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Apis Mellifera Venom and Melittin Block neither NF-B-p50-DNA Interactions nor the Activation of NF-B, Instead They Activate the Transcription of Proinflammatory Genes and the Release of Reactive Oxygen Intermediates¹

Ludwig Boltzmann Institute for Rheumatology and Balneology, Vienna, Austria

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Many alternative treatment approaches, originating from Asia, are becoming increasingly popular in the Western hemisphere. Recently, an article published in a renowned journal reported that venom of apis mellifera (bee venom (BV)) and melittin mediate immune-modulating effects by blocking the activation of the transcription factor NF-B. Such a modus operandi would corroborate the many claims of beneficial effects of BV treatment and give immediate credit to this form of therapy. Fibroblast-like synoviocytes from rheumatoid arthritis patients and dermal fibroblast cells and white blood cells from healthy volunteers were used to study the effects of BV and melittin on the activation of NF-B and a series of genes that are markers of inflammation. EMSAs demonstrate that neither BV nor melittin blocked IL-1-induced NF-B activation; neither did they affect phosphorylation or degradation of IB. Contrary to published data, even high concentrations of BV and melittin were without any effect on NF-B-p50-DNA interactions. More importantly, in fibroblast-like synoviocytes, but also in dermal fibroblasts as well as in mononuclear cells exposed to BV or melittin, mRNA levels of several proinflammatory genes are significantly increased, and Western blot data show elevated cyclooxygenase-2 protein levels. Furthermore, exposure to BV higher than 10 µg/ml resulted in disintegration of all cell types tested. In addition, large quantities of oxygen radicals are produced in a dose-dependent manner in leukocytes exposed to BV. Taken together, data presented in this work do not corroborate an earlier report regarding the effectiveness of BV as an inhibitor of the transcription factor NF-B.

	Introduction

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Utilization of venoms of various organisms has a long history in traditional as well as alternative medicine. With few exceptions, such as the use of cobra venom factor in experimental transplantation research, therapies based on zootoxins have to date not been widely used nor accepted by mainstream medicine. Lately, however, a series of articles were published, some in highly regarded journals, describing beneficial effects of the venom of apis mellifera (1, 2, 3, 4, 5).

Unconfirmed circumstantial evidence for the favorable effects of bee venom (BV)³ on a wide variety of ailments goes back for centuries. With the increasing popularity of alternative medicine and the adoption of Eastern medicine in the Western hemisphere, BV therapy has increasingly gained acceptance among physicians in Europe and in the U.S. BV has been reported to be a possible remedy for many ailments ranging from multiple sclerosis, asthma, polyneuritis, and neuralgia, to malaria, wound healing, and epilepsy. The list of ailments that reportedly can be ameliorated by BV is extensive; most dominant and persistent, however, are reports describing the usefulness of BV for the treatment of various kinds of rheumatism (3, 4, 5). For the most part, reports on the various effects of BV originate in Asia and Eastern Europe, where this form of therapy, alone or in combination with other forms of treatment, seemingly enjoys widespread use (2).

The first basic studies regarding isolation, identification, composition, and pharmacological effects of the components of BV were conducted in the 50s and 60s of the last century. Such analyses revealed that 88% of venom is water. The vast majority of the remaining content is melittin, which accounts for 50–60% of the BV dry weight. Among other protein and peptide contents are pamine (1–3%), mast cell-degranulating peptide (1–3%), secapin (1–2%), procamine (0–1%), and adolapin (0.1–0.8%). Protease inhibitor content reportedly ranges from 0.1 to 15% of BV dry weight. Dominant among the enzymes that have been found to be present in BV is phospholipase A₂ (10–12%), followed by hyaluronidase (1–3%), acid phosphomonoesterase (1%), lysophospholipase (1%), and -glucosidase (0.6%). Histamine (0.5–2%), dopamine (0.2–1%), and noradrenaline (0.1–0.7%) are among the physiologically active amines. The remaining dry weight content is composed of glucose and fructose (2%), phospholipids (5%), aminobutyric acid (0.5%), and -amino acids (1%) (see Ref. 6 and citations in this work).

Speculations as to the mode of action of BV therapy abound and range from blocking undesired enzyme reactions to acting as a booster for the immune system. However, until recently, no clear modus operandi has been described that might explain the many anti-inflammatory effects associated with BV. This has changed with the very recent demonstration that BV, but also melittin, acts by preventing the activation of the transcription factor NF-B (1). This transcription factor is of utmost importance in inflammation in that the up-regulation of most proinflammatory genes depends on the activation of this factor (7). Such a mode of action would put BV and melittin among the most potent anti-inflammatory compounds and provide a rationale for their use in diseases that are characterized by unfettered activation of proinflammatory genes, such as rheumatoid arthritis. This reported observation, however, is surprising considering the composition of BV. Aside from histamine and MCP that account for all of the obvious undesired effects, most of the components of BV have clearly been associated with detrimental effects. Dopamine, among other undesired properties, affects the heart rate and increases the blood pressure (8); apamin can act as a potent neurotoxin (9, 10), but also functions as a K⁺ channel blocker that interferes with NO-induced contractile activity of the myometrium (11). Hyaluronidases degrade, among other things, the protective hyaluronan coating of cells and contribute to the generation of undesired hyaluronan fragments (12). Phospholipase A₂, having antimicrobial hydrolytic activity, takes part in an array of biological processes ranging from homeostasis of cellular membranes to the formation of lipid mediators such as eicosanoids and other arachidonic acid metabolites (13), and therefore also acts as a potent activator of inflammation (14). Melittin, the main constituent of BV, is known to possess lytic activity that is due to its ability to insert into phospholipid layers (15).

