Naunyn-Schmiedeberg’s Arch Pharmacol (2005) 371: 122–132 DOI 10.1007/s00210-004-1013-7
ORIGINA L ARTI CLE
Henrietta Szappanos . Gyula Péter Szigeti . Balázs Pál . Zoltán Rusznák . Géza Szűcs . Éva Rajnavölgyi . József Balla . György Balla . Emőke Nagy . Éva Leiter . István Pócsi . Florentine Marx . László Csernoch
The Penicillium chrysogenum-derived antifungal peptide shows no toxic effects on mammalian cells in the intended therapeutic concentration Received: 30 August 2004 / Accepted: 1 December 2004 / Published online: 9 February 2005 # Springer-Verlag 2005
G. Balla . E. Nagy Department of Neonatology, MHSC, University of Debrecen, P.O. Box 22 Debrecen, Hungary, 4012
ies, however, have pointed to a membrane-perturbing effect of these antifungal compounds, apparent as a potassium efflux from affected fungal cells. If present on mammalian cells, this would severely hinder the potential therapeutic use of these molecules. Here we studied the effects of the P. chrysogenum-derived antifungal peptide (PAF) on a number of mammalian cells to establish whether the protein has any cytotoxic effects, alters transmembrane currents on excitable cells or activates the immune system. PAF, in a concentration range of 2–100 μg/ml, did not cause any cytotoxicity on human endothelial cells from the umbilical vein. Applied at 10 μg/ml, it also failed to modify voltage-gated potassium channels of neurones, skeletal muscle fibers, and astrocytes. PAF also left the hyperpolarization-activated non-specific cationic current (Ih) and the L-type calcium current unaffected. Finally, up to 2 μg/ ml, PAF did not induce the production of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α. These results suggest that PAF should have only minor, if any, effects on mammalian cells in the intended therapeutic concentration range.
É. Leiter . I. Pócsi Department of Microbiology and Biotechnology, Faculty of Sciences, University of Debrecen, P.O. Box 22 Debrecen, Hungary, 4012
Keywords Potassium current . Calcium current . Patch clamp . Cytotoxicity . Inflammatory action . Antifungal proteins
F. Marx Department of Molecular Biology, Medical University of Innsbruck, Innsbruck, Austria
Introduction
Abstract Certain filamentous fungi, such as the penicillinproducing strain Penicillium chrysogenum, secrete small, highly basic and cysteine-rich proteins with antifungal effects. Affected fungi include a number of important zoopathogens, including those infecting humans. Recent stud-
H. Szappanos . G. P. Szigeti . B. Pál . Z. Rusznák . G. Szűcs Department of Physiology, RCMM, MHSC, University of Debrecen, P.O. Box 22 Debrecen, Hungary, 4012 É. Rajnavölgyi Department of Immunology, RCMM, MHSC, University of Debrecen, P.O. Box 22 Debrecen, Hungary, 4012 J. Balla Department of Medicine, MHSC, University of Debrecen, P.O. Box 22 Debrecen, Hungary, 4012
L. Csernoch Cell Physiology Research Group of the Hungarian Academy of Sciences, University of Debrecen, P.O. Box 22 Debrecen, Hungary, 4012 L. Csernoch (*) Department of Physiology, Medical and Health Science Center, University of Debrecen, P.O. Box 22 Debrecen, Hungary, 4012 e-mail:
[email protected] Tel.: +36-52-416634 Fax: +36-52-432289
Filamentous fungi secrete a large number of proteins which might be part of their defense mechanism. Recently, a number of reports have focused on antifungal proteins secreted by Penicillium chrysogenum (Marx et al. 1995), Penicillium nalgiovense (Geisen 2000), Aspergillus niger (Lee et al. 1999), and Aspergillus giganteus (Wnendt et al. 1994). These peptides, while unrelated to cysteine-rich antimicrobial proteins from other species (Lehrer and Ganz 1996; Dimarcq et al. 1998; Fritig et al. 1998; Garcia-Olmedo et al. 1998), have a high homology including their amino acid sequence and relatively basic
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nature (Marx 2004; Theis and Stahl 2004). They were shown to inhibit the growth of different fungi (Wnendt et al. 1994; Marx et al. 1995; Lee et al. 1999; Geisen 2000; Theis et al. 2003), including human pathogens. Since peptides derived from various ascomycetes have long been used in human therapy, this latter finding clearly identifies these antifungal proteins as possible candidates for future antifungal drug development. While their structure and the pathway of their synthesis have been studied in detail (Wnendt et al. 1994; Marx et al. 1995), little is known about how these antifungal proteins exert their detrimental effects on target organisms. Kaiserer et al. (2003) have recently reported that the antifungal protein derived from P. chrysogenum (PAF) triggers the formation of reactive oxygen intermediates in A. niger together with reducing its metabolic rate. Besides the reduced metabolism, PAF was found to induce an efflux of potassium from cultured A. nidulans having intact hyphal tips, indicating a specific efflux pathway for potassium ions (Kaiserer et al. 2003). These effects are alarming for a potential therapeutic use if present on mammalian cells. Furthermore, cells of the human innate immune