-PCR analysis to confirm the presence of the mutated cDNA in cells derived from patients carrying the V2168M RYR1 mutation. A, lanes 1 and 3) and 338 bp (panel B, lanes 1 and 3), respectively. Digestion of the PCR-amplified genomic DNA with MslI gives rise to a band of ϳ192 bp (panel A, lanes 2 and 4). An extra band of ϳ196 bp (*) is present after digestion of the amplified genomic DNA from EBV-immortalized cells from a patient carrying the V2168M mutation (panel A, lane 4). Digestion of the PCR-amplified cDNA fragment with MslI yielded no additional bands when RT-PCR was performed on cDNA obtained from EBV-immortalized cells from a healthy volun- We next tested the sensitivity of EBV-immortalized B-cell lines from healthy controls and from the 4 MHS subjects carrying the V2168M RYR1 gene mutation, to the RYR-activator 4-chloro-m-cresol. This compound has been shown to activate type 1 and 2 RYR in isolated muscle vesicles but has no effect on type 3 RYR (20, 29 -31). Fig. 3A shows that the addition of 300 M 4-chloro-m-cresol causes an increase in the [Ca 2ϩ ] i of the B-cells. This effect is abolished by pre-treatment with thapsigargin an inhibitor of SERCA type CaATPase, indicating that the intracellular stores that are endowed with the RYR also contain this ATPase (Fig. 3B). We also tested the specificity of 4-chloro-m-cresol by adding it to HL60 cells, a human myelomonocytoid cell line. In this case treatment with 4-chlorom-cresol failed to elicit release of Ca 2ϩ from intracellular stores (Fig. 3C), confirming the specificity of this agonist and the lack of functional RYR1 calcium release channels in this myelomonocytoid cell line.
Malignant hyperthermia has been shown to affect the functional characteristics of the skeletal muscle calcium release channel; in particular, most mutations in the RYR1 gene cause a shift in the dose-response curve to RYR agonists, to a lower agonist concentration. Therefore, we examined the dose-response curve of 4-chloro-m-cresol-induced calcium release from intracellular stores of EBV-immortalized lymphocytes from the 4 MHS individuals carrying the V2168M mutation in the RYR channel.
We first calculated the size of the thapsigargin-sensitive calcium pools; the mean (Ϯ S.D.) change in fluorescence was 0.87 (Ϯ 0.19, n ϭ 11) fluorescence units, for controls and 0.74 (Ϯ 0.13, n ϭ 20) fluorescence units for cells carrying the V2168M mutation. Fig. 4 shows a representative trace of the peak [Ca 2ϩ ] i induced by 400 nM thapsigargin. Considering the small day-today variations in the experimental conditions and potential differences linked to the use of different cell lines however, we normalized our results by calculating the mean increase in [Ca 2ϩ ] i induced by different 4-chloro-m-cresol concentrations, as a percentage of the total amount of calcium released by 400 nM thapsigargin. We next performed a dose-response curve to 4-chloro-m-cresol by averaging the mean amount of calcium released from each of the cell lines established from the 4 patients harboring the V2168M mutation and from the 4 healthy volunteers. Our results show that the capacity of 4-chloro-m-cresol to induce Ca 2ϩ release was significantly different in the EBVimmortalized B-cells from control and MHS individuals ( Fig. 5; repeated measurement ANOVA, p ϭ 0.018) and the EC 50 for 4-chloro-m-cresol was shifted from 750 M in cells from control individuals to 450 M in cells from MHS individuals.
