Introduction

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Historically, based on R. Steiner's (1920) anthroposophical treaties, mistletoe extracts gained increasingly common acceptance as an alternative method in tumor therapy, despite lacking proof of its efficacy. Three different lectins have been identified as putative active principles in aqueous mistletoe extracts. Mistletoe lectin (ML)-1 binds specifically galactoside structures (Luther et al., 1980; Franz et al., 1981; Olsnes et al., 1982) and consists of two A-chains and two B-chains linked by a disulfide bond. The binding to D-galactose occurs via the B-chain (mol. wt. = 34 kDa), and the A-chain (mol. wt. = 29 kDa) is supposed to act as an RNA-N-glycosidase of 28 S rRNA of the ribosome and therefore disturbs the ribosomal protein biosynthesis (Endo et al., 1988). ML-2 can bind to either N-acetyl-D-galactosamine or D-galactose, whereas ML-3 is specific for N-acetyl-galactosamine.

Various preparations of mistletoe extracts are available as pharmaceuticals with varying compositions that depend on the methods of preparation, the tree source, and the time of harvest (Gabius et al., 1994). To make standardization of therapy possible, a mistletoe extract standardized for ML-1 (Lektinol) was introduced. As the standard regimen (Hajto et al., 1989; Beuth et al., 1995), a biweekly treatment with s.c. injections of 1 ng of ML-1/kg of body weight is recommended. This treatment is supposed to stimulate the immune system of cancer patients.

In tumor patients subjected to this treatment regimen with Lektinol, statistically significant increases of helper T-cells, CD-25-positive lymphocytes, and natural killer cells (i.e., cells that are known to play a role in the control of tumor growth) were reported. Enhanced expression of interleukin-2 receptors and human leukocyte antigen D-related-antigens on peripheral blood T-lymphocytes was also observed, which could be interpreted as markers of activation (Beuth et al., 1995).

In animal studies in mice and rats, mistletoe extract treatment caused an increase in thymus weight (Beuth et al., 1991). In tumor models, a reduction in the number of metastases and the tumor growth was seen (Beuth et al., 1991). Rabbits, treated with 0.79 ng of ML-1/kg, showed an enhancement in natural killer cell activity and in the frequency of large granular lymphocytes. An acute phase response in the first 48 h and a moderate fever reaction was also observed (Beuth et al., 1991).

Mistletoe lectins were also described as inductors of apoptosis. In the presence of ML-1, human mononuclear cells and different cell lines (Janssen et al., 1993; Büssing et al., 1996; Möckel et al., 1997) undergo apoptosis and show typical apoptotic alterations, such as cell shrinkage, chromatin condensation, and DNA laddering. Recently, it was shown that ML-1 induces apoptosis in a receptor-independent pathway via cytochrome c release from mitochondria (Bantel et al., 1999).

Additionally, mistletoe lectins could activate immune responses by modulating the complex network of cytokines that regulate leukocyte functions. In vitro ML-1 caused an increased secretion of TNF-, IL-1, and IL-6 from isolated human mononuclear cells, and both the mistletoe preparation Lektinol and the isolated mistletoe lectins stimulated IL-1 and IL-6 release from skin bioassay in vitro (Hajto et al., 1990; Joller et al., 1996; Ribéreau-Gayon et al., 1996; Möckel et al., 1997).

The putative immunomodulatory effects of mistletoe extracts used in cancer therapy have been under debate for almost a century. In the present study, we critically investigated possible immunomodulatory effects of mistletoe lectins. The modulation of cytokine release in LPS-stimulated human whole blood has proven a valuable in vitro model to study immunomodulatory properties of substances (Hartung et al., 1996). We used this approach to test the potency of the mistletoe preparation Lektinol to induce cytokines in human whole blood and to modulate the cytokine network in LPS-stimulated human whole blood.

	Materials and Methods

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Mistletoe Preparations. The standardized mistletoe preparation, Lektinol, was obtained from Madaus AG (Cologne, Germany). The following lots of Lektinol were used during this study: 614758, 728473, and 730475. Standardization (30 ng of bioactive mistletoe lectin/ml) had been performed by measuring the carbohydrate binding activity to asialofetuin in relation to ML-1 by an enzyme-linked lectin assay (Vang et al., 1986). Briefly, microtiter plates were coated with asialofetuin (0.1 mg/ml) and blocked with bovine serum albumin. The ML-1 reference solution, in a concentration range of 10 to 150 ng/ml, and Lektinol were incubated at 37°C. Detection was performed using a polyclonal anti-mistletoe lectin antiserum from goat (obtained from Prof. Dr. U. Pfüller, Institute of Phytochemistry, University of Witten/Herdecke, Witten, Germany) and an anti-goat immunoglobulin peroxidase conjugate from rabbit (Sigma, Deisenhofen, Germany). The peroxidase bound to the plate was visualized by the substrate o-phenylenediamine and determined photometrically on an ELISA reader set to the wavelength of 492 nm versus 690 nm. Lektinol contains 0.092 mg/ml of aqueous mistletoe extract with the stabilizers polyvidone and edetate disodium.