In contrast, at least some of the components of BV have been reported to exert effects that might be of benefit. Aminobutyric acid has been associated with protection from the development of diabetes (16), although this substance is better known as a highly neuroactive substance. MCP, with its potent histamine-releasing activity, has been attributed with certain anti-inflammatory effects (17). In summary, for all the aforementioned reasons, one would expect the net effect of BV treatment to tip toward an unfavorable outcome. Therefore, it seems surprising that BV could block the activation of the crucial transcription factor NF-B. We therefore decided to revisit and to further investigate the compounds of BV as well as the mechanisms that mediate its effect on NF-B activation. In this study, we present in vitro data, resulting from studying the effects of BV and melittin on the activation of NF-B and mRNA levels of a selected group of proinflammatory genes in several cell types as well as on oxygen radical release by leukocytes.

	Materials and Methods

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Reagents

If not stated otherwise, reagents, e.g., PMA, melittin, and BV, were from Sigma-Aldrich. IL-1 and TNF- were purchased from Strathmann Biotec. Abs for p38, ERK, and JNK used in Western blots were from Cell Signaling Technology. Abs used in EMSA supershift experiments were from Santa Cruz Biotechnology. Oligonucleotides resembling consensus sequences for NF-B, AP-1, etc., were from Promega. The Ab recognizing cyclooxygenase-2 (COX-2) as well as the PGE₂ ELISA were from Cayman Chemical. The 2',7'-dichlorofluorescin-diacetate (DCFH-DA) and calcein-AM were from Molecular Probes.

Cell culture

A series of human fibroblast-like synoviocytes (FLS) were used in these experiments. Information regarding the origin of cells, passage numbers, culture conditions, etc., of FLS has been provided in detail elsewhere (18). White blood cells were isolated from freshly drawn blood of healthy volunteers. Hep G₂ cells were purchased from the European Collection of Cell Cultures, and normal dermal fibroblast cells (SF) were from LGC-Promochem.

EMSA

Preparation of nuclear extract, execution of EMSA, as well as EMSA specificity control experiments have been described in detail elsewhere (18, 19). In short, the double-stranded oligonucleotides used in all experiments were end labeled using T4 polynucleotide kinase and [-³²P]ATP. After labeling and purification by chromatography, 5 µg of nuclear extract was incubated with 100,000 cpm of labeled probe in the presence of 1.5 µg of poly(dI-dC). The resulting mixture was separated on a native 6% polyacrylamide gel. For specific competition, 7 pmol of unlabeled NF-B oligonucleotides was included. For nonspecific competition, 7 pmol of the double-stranded nucleotides was used. For supershift assays, 1 µl of specific supershift Abs (Santa Cruz Biotechnology) was added to the nuclear extract 15 min before the addition of the labeled probe.

Western blot experiments

SDS-PAGE and Western blotting were conducted essentially as described (18). In short, cells were washed twice in ice-cold PBS and subsequently dissolved in SDS sample buffer without bromphenol blue (62.5 mM Tris/HCl (pH 6.8), 2% w/v SDS, 10% glycerol, 50 mM DTT). Proteins were quantitated, separated (8 or 10% polyacrylamide mini gels), blotted, and stained with indicated Abs. Proteins were made visible using RenaissancePlus (PerkinElmer Life Science) and Kodak BioMax MR films or the chemiluminescence detection device GeneGnome (Syngene). In Western blot experiments, lower concentrations of proteins were loaded on separate gels that served as controls for loading and protein transfer (loading controls (LC)). Such blots were stained with an Ab recognizing tubulin, or were stained with Ponceau red.

Real-time RT-PCR, data analysis, and quality controls

Gene expression in FLS was measured by real-time RT-PCR on a Mx3000P (Stratagene), using SYBR Green as reporter fluorophore for quantitating mRNA levels (18). To normalize the amount of total RNA present in each reaction, mRNA levels of hypoxanthine phosphoribosyltransferase and/or actin were used. Results are expressed as relative threshold cycle (CT values) (CT values of mRNA levels in stimulated minus CT values of a given gene in resting cells). Standard curves were generated for each gene using serial dilutions of RNA isolated from stimulated or unstimulated FLS as controls for amplification efficiency. Basal mRNA expression levels in unstimulated FLS were chosen to represent 1x expression of a given gene. Most primer sequences, amplification curves, and equations for the calculation of hypoxanthine phosphoribosyltransferase have been reported elsewhere (18, 20, 21, 22). The remaining sequences of primers used in this study were as follows: MCP-1 forward, 3'-ctg cag ctc tgt gtg aag g-5'; MCP-1 reverse, 3'-aat ttc tgt gtt ggc gca gt-5'; IL-1 forward, 3'-tca gca cct ctc aag cag aa g-5'; IL-1 reverse, 3'-ccc tag gga ttg agt cca ca g-5'; TNF- forward, 3'-cct caa cct ctt ctg gc tca g-5'; TNF- reverse, 3'-agg ccc cag ttt gaa ttc tt g-5'; IL-6 forward, 3'-cac aag tcc gga gag gag ac g-5'; and IL-6 reverse, 3'-cag aat tgc cat tgc aca ac g-5'.