system express pattern recognition receptors, which are specific for various microbial compounds. The highly conserved family members of toll-like receptors (TLR) are expressed on neutrophils, monocytes, macrophages, and dendritic cells circulating in peripheral blood and respond to minute concentrations of cell wall components derived from bacteria and/or fungi (Barton and Medzhitov 2002). Ligandinduced activation through TLR results in the rapid production of pro-inflammatory cytokines, such as IL-6, IL-1β, TNF-α, and IL-8, which initiate inflammation and the acute phase response (O’Neill 2002). Proteins of microbial origin or their contamination with pg/ml amounts of TLR ligands, such as lipopolysaccharides (LPS) of Gram (−) bacteria (Triantafilou and Triantafilou 2002) or fungal components (Bellocchio et al. 2004; Braedel et al. 2004), may induce severe inflammatory responses. We therefore set out to explore the potential toxic effects of PAF on a wide variety of mammalian cells, specifically focusing on potassium currents, cell toxicity and inflammatory effects. Here we report that PAF, applied at 10 μg/ml, a concentration which was shown to induce potassium efflux from target fungi, failed to modify the potassium currents of all cells examined. In addition, PAF neither affected the total membrane current of hippocampal neurones nor changed the L-type calcium current of skeletal muscle fibers. In the concentration range of 2–100 μg/ml, where it was shown to severely inhibit the growth of a number of filamentous fungi, PAF had no toxic effects on human endothelial cells. Finally, PAF had only little, if any, effects on the production of IL-6, IL-8, and TNF-α, indicating that its inflammatory effects should be minor. These results establish PAF as a potential new antifungal therapeutic agent.
Materials and methods Preparation of tissue and cell samples Preparation of brain slices The basic steps of the preparation were the same as described earlier (Rusznák et al. 1997). Briefly, 7- to 14-day-old Wistar rats were decapitated, and 150- to 200-μm-thick, sagittal slices of the cochlear nucleus were cut by employing a Campden vibratome (Campden Instruments, Loughborough, UK). The slices were maintained in an incubation chamber containing normal artificial cerebrospinal fluid (aCSF; in mM: NaCl, 125; KCl, 2.5; glucose, 10; NaH2PO4, 1.25; NaHCO3, 26; CaCl2, 2; MgCl2, 1; myo-inositol, 3; ascorbic acid, 0.5; sodium pyruvate, 2). The pH was 7.2 when gassed with 95% O2/5% CO2; its osmolarity was set to 320 mOsm/l. During the electrophysiological experiments, the slices were continuously perfused (∼1 ml/min) with gassed aCSF solution. In some cases the extracellular solution contained 1 μM tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel) during the recordings, to prevent the activation of voltage-gated Na+ channels. Isolation of hippocampal neurones The neurone isolation procedure was similar to that described earlier (Rusznák et al. 2001). Briefly, after the decapitation of the 6- or 9day-old rat the brain was removed, and the hippocampus separated. The enzymatic dissociation was achieved by using normal aCSF containing 0.03 mg/ml collagenase (type IA) plus 0.12 mg/ml pronase (type XIV), for 50 min at 31°C. The enzyme treatment was terminated by transferring the tissue to normal aCSF containing 1 mg/ml trypsin inhibitor (type I-S). Neurones were isolated by gentle trituration with fire-polished Pasteur pipette in HEPES buffered aCSF, containing (in mM): NaCl, 135; KCl, 3; glucose, 10; HEPES, 10; sucrose, 30; CaCl2, 2; MgCl2, 1. After dissociation, the cells were allowed to settle for 30 min prior to the experiments. Enzymatic isolation and tissue culturing of astrocytes Astrocyte cultures were prepared from the hippocampus of the rat. The preparation of the hippocampus was performed in ice-cold dissecting medium (D1; in mM: NaCl, 136; KCl, 5.2; Na2HPO4·H2O, 0.64; KH2PO4, 0.22; glucose, 16.6; sucrose, 22; HEPES, 10; plus 0.06 U/ml penicillin and 0.06 μg/ml streptomycin), followed by its enzymatic dissociation in D1 solution, containing trypsin (0.025 g/ml; 30 min, 37°C). At the end of the incubation period the tissue pieces were transferred to minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS) for 5 min (room temperature). Individual cells were separated by gentle agitation with fire polished Pasteur pipettes. The cell suspension was then diluted to 100,000 cells/ml, and 0.5 ml of this suspension was transferred onto cover-slips situated in 12 wells of a 24well tray (marginal wells were not used in order to reduce the risk of infection). Cells were allowed to grow at 37°C in a 5% CO2 atmosphere. The feeding medium (MEM, supplemented with 10% FCS) was changed on the fol-
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lowing day, and on every other day, thereafter. Four- to 5day-old (∼70–80% confluent) cultures were employed for the electrophysiological experiments.
lected by centrifugation at room temperature (1,500 rpm, 23 min) and stored at −70°C until cytokine determination.