We next carried out two sets of experiments: first, we examined whether stimulation of RYR by caffeine and 4-chloro-mcresol caused the release of IL-1␤ and IL-6 from the EBVimmortalized B-cell lines. Under our experimental conditions, the amount of cytokines released by these cells was barely detectable. Thus, we examined the effect of RYR agonists on IL-1␤ and IL-6 release from peripheral blood mononuclear leukocytes. We also examined whether the effect of the RYR agonist could be blocked (i) by dantrolene, an inhibitor of the type 1 RYR (32,33) and (ii) by chelating intracellular calcium ions with BAPTA. We only investigated "early" cytokine release (after 60 min) as the symptoms of an MH crisis almost always occur within the first hour after contact with trigger agents. Experiments were first performed on whole peripheral blood mononuclear leukocytes from control individuals. Fig. 6 shows that under our experimental conditions both caffeine (panel A) and 4-chloro-m-cresol (panel B) caused a dose-dependent increase in interleukin production in cells from control individuals; the amount of IL-1␤ released was ϳ5-fold higher than that of IL-6 released. This increase appears to be calcium-dependent because depletion of intracellular calcium stores with BAPTA inhibited the release of IL-1␤ induced by 10 mM caffeine and 400 M 4-chloro-m-cresol (Fig. 7, crossed boxes). Furthermore, 20 M dantrolene abolished the stimulation of IL-1␤ release by 10 mM caffeine and 400 M 4-chloro-m-cresol, supporting the involvement of the RYR Ca 2ϩ channel in this process (Fig. 7,  hatched boxes).
Finally, we compared the amount of IL-1␤ released after addition of the two RYR agonists by peripheral blood mononuclear leukocytes from 4 healthy donors and 4 individuals carrying the RYR1 MH-linked mutation. Fig. 8 shows the percentage increase in IL-1␤ released, induced by treating leukocytes with the indicated concentrations of caffeine (panel A) and 4-chloro-m-cresol (panel B) for 60 min at 37°C. There was considerable between-subject variability in the amount of IL-1␤ released into the medium. We thus normalized the values by calculating the amount of IL-1␤ released after incubating the leukocytes from each individual, for 60 min at 37°C with carrier alone; this value was considered 100%, and the increase in interleukin released by caffeine and 4-chloro-mcresol was calculated. Repeated measurements ANOVA revealed significant differences in the amount of IL-1␤ released after addition of caffeine and 4-chloro-m-cresol, between individuals carrying the V2168M mutation and the control group (p ϭ 0.0034 and p ϭ 0.0026, respectively).

DISCUSSION
The clinical signs of an impending MH crisis are highly variable and include muscle rigidity, metabolic acidosis, rhabdomyolysis, tachycardia, and an increase in body temperature (1,2). Investigations into the molecular mechanism of MH have led to the hypothesis that this is a disorder of skeletal muscle excitation-contraction coupling and that the clinical signs are a result of the hypermetabolic state caused by alterations in the mechanism regulating the myoplasmic calcium concentration. The aim of the present report was to establish whether MHassociated mutations in the RYR1 gene could also be associated with an increase in body temperature via an alteration of thermoregulatory mechanisms. Body temperature is controlled by the thermoregulatory centers localized in the anterior hypothalamus. Increases in body temperature may be caused either (i) by a "peripheral" mechanism, involving an increase of thermogenesis by skeletal muscle contraction (shivering) and/or a decrease of thermodissipation, or (ii) by a central mechanism mediated by endogenous pyrogens. Phagocytic cells produce and release the major endogenous pyrogens IL-1, IL-6, tumor necrosis factor-␣, and interferon-␣, although endothelial cells, fibroblasts, myoblasts, and B-lymphocytes are also capable of producing, among others, IL-␣ and IL-␤.