Membrane integrity assay

RBC were washed three times and resuspended to a final concentration of 2 x 10⁷ RBC/ml in HBSS. Subsequently, aliquots (90 µl/well) were transferred to 96-well plates. Serial dilutions of melittin or total BV (10 µl/well) were added in triplicates; thereafter, plates were transferred to 37°C. At indicated intervals, OD was measured at 490 nm. Total lysis, expressed as 0% cell membrane integrity in this type of experiment, was induced by H₂O. Furthermore, OD readings at 490 nm of cells kept in PBS were considered to represent 100% intact cells. Lysis (loss of hemoglobin) was also confirmed by microscopy. FLS membrane integrity was assessed by the loss of calcein. FLS were washed with PBS or HBSS and incubated with calcein-AM at 4°C for 30 min, washed again three times, and exposed to BV and/or melittin. Loss of calcein was monitored on a fluorimaging device (FluorImager-595; Amersham Biosciences).

Respiratory burst measurements

Oxidative products in white blood cells were measured using a published, nondestructive method that allows for continuous measurement of oxygen radicals using DCFH-DA (23). This method was modified in that 24-well tissue culture plates were used and fluorescence of dichlorofluorescin was monitored on the FluorImager-595. Polymorphonuclear cells (PMN) and mononuclear cells (MNC) were isolated according to the method provided by the supplier of histopaque-1077 and histopaque-1119 (Sigma-Aldrich). DCFH-DA-loaded PMN (2.5 x 10⁵/well) and MNC (1.5 x 10⁵/well) were resupended in HBSS (with CaCl and MgCl) and incubated with and without serial dilutions of BV and melittin at 37°C. For the measurements of DCFH-DA oxidation, plates were transferred to a fluorescence imaging device FluorImager-595. In some cases, oxidation of DCFH-DA was also analyzed on a flow cytometer (FACScan; BD Biosciences).

Quality controls and statistical analysis

Basal mRNA expression levels in unstimulated FLS were chosen to represent 1x expression of a given gene. All experiments were done at least three times. Amplification curves and quality controls for RT-PCR have been reported elsewhere (18, 22, 24). Statistical analysis was done using the nonparametric Wilcoxon-Mann-Whitney U test. Value of p 0.05 is considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Neither BV nor melittin has any effect on IL-1-induced phosphorylation or degradation of IB

Degradation of IB is in most cases prerequisite for the activation of NF-B. For this reason, BV and melittin were used to investigate whether the reported inhibitory effect of these compounds on NF-B activation is due to the blockage of IB phosphorylation and/or IB degradation. IL-1 induces many proinflammatory genes in FLS and has been shown to act as a potent stimulus for NF-B activation in FLS (22, 24). FLS were left untreated medium (MED) or exposed to BV (5 and 0.5 µg/ml) or melittin (2.5 and 0.25 µg/ml) for 30 min. After this period, IL-1 was added at a concentration of 2.5 ng/ml. IL-1-induced phosphorylation of IB was monitored at 5 min, and the presence of IB protein in total protein extract was monitored by Western blot analysis after 20 min of exposure to IL-1. Shown in Fig. 1 are data that demonstrate that at the concentrations used, BV had no effect on IL-1-induced IB degradation. However, as shown in the left panel of Fig. 1, preincubation with pyrrolidine dithiocarbamate (50 µM, 30 min), a known NF-B inhibitor, completely blocked IL-1-induced degradation of IB.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. IL-1 induces complete and rapid degradation of IB, an event that is not influenced by preincubation with BV. FLS were left untreated (MED) or pretreated with BV (5 and 0.5 µg/ml) or pyrrolidine dithiocarbamate (50 µM). After 30 min, IL-1 (2.5 ng/ml) was added and cells were incubated for an additional 20 min. LC, Indicates a blot that serves as a control for equal loading and protein transfer.

BV and melittin do not inhibit IL-1-induced activation of NF-B in FLS, nor do they compete for p50-DNA interactions