Isolation and voltage clamp of rat skeletal muscle fibers Skeletal muscle fibers isolated enzymatically from the extensor digitorum communis muscles of rats were mounted into a double Vaseline gap chamber as described earlier (Szentesi et al. 1997). Briefly, rats were anaesthetized and killed by cervical dislocation. The muscles were removed and were treated with collagenase (Sigma, St. Louis, MO, USA; Type I) for 60–90 min at 37°C. The isolated fiber was transferred into a recording chamber filled with relaxing solution containing (in mM) K-glutamate, 150; MgCl2, 2; HEPES, 10; and EGTA, 1. Fiber segments in the two end pools were permeabilized by a brief exposure to 0.01% saponin. After completing the permeabilization, the solutions were exchanged to internal solution in the openend pools (containing in mM Cs-glutamate, 120; MgCl2, 5.5; Na2-ATP, 5, Na-phosphocreatine, 10; glucose, 10; HEPES, 5; and EGTA, 5) and to external solution in the middle pool (containing in mM TEA-CH3SO3, 140; MgCl2, 2; HEPES, 5; tetrodotoxin, 0.0003’ and 3,4-diaminopyridine, 1). For calcium current measurements 10 mM CaCl2 was added to the external solution and TEACH3SO3 was reduced appropriately, while the EGTA concentration in the internal solution was increased to 20 mM with reducing Cs-glutamate. For potassium current measurements the internal solution contained 120 mM K-glutamate instead of Cs-glutamate and the external solution was modified to have 140 mM N-methyl-D-glucamine instead of tetraethyl ammonium (TEA), and 4 mM MgCl2 instead of 2 mM. All solutions were adjusted to pH 7.2 and 300 mOsm/l.
Functional measurements
Endothelial cell isolation and culture Human umbilical vein endothelial cells (HUVECs) were removed from human umbilical vein by exposure to dispase II (Boehringer Mannheim, Vienna, Austria) as described earlier (Balla et al. 1993). Cells were cultured in medium 199 containing 15% fetal bovine serum (both from Life Technologies, Vienna, Austria), supplemented with L-glutamine, sodium pyruvate, endothelial growth factor, a mixture of penicillin streptomycin (100 U/ml each), and 5 U/ml heparin (Biochemie, Vienna, Austria). Endothelial cells were identified as described earlier (Balla et al. 1993). Activation of white blood cells Fresh blood samples of healthy donors were obtained from the Regional Blood Transfusion Center. Blood clotting was inhibited by citratebuffered dextrose. Three milliliters of blood were incubated with 300 μl of the test samples at 0.2–20 μg/ml final concentrations of PAF. Negative controls were incubated with 300 μl saline. Positive control samples were incubated with 300 μl LPS (E. coli 0128, Difco Laboratories, Sparks, MI, USA) solutions to give a final concentration of 0.1 μg/ml. Samples were incubated for 1 h at room temperature on a shaker and the in vitro culture was continued for 24 h at 37°C in a CO2 incubator (5% CO2). Plasma was col-
Recording of ionic currents in neurones and astrocytes Whole-cell patch-clamp pipettes were fabricated from thinwalled borosilicate glass (Clark Electromedical Instruments, Reading, UK), and filled with a solution containing (in mM): KCl, 120; HEPES, 40; MgCl2, 1; EGTA, 10; Na3GTP, 0.5; MgATP, 2. The resistance of these patch pipettes varied between 2 and 4 MΩ when filled with the pipette solution. The series resistance was usually between 3 and 7 MΩ and it was compensated by 40–80%. The series resistance was kept as constant as possible during the measurements by occasional light suction or repositioning of the electrodes. For the whole-cell voltage-clamp experiments an Axopatch 200A patch-clamp amplifier was used, together with a DigiData 1200 interface (Axon Instruments, Foster City, CA, USA). Data acquisition and analysis were performed by employing the pClamp 6.0 software. Unless otherwise stated, cells were incubated in PAF