We first carried out a set of experiments to confirm the results of Sei et al. (23) and unequivocally demonstrate the expression of type 1 RYR in EBV-immortalized B-cell lines. RT-PCR analysis revealed the presence of the mRNA encoding skeletal muscle type 1 RYR in B-lymphocytes. The presence of mRNA strongly suggests but does not necessarily prove the existence of the protein product. This issue was confirmed by immunoblotting, which revealed a protein band referable to the RYR in the microsomal fraction of B-lymphocytes. The expression of type 1 RYR in B-cells was also confirmed by genotype analysis of patients carrying the V2168M mutated RYR1 allele in their B-lymphocytes. The EBV-immortalized B-lymphocytes thus offer an interesting tool to investigate the functional effects of "natural" mutations in the RYR1 gene. To trigger calcium release from the intracellular store of EBV-immortalized B-cells, we used 4-chloro-m-cresol, a RYR type 1-specific agonist, which has been used to characterize MH-linked mutation (34 -37). Our results show that EBV-immortalized lymphocytes from normal donors are sensitive to 4-chloro-m-cresol, and that the presence of the V2168M mutation in the RYR1 gene decreases the EC 50 of 4-chloro-m-chresol-induced calcium release ϳ2-fold (750 M versus 450 M for control and MHS-EBV cells, respectively). We would like to point out that the EC 50 of 4-chloro-m-cresol-induced calcium release in EBV-transformed lymphocyte is similar to that described previously in other experimental models (34 -37). Thus, treatment with Epstein-Barr virus does not dramatically interfere with the pharmacological sensitivity of the RYR.
The most interesting result of the present report concerns the involvement of the RYR on IL-1␤ release from the peripheral blood mononuclear leukocytes of MHS and control individuals. Caffeine and 4-chloro-m-cresol are considered specific activators of RYR1, although the latter compound has been shown to be specifically active on RYR type 1 and 2 (20, 29 -31). We believe that caffeine and 4-chloro-m-cresol are eliciting IL-1␤ release by stimulating the RYR of circulating B-lymphocytes. However, because cells of the immune system work in an integrated network and communicate with each other via cytokine production, both in vivo and under our experimental conditions, we cannot exclude the possibility that activated B-lymphocytes may in turn activate other cell types (monocytes, endothelial cells, fibroblasts, etc.) to also release IL-1␤. We also assayed whether RYR agonists affect the release of other cytokines such as interferon-␥ or tumor necrosis factor-␣, but the amounts of these cytokines present in the supernatant of stimulated peripheral blood leukocytes was barely detectable (results not shown). As to the EBV-immortalized cell lines, the amounts of IL-1␤ released was barely detectable, a result consistent with the fact that synthesis and production of cytokines by B-lymphocytes may be restricted to specific stages of differentiation (38).
We found that maximal stimulating concentrations of 4-chloro-m-cresol and caffeine caused the release of approximately 100 pg of IL-1␤/10 6 cells. The amount of IL-1 produced by circulating B-lymphocytes in a normal individual could therefore be approximately 350 ng (0.7 ϫ 10 9 cells ϫ 5 liters of blood). Because IL-␤ is the most pyrogenic cytokine, inducing temperatures of 39°C in response to doses as small as 1-10 ng/kg body weight (25), the theoretical quantity of IL-1 produced is well within the limit necessary to increase body temperature via a central mechanism.
MH reactions can be triggered by inhalative anesthetics, exercise, and stress, and several laboratories have reported that anesthetic agents, as well as sustained exercise activity, increase the plasma level of pro-inflammatory cytokines (39 -42). Because human myoblasts have been shown to behave as immunologically active cells during inflammation, producing, among others, cytokines of the monocyte-macrophage cell lineage (43), it may be that in skeletal muscle cells cytokine secretion is influenced by elevations in the [Ca 2ϩ ] i . The RYR1 calcium channel plays a central role in controlling the myoplasmic [Ca 2ϩ ]; thus, our results imply that the muscle cells from MHS individuals may also produce more IL-1, an additional factor that may contribute to the increase in body temperature. Dantrolene, an inhibitor of the RYR1, is a life-saving compound used by clinicians to revert MH reactions and neuroleptic malignant syndrome (21,44,45). This pharmacological agent has also been shown to decrease plasma and tissue concentrations of inflammatory cytokines in septic animals and improve their survival (46 -49). Thus, by blocking the RYR, dantrolene may also block cytokine production by muscle cells and lymphocytes and, in this way, help re-establish physiological body temperature.