FLS were cultured in 10-cm-diameter culture dishes until highly confluent. Medium was changed and cells were left either untreated (MED), exposed to BV (5 and 2.5 µg/ml), or pretreated with melittin (2 and 0.2 µg/ml) for 45 min. Thereafter, where indicated, IL-1 (2.5 ng/ml) was added and FLS were placed into the incubator for another 45 min. Nuclear protein extract was prepared, as described previously (19). As shown in the representative experiment in Fig. 2A, FLS treated with IL-1 readily respond with activation and subsequent translocation of NF-B. Also shown in this figure is that neither BV nor melittin activated NF-B. More importantly, however, neither BV nor melittin had any effect on IL-1-induced NF-B-DNA interactions. It is worth mentioning that, initially, a series of similar experiments were performed in which BV and melittin were added simultaneously with IL-1; only after such experiments did not demonstrate any significant effect on IL-1-induced NF-B activation did I resort to a period of preincubation, a procedure that usually is opted for in similar inhibitory studies.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. EMSA demonstrating that neither BV nor melittin prevents NF-B-DNA interactions. A, FLS were left untreated or pretreated with 5 and 0.5 µg/ml BV or with 2 and 0.2 µg/ml melittin. Where indicated, 30 min afterward IL-1 was added. Cells were harvested after an additional 45 min. Competition experiments are shown in B. NF-B and AP-1 oligonucleotides were preincubated with the indicated amounts of BV and melittin or Abs directed against p50 and p65; 30 min later, nuclear protein extract was added. After an additional 20 min, the resulting mixtures were separated by gel electrophoresis. Shown in C are a series of control experiments demonstrating EMSA specificity and the activation of the classic NF-B complex p50/p65 in FLS stimulated with IL-1.

As shown in this figure, even high concentrations such as the 10 µg/ml BV and the 5 µg/ml melittin were without any effect. The label "w/o" indicates the lane in which protein extract of IL-1-stimulated FLS was loaded. In the lanes labeled "p50" and "p65," Abs recognizing p50 and p65 were added to the reaction mixture. The lane labeled "p50" also serves to demonstrate the presence of sufficient amount of p50 in the nuclear protein extract of FLS.

Identical experiments were done using oligonucleotides resembling the AP-1 consensus sequence. As shown in the lower section of Fig. 2B, similar to NF-B, BV and melittin were also without effect on AP-1-DNA interactions.

Shown in Fig. 2C are data from control experiments that demonstrate that exposure of FLS to IL-1 results in the activation and translocation of the classical NF-B complex p50/p65. This figure also demonstrates the specificity of the EMSA experiments in that only unlabeled NF-B oligonucleotides could compete for NF-B-DNA interactions, and that the same concentration of unlabeled AP-1 and CRE oligonucleotides was without effect. Shown on the right panel of Fig. 2C are supershift experiments that again demonstrate the presence of p50 and p65, but also the absence of p52 and c-Rel in the shifted complex. Specific SP-1 Abs were added as an additional specificity control in the lane labeled "SP-1."

No significant activation of NF-B by BV and melittin

In most cases, 30–90 min of activation is sufficient for activation and translocation of NF-B. Because BV and melittin act as potent inducers of MAPK, in many cases a prerequisite for the activation of NF-B, it was tested whether BV or melittin might activate NF-B only if exposed for prolonged periods. Such experiments reveal that exposure of FLS to BV and melittin did not result in significant activation of NF-B, even when exposed to these two reagents for up to 7 h (data not shown).

In all EMSA experiments, the label "NS" indicates a band that resembles an non-NF-B protein that also binds to consensus NF-B oligonucleotides. "Free Pr." indicates the position of unbound ³²P-labeled oligonucleotides.

MAPK are activated by BV and melittin within minutes

Phosphorylation of MAPK is a well-documented feature of cell activation, and many anti-inflammatory reagents exert their effects by blocking MAPK. To test whether BV and melittin might affect MAPK activation, Western blot experiments were performed. FLS were treated with BV for times ranging from 0 to 3 h. Total proteins were extracted and adjusted where needed. Equal concentrations of protein were separated by gel electrophoresis. Blots, resulting from SDS-PAGE, were stained with Abs recognizing the phosphorylated forms of p38, ERK, and JNK. As shown in Fig. 3A, exposure to BV (5 µg/ml) induced phosphorylation of all three MAPK in a time-dependent manner. Shown on the bottom of Fig. 3A (labeled "Tubulin") is one example of control experiments demonstrating equal loading.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. BV and melittin induce the phosphorylation of all three MAPK, but not the degradation of IB. FLS were exposed to either 5 µg/ml BV or to 2.5 µg/ml melittin for times (in minutes) indicated on top of these figures. As indicated on the right, blots were stained with Abs specific to the phosphorylated forms of MAPK (p38, JNK, ERK) and for IB. One blot serving as a LC is included as well (Tubulin; A).

Induction of COX-2 and IL-8 by BV and melittin

Next, because MAPK are activated by BV and melittin, it was tested whether genes that are associated with inflammation are induced rather than blocked by exposure to BV and melittin. Real-time RT-PCR was used to monitor the response of FLS over a wide time range. FLS were stimulated with BV and melittin from times ranging from 1 to 24 h. Among the genes tested were the cytokines IL-1, IL-8, and TNF- and the adhesion molecules VCAM-1 and ICAM-1. Also tested were changes in mRNA levels of hyaluronansynthase-1 (HAS1) and COX-2; the metalloproteinases MMP-1, MMP-2, and MMP-9; as well as the stress response genes heat shock protein-70 and heat shock protein-32; and genes involved in apoptosis, such as BAX, BAD, and BCL-2. Interestingly, with the exception of COX-2 and IL-8, no significant changes in mRNA levels of the above genes were noted in FLS in response to BV and melittin treatment. Shown in Fig. 4A is a comparison of the effects of IL-1 (2.5 ng/ml), BV (5 and 1.66 µg/ml), and melittin (2.5 and 0.8 µg/ml) treatment on the mRNA levels of the genes COX-2 and IL-8 in FLS. Compared with untreated cells (MED), at each time point analyzed, IL-1-induced mRNA levels for IL-8 and COX-2 were significantly higher (n = 3, p 0.05). More importantly, treatment of FLS with BV (5 µg/ml) and melittin (2.5 µg/ml) for 12 and 24 h also resulted in significantly elevated mRNA levels of IL-8 and COX-2 (n = 3, p 0.05).