for at least 5 min prior to the measurements. Recording of ionic currents in isolated rat skeletal muscle fibers The experimental setup and data acquisition have been described in detail in earlier reports (e.g., Csernoch et al. 1999). Fibers were voltage-clamped and the holding potential was set to −100 mV. All experiments were performed at 16–18°C. Calcium current (ICa) was measured using 800-ms depolarizing voltage pulses exploring the −50 to +60 mV voltage range. Potassium current (IK) was measured using 100- or 200-ms depolarizing voltage steps exploring the −80 to +40 mV voltage range. The linear capacitive component was subtracted by applying 20 mV hyperpolarizing pulses (Szentesi et al. 2001). Cells were incubated in PAF for at least 5 min prior to the measurements. Endothelial cell cytotoxicity assay Endothelial cell monolayers were grown in 24-well tissue-culture plates. Prior to exposure to PAF, cells were washed twice with Hanks balanced salt solution (HBSS, Life Technologies). Cells were treated with PAF at concentrations from 2 to 100 μg/ ml in HBSS and in medium 199. After incubation for 7 and 16 h, respectively, the test solutions were replaced with 550 μl 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium-bromide (MTT) in HBSS and cells were incubated for additional 6 h (Mosmann 1983). The amount of reduced MTT was measured spectrophotometrically at 570 nm after the formazan was dissolved in 100 μl 10% sodium dodecyl sulfate and 500 μl hot isopropanol containing 20 mM HCl. Cytokine determination Measurement of soluble IL-6, IL8 and TNF-α cytokine concentrations in the plasma samples was performed by ultrasensitive double sandwich enzyme immunoassays (ELISA) according to the manu-
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facturer’s instructions (Biosource International, Camarillo, CA, USA). Cytokine concentrations were calculated from the linear range of the standard curves determined in all titrations. The results were expressed as pg cytokine/ml detected in the undiluted individual plasma samples. The optimal dilution of the negative and positive controls was determined in preliminary experiments. The inflammatory effect of the test samples was characterized by the relative increase in cytokine concentration compared with the basal level detected in the untreated control plasma. Cytokine concentrations measured in the LPS-treated blood samples were used as positive controls and reflected the magnitude of the pro-inflammatory response in the different donors. Protocols and chemicals—purification of PAF P. chrysogenum NCAIM 00237 was grown in a sucrose (20 g/l)–NaNO3 (3 g/l) minimal medium for 72 h at 25°C with shaking (4.2 Hz; Marx et al. 1995). Mycelia were removed with centrifugation and the low molecular weight protein fraction of the supernatant was separated in Amicon Stirred Cells (volume=50 ml, Biomix PBTK ultrafiltration discs, size exclusion limit Mr=30,000; Millipore, Billerica, MA, USA). PAF was purified with ion exchange chromatography on a CM Sephadex Fast Flow column (2×18 cm, equilibrated with 50 mM sodium phosphate buffer, pH 6.6, flow rate 1 ml/min, t=4°C, NaCl gradient 0.05–1.0 M; Amersham-Pharmacia, Uppsala, Sweden). The quality of the preparation was always checked with SDS-PAGE on pre-cast Novex 16% Tris/glycine gels (Invitrogen Life Technologies, Carlsbad, CA, USA), where protein bands were visualized with Coomassie Brilliant Blue R staining. All protocols followed the guidelines put forth by the Declaration of Helsinki and have been approved by the Institutional Ethics or Institutional Animal Care committees. Chemicals, unless otherwise stated, were purchased from Sigma and were of analytical grade. Averages were expressed as means±standard error (SE) of the mean. Significance between groups of data was assessed using Student’s t test.