View larger version (44K): [in this window] [in a new window]   FIGURE 4. The genes COX-2 and IL-8 are activated by melittin and BV. FLS were exposed to BV, melittin, or IL-1 (2.5 ng/ml) for indicated lengths of time. Shown in A are data of RT-PCR experiments that demonstrate that both IL-8 and COX-2 mRNA levels are significantly elevated in FLS treated with either BV (5 µg/ml) or melittin (2.5 µg/ml) for 12 and 24 h (n = 3, p 0.05). Also shown is that IL-1 treatment resulted in significantly higher mRNA levels of COX-2 and IL-8 at all time points measured (n = 3, p 0.05). Shown in B are Western blot experiments demonstrating changes in COX-2 proteins in FLS that were exposed to BV and melittin for 12 and 24 h. BV (5 and 0.5 µg/ml), melittin (Mel) (2 and 0.2 µg/ml), or IL-1 was used for times indicated on the left. LC, Marks a blot that serves as LC. A and B, MED refers to cells that were left untreated.

Severe disruption of cell membrane integrity by BV and melittin

Melittin, which accounts for 50% of the dry weight of BV, has been shown to affect cell membrane integrity. In addition, it has been noted in preliminary dose-finding experiments on FLS that concentrations of BV higher than 10 µg/ml eventually will affect cell viability. To test the effects on cell membrane integrity, a RBC lysis test was established in which washed RBC were kept in PBS; exposed to BV, melittin, or H₂0; and in which OD readings at 490 nm were used as an indicator of membrane integrity. BV at final concentrations ranging from 0.45 to 1000 µg/ml was added, and the retention of hemoglobin in RBC was measured at intervals ranging from 4 to 120 min. Complete loss of membrane integrity (0% intact cells) was induced by H₂O, and OD readings from cells kept in PBS were considered 100% intact. As shown in Fig. 5A, exposure of RBC to concentrations of BV higher than 10 µg/ml resulted in nearly instantaneous loss of membrane integrity. Such effects are clearly dose and time dependent in that after 120 min, even concentrations as low as 0.45 µg/ml resulted in the loss of 40% of hemoglobin.

View larger version (40K): [in this window] [in a new window]   FIGURE 5. Membrane integrity is severely affected by BV. Shown in A are the results of an RBC lysis test demonstrating that exposure of RBC to BV results in dose- and time-dependent loss of hemoglobin. Complete loss of membrane integrity (0% remaining hemoglobin) was achieved by H2O treatment, and 100% membrane integrity was defined by the hemoglobin values measured at 4 min in RBC kept in PBS. Values given in the legend on the right side of this figure are BV in µg/ml, and measurements were done in triplicates. Shown in B are experiments that demonstrate BV-induced leaking out of the fluorescence dye calcein as a consequence of BV treatment. A, Resulted from data obtained 15 min after addition of BV, and B from data acquired after 60 min of exposure to BV at 37°C. Shown in C are data resulting from measuring fluorescence in aliquots of supernatant of plates incubated for 15 min. Numbers 1–6 represent the following: untreated FLS (1), 10 µg/ml BV treated (2), 5 µg/ml BV treated (3), 2.5 µg/ml BV treated (4), 1.25 µg/ml BV treated (5), and Nonidet P-40 treated (6).

If melittin was used instead of BV, approximately one-half of the concentration was needed to obtain very similar effects to those produced by BV in these types of assays (data not shown).

Shown on the right in Fig. 5 are actual fluorescence scanner pictures; shown on the left are graphs resulting from computation of the fluorescence readings in wells 1–6. Relative fluorescence units (RFU) are given on the y-axes of each graph.

Cell death by apoptosis and necrosis

Because membrane integrity seemed severely affected by both BV and melittin, it was decided to investigate whether these compounds induce massive cell death within a relevant time frame. FLS were exposed to BV (5 and 10 µg/ml) for up to 4 h, after which DNA was extracted with the help of a DNA isolation kit from Qiagen. Shown in Fig. 6 are data that demonstrate that although there are signs of apoptosis as well as necrosis in FLS, exposure of these cells to 5 µg/ml BV does not lead to massive cell death, because most DNA is intact and the typical 180-bp fragments are largely absent. As a control for the classical apoptotic DNA ladder formation, DNA from blood stored at 4°C for 14 days was isolated and separated as well (lane CTLR).

View larger version (89K): [in this window] [in a new window]   FIGURE 6. Signs of necrosis and apoptosis in FLS exposed to BV. FLS were treated with 5 and 10 µg/ml BV for 4 h. Although one fragment of 180 bp is present in BV-treated FLS, indicating BV-induced apoptotic events, the majority of the DNA fragments made visible by gel electrophoresis resemble a pattern of necrotic DNA. SM, Marks the position of the size markers, and shown in the lane labeled "CTRL" is a control experiment showing a classical apoptotic DNA pattern.