Results Ionic currents on neurones in brain slices In the first step of the experiments the effects of PAF were investigated on the hyperpolarization-activated nonspecific cationic current (Ih) of the bushy cells situated in brain slices prepared from the ventral cochlear nucleus. The detailed characterization of this current component was performed in a previous study (Cuttle et al. 2001). In the present experiments 2-s hyperpolarizing pulses were delivered from a holding potential of −60 mV in 10-mV steps, to a maximum hyperpolarization of −140 mV. As Fig. 1A indicates, the time course of the current was not affected in the presence of 10 μg/ml PAF. Although a slight decrease in the Ih amplitude can be noticed in Fig. 1A, a statistically
significant difference could not be substantiated when the data from all cells were pooled, as demonstrated by the current–voltage relations of the instantaneous (measured just after the capacitive transient) and steady-state (determined at the end of the hyperpolarizing voltage step) current components (n=7). Note that in order to avoid the error introduced by the different size of the cells investigated, current density was calculated and plotted. In the next step of the experiments the depolarizationactivated current components were investigated in slices. In these cases a holding potential of −60 mV was applied and 200-ms voltage steps were delivered starting from −80 mV up to a maximum depolarization of +40 mV. No channel blockers were employed in these experiments. Although this experimental approach does not allow the detailed investigation of the Na+ currents, the records demonstrated in Fig. 1B do not imply any change of the amplitude of the Na+ current. The amplitude of the depolarization-activated outward current was not affected significantly either, as indicated by both the individual current traces and the normalized current–voltage relationships (n=12). The latter observation can be clearly assessed on the bases of the data presented in Fig. 1C. In this (and similar) experiment a single, 200-ms depolarizing voltage step was applied from a holding potential of −60 to 0 mV depolarization in every 10 s. TTX was regularly employed in these experiments. After recording the control current traces (and making sure, therefore, that the seal and the current were stable enough) the application of 10 μg/ml PAF was commenced. The cell was exposed to the drug for 5 min then a wash-out period followed. As the current traces (recorded prior to and during the application of PAF) as well as the time course of the current amplitude indicate, no major change was induced by the presence of the drug. Ionic currents on isolated neurones and astrocytes in culture The effect of PAF on the voltage-gated currents of neurones was investigated in a more conventional experimental design as well. In these cases enzymatically separated hippocampal neurones were employed. The pulse protocol was similar to that applied in conjunction with Fig. 1B, but the holding potential was set to −90 mV. To exclude the interfering effects of the Na+ currents, TTX was added to the bath solution. As Fig. 2A demonstrates, PAF did not influence the depolarization activated K+ currents of the hippocampal neurones. Similar experiments were carried out on four more cells, and the normalized, pooled data are presented in Fig. 2B, indicating that neither the activation thresholds of these components nor their amplitudes was affected by PAF application. To have an overview of the potential effects of PAF on the function of the central nervous system, its effects on astrocytes maintained in tissue culture were also tested. Figure 3A shows that the drug did not seem to induce any change in the amplitude of the currents present on the
126 Fig. 1 Effect of Penicillium chrysogenum-derived antifungal peptide (PAF) on bushy cells situated in brain slices. A Effects on the hyperpolarizationactivated non-specific cationic current (Ih) of the bushy cells. The holding potential (VH) was set to −60 mV and 2-s hyperpolarizing voltage steps were delivered to a maximum hyperpolarization of −140 mV in 10-mV steps. Actual current recordings from the same cell are presented prior to (control) and during (PAF) the application of 10 μg/ml PAF. The bottom panel presents pooled data (mean ± SE; n=7) from similar experiments. Here and in all subsequent figures current amplitudes in pooled data were normalized to the cell capacitance. Squares indicate the instantaneous, while circles the steady-state current–voltage relations (here and in all subsequent figures filled symbols control, open symbols PAF application). B Effect of PAF on the depolarization-activated currents of bushy cells. VH=−60 mV was used and 200-ms depolarizing voltage steps were employed to a maximum depolarization of +30 mV in 10-mV steps. Current recordings from the same cell are presented prior to (control) and during (PAF) the application of 10 μg/ml PAF. For clarity only every second trace is shown. Bottom panel displays pooled data from similar experiments (n=12). Triangles indicate the peak, while squares the steady-state current– voltage relations. C The cell was depolarized to 0 mV for 200 ms (VH=−60 mV) once every 10 s. The current amplitude was measured and plotted as the function of time. Open symbols demonstrate current values obtained during the application of the drug. Current traces are representative recordings from before and during PAF application.
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indicating no major change in the voltage dependence. Pooled data from six experiments on the voltage dependence of peak current (Fig. 4C) confirmed this observation. Although IK was slightly, but not significantly, smaller at certain voltages in the presence of PAF, overall, the drug had no major effects on the membrane potential dependence of IK in skeletal muscle. L-type calcium currents in skeletal muscle To complement the observation on potassium currents, measurements were carried out to characterise the L-type calcium current on skeletal muscle cells. Figure 5A presents families of current records under conditions where all but the calcium channels were blocked. The current under control conditions (Fig. 5Aa) activated at −20 mV, had a slow rising phase and displayed a very slow inactivation, all characteristic of the L-type calcium channels. PAF, in a concentration of 10 μg/ml (Fig. 5Ab), neither altered the voltage at which the current first appeared nor did it modify the kinetics of current activation. On the other hand, the amplitude of the current was slightly suppressed at all voltages. Since the L-type current is known to “run-down” (McDonald et al. 1994), mea-
Fig. 2 Effect of PAF on the depolarization-activated currents of enzymatically isolated hippocampal neurones. A The 200-ms depolarizing voltage steps were employed from VH=−90 mV, to a maximum depolarization of +40 mV in 10-mV steps. Current recordings from the same cell are presented prior to (control) and during (PAF) the application of 10 μg/ml PAF. B Pooled data collected from similar experiments (n=5). Triangles indicate the peak, while squares the steady-state current–voltage relations.