MNC and PMN are cells that play a key role in the propagation of inflammatory responses. For this reason, it was thought that these cell types might be suited to further assess the pro- or anti-inflammatory effects of BV. Release of oxygen intermediates is a measure for cell activation; therefore, MNC and PMN were separated and subsequently exposed to BV at concentrations ranging from 0.625 to 10 µg/ml. Shown in Fig. 7 is one of three experiments conducted in triplicates. Compared with PMA, a potent inducer of the respiratory burst, BV at concentrations of 5 and 10 µg/ml induced both cell types to produce larger quantities of oxygen intermediates than PMA. Furthermore, even concentrations as low as 1.25 µg/ml BV induced significantly more oxygen radicals than can be measured in resting, nontreated cells (Contr.) (n = 3; p 0.05). RFU are given on the y-axes, and culture conditions (BV in µg/ml) are indicated on the x-axes. The upper panel refers to data obtained from PMN, whereas the lower panel depicts data obtained from MNC. The label "Contr." indicates fluorescence values of untreated cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. BV activates PMN and MNC to produce large quantities of reactive oxygen intermediates. Cells were labeled with the fluorescence dye DCFH-DA and left untreated (Contr.) or treated with PMA (25 ng/ml), or exposed to BV at concentrations ranging from 0.625 to 10 µg/ml for 30 min. Emitted fluorescence (RFU) was quantitated using ImageQuant software and plotted.

BV prevents neither TNF--induced IB degradation nor NF-B-DNA interactions

It could be argued that the effect of BV might be restricted to certain stimuli or limited to specific cell types. To further investigate the potential of BV as an inhibitor of NF-B activation, a series of EMSA and Western blot experiments were performed. FLS, SF, as well as MNC, and, in some cases, macrophages were used and left either untreated or pretreated with concentrations of BV ranging from 0.5 to 5 µg/ml. After a 30-min preincubation period, TNF- (2.5 ng/ml) was added where indicated. Cells were harvested after an additional 60-min incubation period. Such experiments revealed that up to the highest concentration used (5 µg/ml), BV does not interfere with TNF--induced NF-B activation. Similar to the outcome of EMSA experiments using IL-1 as stimulus, nowhere was TNF--induced NF-B-DNA binding significantly affected. Levels of NF-B-DNA complexes were reduced neither in FLS, SF, MNC, nor macrophages.

As shown in the EMSA data presented in Fig. 8A, all cell types tested readily responded to TNF- treatment with the activation and translocation of NF-B. More importantly, as a comparison of the cells treated with TNF- only (lane TNF) with cells treated with BV (5 µg/ml) before exposing these cells to TNF- shows (lane BV 5 + TNF), in none of these experiments did BV treatment result in reduced NF-B-DNA binding. The label "MED" refers to control experiments in which cells were left untreated.

View larger version (28K): [in this window] [in a new window]   FIGURE 8. EMSA experiments demonstrating that BV does not interfere with TNF--induced NF-B activation. The effectiveness of BV as inhibitor of TNF--mediated NF-B activation was tested by EMSA in three different cell types (FLS, MNC, and SF). One representative EMSA experiment is shown in A. Cells in the lane BV 5 + TNF were pretreated with 5 µg/ml BV for 30 min, followed by addition of TNF- (2.5 ng/ml) for an additional 60 min. Cells in lane TNF were exposed only to TNF- (2.5 ng/ml) for 60 min, whereas cells in lane MED were left untreated. BV treatment does not affect TNF-induced IB degradation. Data in B demonstrate that BV has no effect on TNF-induced IB degradation. FLS (upper section; A) and SF (lower panel; B) were pretreated with BV for 30 min, followed by exposure to TNF- for 15 min. LC refers to blots that were stained with tubulin as an additional LC.

BV itself activates and does not prevent transcriptional activation of proinflammatory genes in TNF--stimulated cells

Next, FLS as well as SF were used to investigate whether BV might affect TNF--induced action at the transcriptional level. Such cells were left untreated or were pretreated with BV (0.5 and 5 µg/ml) for 30 min. Subsequently, TNF- (2.5 ng/ml) was added to induce gene transcription. Representative data of experiments using SF are shown in Fig. 9A. Such experiments reveal that BV neither affects TNF--induced transcriptional activation of IL-1, COX-2, VCAM-1, nor that of HAS1, another gene that depends on NF-B for its activation. Also, no inhibitory effect of BV could be seen in identical experiments using TNF- to induce the same genes in FLS (data not shown). However, analogous to data obtained using FLS, significantly higher mRNA levels of several genes could be observed in SF that were exposed to BV only. Although BV (0.5 µg/ml) was not sufficient to significantly increase any of the monitored mRNAs, 5 µg/ml BV for 8.5 h resulted in significant higher levels of HAS1 and COX-2, and IL-1 levels of mRNA.