astrocytes maintained in tissue culture. It is worth noting that the current configuration presented in Fig. 3A indicates that these measurements were conducted on GluT type astrocytes (Matthias et al. 2003). The normalized current densities from 12 astrocytes also demonstrate that PAF did not exert significant effect on the ionic currents presented by glial cells. Potassium currents in skeletal muscle To complete the evaluation of the effects of PAF on potassium currents, skeletal muscle fibere from the rat were voltage clamped and the current through the delayed rectifier potassium channel was measured. Figure 4 presents families of current records before (Fig. 4A) and after (Fig. 4B) the addition of 10 μg/ml PAF. As evidenced from the figure, the drug neither affected the time course nor the amplitude of IK. Currents displayed a peak at around 30 ms after the onset of the pulse and showed a slow inactivation characteristic of skeletal muscle. Currents started to activate between −70 and −60 mV both in control and after the addition of PAF,
Fig. 3 Effect of PAF on the currents of astrocytes maintained in tissue culture. A The 200-ms voltage steps were delivered between −80 and +20 mV in 10-mV steps from a holding potential of −60 mV. Current recordings from the same cell are presented prior to (control) and during (PAF) the application of 10 μg/ml PAF. B Pooled data from similar experiments (n=12).
128 Fig. 4 Effect of PAF on K+ currents of isolated skeletal muscle fibers. A Potassium currents recorded from a fiber in response to 200-ms depolarizing voltage steps exploring the −80 to 0 mV voltage range in 10-mV increments (VH=−100 mV). B Potassium currents from the same cell following the application of 10 μg/ml PAF. Currents were normalized to cell capacitance. C Pooled data (n=6) presenting the current– voltage relationship for the amplitude of the currents.
surements were repeated after the removal of the drug (Fig. 5Ac). Current traces were similar in time course but smaller in amplitude as compared with those measured either under control conditions or in the presence of PAF, indicating that the suppression was indeed due to the “rundown” of the current. Fig. 5 Effect of PAF on L-type Ca2+ currents of isolated skeletal muscle fibers. A Calcium currents recorded in response to 800-ms depolarizing voltage steps exploring the −20 to +50 mV voltage range in 10-mV increments (VH=−100 mV). Calcium currents from the same cell Aa before, Ab following the application and Ac removal of 10 μg/ml PAF. Currents were normalized to cell capacitance. B Pooled data (n=4) presenting the current–voltage relation of the amplitude of the currents (filled symbols average of control and wash, open symbols PAF application).
To characterize the voltage dependence of L-type calcium current activation, data in control and after wash were first averaged for each individual cell. This averaged value together with the corresponding value in the presence of PAF were plotted as a function of the membrane potential during the test pulse. Pooled data from four
129 Table 1 Effects of Penicillium chrysogenum-derived antifungal peptide (PAF) on umbilical vein endothelial cells
Norm. A570*
2 μg/ml PAF
5 μg/ml PAF
10 μg/ml PAF
30 μg/ml PAF
50 μg/ml PAF
100 μg/ml PAF
1.05±0.05
1.01±0.07
1.04±0.04
1.05±0.03
1.05±0.04
1.08±0.10
*Absorbance in an 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium-bromide (MTT) colorimetric assay at various PAF concentrations normalized to that measured in the absence of the drug
fibers, calculated as described above, are displayed in Fig. 5B, revealing no significant difference between values in control and in the presence of the drug. PAF, thus, affected neither the kinetics nor the voltage dependence of the L-type calcium current in skeletal muscle.
changes were significantly smaller than that induced by LPS at a 20-fold lower concentration. Taken together, these data suggest that PAF has minor, if any pro-inflammatory action.