View larger version (37K): [in this window] [in a new window]   FIGURE 9. BV does not prevent TNF--induced gene activation. Shown in A are data demonstrating that in TNF--stimulated FS, levels of mRNA are not diminished when these cells are pretreated with BV. Neither 0.5 nor 5 µg/ml has any suppressive effects on induced mRNA levels of the genes tested. However, exposing SF to the higher of the two concentrations of BV (5 µg/ml) resulted in significantly elevated levels of HAS-1, COX-2, and IL-1 mRNA (n = 3; p 0.05). Shown in B are effects of BV on steady-state mRNA levels of several genes in MNC induced by TNF- or IL-1. As shown here, both stimuli activated a series of genes; however, neither 0.5 nor 5 µg/ml BV had any significant inhibitory effect on the genes tested. However, in MNC exposed for 8.5 h to 5 µg/ml BV, significantly increased mRNA levels of TNF-, IL-1, and COX-2, but not IL-6, were noted.

	Discussion

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From histamine, involved in the sometimes deadly allergic responses to a variety of nerve toxins, to phospholipase A₂, which degrades cell membrane phospholipids, to melittin, a highly lytic molecule, BV contains many compounds that live up to its name and characterization as a venom. The venom sac of bees contains 200–300 µg of venom, but in most cases not all of it is injected into victims. Although the list of reported beneficial effects of bee sting treatment is long, most studies were done under conditions that do not fulfill meticulous Western medical standards and seem to be based primarily on anecdotal evidence. For this reason, and due to the absence of controlled trials, a final judgment of the effectiveness of BV therapy is still lacking. Nonetheless, BV treatment is increasingly gaining popularity and followers in the Western medical community.

The recently reported demonstration of BV and melittin as potent inhibitors of NF-B (1) would support the many claims that BV and/or melittin can indeed be used as an effective anti-inflammatory remedy. This would also give credit to this form of therapy for the treatment of arthritis, because people suffering from the many forms of this disease are the main target for the increasingly promoted and popular BV therapy, a form of treatment that is no longer restricted to supporters and followers of alternative medicine or to patients in countries with limited access to conventional medicine.

As said above, an effect of BV and melittin, the compound that accounts for 50% of BV dry weight, on NF-B activation is surprising because there is a vast body of literature that describes properties of melittin that are identical with the observations made in the course of the experiments described in this study. Melittin has been shown to possess detergent-like actions (25). Melittin shares its amphipathic properties with a series of peptides that are characterized by their capacity to disturb cell membrane bilayer integrity, either by creation of defects, disruption, or through pore formation (25). The resulting opening in the lipid bilayer leads to the collapse of the transmembrane electrochemical gradients, events that provide an explanation for the reported destructive activities of the class of channel forming, amphipathic peptides (26). Although some of these amphipathic peptides are currently being investigated due to their potential as a novel class of antibiotics, melittin has long ago been shown to be indiscriminate in that it lyses both bacteria as well as eukaryotic cells (27). Such descriptions are in accordance with observations made during experimentation for this work. No cell type tested (MNC, PMN, Hep G₂, dermal fibroblast cells, FLS) was able to maintain cell membrane integrity at BV concentrations higher than 10 µg/ml. In the experiments presented in this work, RBC, the cell type most sensitive to BV and melittin, disintegrated within seconds at this concentration. Others, investigating viability and cell membrane integrity of the lymphoblastoid cell line Hmy2, demonstrated that concentrations as low as 3.5 µM affected cell viability significantly (28). Similarly, Pratt et al. (29) reported that only concentrations of melittin lower than 2 µM do not disrupt cell membranes of leukocytes. In stark contrast, the reported inhibitory effect on NF-B-p50 was achieved by exposing Raw 264.7 cells to up to 50 µg/ml melittin (equivalent to 17.5 mM) without reporting any detrimental effects on cell viability (1).

In experiments conducted in this laboratory on a number of cell types, no inhibitory effect of BV or melittin on IL-1- or TNF--induced NF-B activation could be observed, even when, as in initial dose-finding experiments, the BV concentrations used were so high that they resulted in massive and near instant cell disintegration. It is also not entirely clear whether the reported inhibition of NF-B-p50-DNA interaction would even result in the inhibition of NF-B-dependent genes because LPS has been shown to activate proinflammatory genes not only by p50/65 heterodimers, but also by the generation of p65 homodimers (30). In addition, NF-B-p50-DNA binding rather than inhibition would seem to be advantageous because NF-B-p50 lacks the trans activation domain of p65 and has been described to exert suppressive effects on the activation of proinflammatory, NF-B-dependent genes (30). It has, for example, also been shown that it is the LPS-induced NF-B-p50 homodimers that are responsible for unresponsiveness of cells to LPS and other NF-B-activating substances (31).

Instead of having anti-inflammatory properties, at least at high concentrations BV as well as melittin seem rather to exert proinflammatory effects. A conclusion that is also supported by the data (Fig. 3) is that, in FLS, both compounds activate all three MAPK, events that are considered to be clear markers of cell activation (32).

Furthermore, COX-2 and IL-8 are significantly up-regulated in FLS treated with BV and melittin. COX-2 activation is clearly undesirable in arthritis patients, a view that is supported by the success of specific COX-2 inhibitors for treating rheumatoid arthritis. IL-8, in contrast, is the principal chemoattractant protein for neutrophils and neutrophil activation, events that normally are equally undesired. In addition to the observations made using FLS, it is noteworthy that BV also failed to block both IL-1- as well as TNF--induced activation of proinflammatory genes in cells other than FLS. On the contrary, not only did BV fail to block NF-B activation in MNC and SF, it actually led to a significant increase in mRNA levels of a number of proinflammatory genes in these cell types.

At first, it might seem contradictory that, as shown in Fig. 5, A and B, FLS clearly become leaky already at relatively low concentrations of melittin, but remain viable and even activate the above mentioned genes at significantly higher concentrations than those resulting in significant loss of calcein, the marker molecule used to assess cell membrane integrity. However, it has been observed that cells can recover and are able to reduce the number of melittin molecules per cell over time (28). Still, the threshold value of melittin compatible with survival of Hmy2 cells is 2–3 orders of magnitudes lower than what has been presented as an inhibitory concentration for NF-B activation (1, 28).

When comparing data gained from experiments in this laboratory with that of Park et al. (1), demonstrating NF-B inhibition by BV and melittin, it is of interest to note that nonstimulated FLS cultured in this laboratory never demonstrated any significant phosphorylation of IB, whereas IB proteins in resting synoviocytes used by Park et al. have been shown to be highly phosphorylated. In addition, whereas IB, a protein that is normally degraded in the cytoplasm within minutes following stimulation, has indeed been shown to be able to shuttle between cytoplasm and nucleus, I am unaware of data demonstrating a high degree of phosphorylation of IB detectable inside the nucleus, as shown by this group in synoviocytes as well as in Raw 264.7 cells (1). Similarly, some of the EMSA experiments shown by Park et al. (1) also demonstrate a high degree of NF-B activation in resting Raw 264.7 cells (Fig. 5A), something that again seems rather unusual for resting, unstimulated non-B cells.

As shown by supershift experiments (Fig. 2B), NF-B-p50 is readily detectable in the shifted DNA-protein complex of stimulated FLS and, contrary to anti-NF-B-p50 Abs, BV does not compete for NF-B-p50-DNA binding in this type of assay. Such data are in accordance with the observation that neither BV nor melittin interferes with IL-1-induced activation of NF-B-dependent genes in FLS. It is not entirely clear why Park et al. (1) concluded from their experiments that BV and melittin prevented DNA-NF-B-p50 interactions. The data provided in their paper are difficult to interpret because EMSA supershift controls for this particular experiment using rNF-B-p50 in a cell-free system are not provided. Such data would be essential to clarify in which of the resolved DNA-protein complexes NF-B-p50 takes part. The dominant lower protein-DNA complex is clearly not affected by BV. In addition, it is also not clear which protein extract was used as a positive (LPS) control because, as stated in the figure legend, rNF-B-p50 was incubated in the absence or presence of BV and melittin in a cell-free system.

Among a series of genes tested, COX-2 and IL-8 in FLS, but also COX-2, IL-1, TNF-, and MCP-1 in MNC, and COX-2, IL-1, and HAS-1 in SF were significantly higher in cells exposed to BV. The observed activation of these genes is significant and of importance; however, it is also modest when compared with the response elicited by IL-1 in this cell type. These data also fit well into the overall picture that a single bee sting will be relatively harmless and that most fatalities result from allergic reactions. Less clear is the significance of the demonstration of large amounts of oxygen radicals released in response to BV. Although there is no doubt that oxygen radicals will contribute to tissue destruction, in vivo they might also amplify negative effects of BV, for example, by increasing IL-8 production, activating collagenases, etc. (33, 34, 35, 36).

Taken together, neither BV nor melittin blocked IL-1- or TNF--induced activation of NF-B. However, both BV and melittin induced the activation of all three MAPK and, although modest in degree, activated a number of genes with documented proinflammatory properties in all cell types tested. Also of importance, BV seems to be among the most potent inducers of oxygen radical release in leukocytes. Furthermore, exposure of cells to BV and melittin leads to near instant cell disintegration at concentrations that are relatively low when compared with the amounts used to show suppression of NF-B-p50-DNA interactions. Therefore, it seems unlikely that BV treatment will be of great benefit, because the presented in vitro data do not support the concept of BV having anti-inflammatory effects.

	Acknowledgment

 
I thank C. Pollaschek for expert technical assistance in performing RT-PCR experiments.

	Disclosures

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The author has no financial conflict of interest.

	Footnotes

 
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.

¹ This work was supported in part by a grant from the Austrian National Bank.

² Address correspondence and reprint requests to Dr. Karl M. Stuhlmeier, Ludwig Boltzmann Institute for Rheumatology and Balneology, Kurbadstrasse 10, 1100 Vienna, Austria. E-mail address: karlms{at}excite.com

³ Abbreviations used in this paper: BV, bee venom; COX-2, cyclooxygenase-2; CT, threshold cycle; DCFH-DA, 2',7'-dichlorofluorescin-diacetate; FLS, fibroblast-like synoviocyte; HAS1, hyaluronansynthase-1; LC, loading control; MED, medium; MNC, mononuclear cell; PMN, polymorphonuclear cell; RFU, relative fluorescence unit; SF, dermal fibroblast cell.

Received for publication September 28, 2006. Accepted for publication April 14, 2007.

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