Cytotoxicity of PAF The data in the previous sections have convinced us that PAF has little, if any effect on various voltage-gated ion channels of mammalian tissues. It was, therefore, of interest to see whether PAF had any cytotoxic effects. To this end, endothelial cells were isolated from the umbilical vein, cultured in 24-well plates and treated with different concentrations of the drug for several hours. Cytotoxicity was assessed using the colorimetric MTT assay by calculating the proportion of measured optical density of PAF-treated and control cells and then expressing the values as percent of untreated cells. Table 1 presents pooled data from three independent experiments (each carried out in triplicates) at concentrations ranging from 2 to 100 μg/ ml. The results in Table 1 show that PAF, in the concentration range tested, had no effects on human umbilical vein endothelial cells in the MTT assay. Effects of PAF on pro-inflammatory cytokine production To assess the inflammatory effect of PAF, whole human blood was incubated with various concentrations of the protein and the concentrations of released IL-6, TNF-α, and IL-8 cytokines were measured. IL-6 concentrations measured in the control samples of the individual donors varied between 2 and 56 pg/ml, with an average concentration of 12±5 pg/ml (n=10). Similar values for IL-8 were 149–1,414 and 776±156 pg/ml (n=8), while for TNF-α they were 1.5–19 and 8.4±3.4 pg/ml (n=5). LPS, in a concentration of 0.1 μg/ml, caused a massive production of all three cytokines tested for each sample (Fig. 6) resulting in several ten-fold (IL-8; Fig. 6B), hundredfold (TNF-α; Fig. 6C) or thousand-fold (IL-6; Fig. 6A) increases. These observations established that all samples were capable of producing a large increase in cytokine production. Penicillium chrysogenum-derived antifungal peptide, on the other hand, failed to induce appreciable release of any of the three cytokines. The relative increase in the production was 1.4±0.7, 1.8±0.4, and 2.6±1.3 for IL-6, IL-8, and TNF-α, respectively, at 2 μg/ml (Fig. 6). All of these
Fig. 6 Effect of PAF on pro-inflammatory cytokine production. A IL-6, B IL-8, and C TNF-α levels were measured after the addition of various concentrations of PAF to blood samples from different donors. Data were normalized to the plasma concentration of the given cytokine before the addition of PAF and are presented as averaged values from different individuals. Lipopolysaccharide (LPS; filled bars in each panel) at a concentration of 0.1 μg/ml was used as a positive control.
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Discussion In this study we explored the effects of the Penicillium chrysogenum-derived antifungal peptide with the question whether or not it has any major toxic effects on mammalian cells. We report that PAF failed to alter any voltage-gated current investigated on any of the cell types tested (neurones, astrocytes and skeletal muscle). These include different potassium currents, the L-type calcium current, the hyperpolarization-activated non-specific cationic current, as well as the total membrane current. In addition, the drug had no detectable cytotoxic effects on endothelial cells from the umbilical vein and had only minor pro-inflammatory action as assessed by its effects on the production of IL-6, IL-8, and TNF-α. These observations suggest that PAF has little, if any toxic effects on mammalian tissues and provide a basis for further investigations that might lead to the introduction of a new class of antifungal drugs into human therapy. Effects on potassium currents Penicillium chrysogenum-derived antifungal peptide and its close analogue, the antifungal peptide (AFP) from A. giganteus, have been shown to induce membrane permeabilization in affected fungi (Kaiserer et al. 2003; Theis et al. 2003) associated with substantial efflux of potassium in the case of PAF. This observation is at least alarming if these molecules are to be used in therapy. It suggests that PAF and its relatives open a pathway through which potassium ions can leave the cell. As discussed by Kaiserer et al. (2003), this effect cannot be attributed to necrosis, rather, the pathway—at least in fungi—seems to be specific for potassium ions, suggesting the involvement of an ion channel. Despite this suggestion, we found essentially no effect on voltage gated potassium channels of neurones, astrocytes and skeletal muscle (Figs. 1–4) in a concentration of 10 μg/ml, previously shown to induce significant potassium efflux from target fungi (Kaiserer et al. 2003). In addition, the holding current in patch-clamp experiments was also unaffected by the drug, indicating that two-poredomain potassium channels—thought to be responsible for the resting K+ conductance of the cells (for review, see e.g., Lesage and Lazdunski 2000)—were also not altered. These observations can be reconciled only if one assumes that the potassium channels of filamentous fungi are substantially different from those in the animal kingdom, or the channels affected in these fungi are of a different family of potassium channels then those tested here. While detailed reports on the similarity of potassium channels in fungi and in mammals have not, to our knowledge, been published, the first cloned K+ channel (NcTOKA) in the filamentous fungus N. crassa (Roberts 2003) belongs to the two-pore-domain potassium channel family, also present in mammalian cells. The electrophysiological properties reported by Roberts (2003) suggest that, at least for this family, K+ channels in the two kingdoms are not that different. It should also be noted here that NcTOKA channels
conduct an inward current at voltages negative to the equilibrium potential for potassium ions (Roberts 2003). Were they to be responsible for the potassium efflux observed previously following the addition of PAF, a depolarization caused by PAF needs to be accounted for. Taken all this, our data suggest that PAF affects another class of potassium channels in target fungi and at this point we can only speculate which class of potassium channels this might be. A possible candidate could be the G-protein coupled channels (Dascal 2001), which would imply that PAF might act on the G-protein, or on the signal transduction pathway, rather than on the potassium channel itself. Nevertheless, the presence of these channels in filamentous fungi remains to be proven. Effects on other ion channels The effects of PAF on two other voltage dependent ion channels were tested specifically. These included the hyperpolarization-activated non-specific cationic current and the L-type calcium current. Both these currents play crucial roles in the cellular function in a wide variety of tissues. Ih may regulate repetitive activation of neurones and it is involved in the pacemaker function of sino-atrial node cells in the heart (Robinson and Siegelbaum 2003). L-type calcium channels, or dihydropyridine receptors (DHPR-s), are responsible for the trigger inward calcium current in cardiac (Bers 2001) and smooth muscle excitation–contraction coupling as well as in excitation– secretion coupling. DHPR-s also serve as voltage sensors in striated muscle (Ríos and Pizarro 1991). Given these diverse and vital functions it is of major importance that PAF was found not to modify these currents. It should, however, be stressed that these channels are not identical in the different cell types. A number of different isoforms of the channel proteins for both the hyperpolarization-activated current and the L-type calcium current have been identified (even if one considers only the pore-forming α1 subunit of the DHPR; Catterall 2000), with a specific tissue distribution. The findings here should, therefore, be treated as an indication only that these channel classes are not affected by the antifungal drug. Although fast voltage-gated sodium channels were not investigated directly in this study, the observation that PAF had very little, if any effects on the early phase of the total membrane current in neurones clearly indicates that these channels are also not affected by the drug. Again, due to isoform differences (Goldin 1999), these data give an indication only that this class of voltage gated channels is not influenced by PAF. However, taken all the individual negative results, they together point in the direction that this antifungal peptide has no direct effects on mammalian voltage gated channels. It should be mentioned here that Kaiserer et al. (2003) have reported that the ionic strength of the solution influences the effect of PAF on target fungi. They have shown that the inhibition of growth observed in the presence of PAF was reduced with increasing salt concen-
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tration. This effect was especially prominent in the presence of MgCl2 or Na2SO4 (0.1 M), where the inhibition was reduced from a control value of more than 90% to approximately 20%. This interaction, if present under our conditions, could render the interpretation of our results difficult. However, NaCl had little effect on the inhibition of growth, since the inhibition was found to be larger than 80% even in the presence of 0.1 M NaCl (Kaiserer et al. 2003). Furthermore, the concentration dependence of the inhibition in NaCl was shallow, indicating that marked changes at higher NaCl concentrations are not expected. Since our measurements were carried out in solutions where the major constituent was NaCl (Mg was present at a concentration of around 1 mM), we believe that this effect of salts, under the conditions used here, had to be minor. Cytotoxic and inflammatory action of PAF Data from affected filamentous fungi pointed towards a specific pathway for potassium efflux following the incubation with PAF. However, based on the results from Theis et al. (2003), a general membrane perturbing effect of a similar antifungal protein was also suggested. While our measurements of the holding current in voltage clamp experiments proved that PAF does not acutely cause an increase in leak current (Fig. 1), long-term detrimental effects, based on these measurements, could not be excluded. The experiments using the endothelial cells from the umbilical vein were designed to answer this specific question. Our data clearly indicate that the incubation with PAF (2–100 μg/ml) for several hours, even at concentrations higher than those used in the voltage clamp studies (10 μg/ ml), does not provoke cytotoxic effects. These observations, on the one hand, suggest that PAF has no detrimental effects on the cell membrane of mammalian cells and, on the other hand, indicate that the effect on target fungi is indeed specific for potassium equilibrium. A protein with the size of PAF is at the limit of being potentially immunogenic in humans. Since the pro-inflammatory cytokines IL-6, IL-8, and TNF-α are produced primarily by activated monocytes—present in the peripheral blood of healthy individuals at approximately 3–5%— they provide a simple way of testing this effect. Furthermore, the rapid secretion of these cytokines in situ results in the production of other cytokines, acute phase proteins and other biologically active compounds, which initiate an inflammatory response. This rapid response of the innate immune system has a pivotal role in regulating acquired immunity but also may cause fatal sepsis syndrome in humans (Lakhani and Bogue 2003). In our in vitro experiments we showed that the basal level of IL-6, IL-8, and TNF-α measured in the individual plasma samples varied, but the relative increase in cytokine levels induced by the test samples was comparable. LPS, as a positive control, induced a strong response in all donors tested indicating that all donors were able to mount a pro-inflammatory reaction. PAF, up to 2 μg/ml, did not
induce an increased production of the pro-inflammatory cytokines IL-6, IL-8 or TNF-α tested in 5–10 independent experiments performed with the blood of individual blood donors. The slight increase in cytokine levels at the highest protein concentration tested (2 μg/ml) may suggest a minimal contamination by residual fungal cell wall components, which might have activated the cells of the innate immunity. Acknowledgements The authors wish to thank R. Öri and I. Varga for their technical assistance. The work was supported by research grants from OTKA (TS040773, T034894, T046067, T034315, and T037473) and the Office for Higher Education Programs (0092/2001) of Hungary. I.P. is a recipient of the Széchenyi István Scholarship. F.M. was supported by the Austrian Science Foundation (FWF grant P-15261) and the University of Innsbruck (grant X8).
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