Limulus Amebocyte Lysate Assay. Contamination by bacterial lipopolysaccharides, as assessed by the Limulus amebocyte lysate assay, was <0.06 endotoxin units/ml for Lektinol at the respective concentrations tested in the human whole blood assays. Limulus amebocyte lysate kits were purchased from Boehringer Ingelheim Bioproducts (Heidelberg, Germany). Limit of detection was 0.06 EU/ml.

Whole Blood Incubations. To study the LPS-induced cytokine release of whole blood, 800 µl of RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 2.5 IU/ml heparin (Liquemin, Hoffmann-La Roche, Grenzach-Whylen, Germany) and 100 IU/ml penicillin/streptomycin (Biochrom) was pipetted into a polypropylene reaction tube (Eppendorf, Hamburg, Germany), and 100 ng of LPS from Salmonella abortus equi (Sigma) and one of the mistletoe preparations were added. Finally, 200 µl of heparinized whole blood (withdrawn in lithium-heparin-S-monovettes from Sarstedt, Nümbrecht, Germany) from healthy volunteers was added (final assay volume, 1 ml), and the tubes were incubated at 37°C and 5% CO₂. After incubation, the tubes were shaken, and blood cells were sedimented by centrifugation (16,000g, 2 min). The cell-free supernatants were stored at 80°C until cytokine measurement.

Cytokine Measurement. Cytokines in the cell-free supernatants were quantified by sandwich ELISA. Antibody pairs for TNF-, IL-1, and IFN- were purchased from Endogen (Munich, Germany), and the antibody pair for IL-10 was purchased from PharMingen (Heidelberg, Germany). ELISA plates (Greiner, Frickenhausen, Germany) were coated overnight at 4°C with 50 µl/well coat antibody in 0.1 M NaHCO₃, pH 8.2. After blocking with 200 µl/well PBS supplemented with 3% bovine serum albumin (Serva, Heidelberg, Germany), pH 7.0, for 2 h at room temperature the plates were washed twice with PBS/0.05% Tween 20. Sample (50 µl/well) and tracer antibody (50 µl/well) in PBS/bovine serum albumin 3% were added and incubated for 2 h for all ELISA from Endogen. For measuring IL-10, 100 µl of sample was incubated for 3 h, then the plate was washed four times, and then 100 µl of tracer antibody was incubated for 2 h. After six wash cycles, plates were incubated for 30 min with streptavidin-peroxidase (Dianova, Hamburg, Germany; 1 µg/ml in PBS/ bovine serum albumin 3%, 100 µl/well). After eight washes, 100 µl/well 3,3',5,5'-tetramethyl-benzidine liquid substrate solution (Sigma) was added and incubated at room temperature for 5 to 30 min. After addition of 50 µl/well stop solution (1 M H₂SO₄), absorption was measured at 450 nm using a reference wavelength of 690 nm. IL-12 was measured using the IL-12 Quantikine kit (R&D Systems, Wiesbaden, Germany), according to the manufacturer's instructions.

Neutralization of Endogenous IL-10. To neutralize the biological effects of released IL-10 in LPS-stimulated whole blood, a monoclonal antibody against IL-10 (Clone 12G8, Endogen) was used. Antibody (10 µg/ml) was added to the whole blood incubation before the addition of blood.

Data Analysis. All data are given as means ± S.E.M. Cytokine release was calculated per milliliter of blood (i.e., corrected for the dilution factor of 5 as 20% blood was used). Statistical analysis for the concentration-effect curves was performed with the Ryan-Einot-Gabriel-Welsch multiple range test. For the time course experiments, the data of each time point were compared by using the Wilcoxon matched-pairs signed ranks test. The Wilcoxon matched-pairs signed ranks test was also used for the experiments with recombinant mistletoe lectin. For the kinetics with anti-IL-10, a paired t test was applied.

	Results

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The stimulation of whole blood with Lektinol, the recombinant B-chain, or the recombinant heterodimer of A- and B-chain of ML-1 alone did not cause any significant cytokine release in a concentration range of 0.01 to 10 ng/ml ML-1 (i.e., up to 33% of the incubation volume) (data not shown). However, in LPS-stimulated blood, Lektinol shifted the LPS-induced cytokine secretion toward a more proinflammatory response. In the presence of Lektinol with 0.05 to 10 ng/ml ML-1, an increased release of TNF- (p < 0.05) was observed compared with LPS control (Fig. 1A). In the concentration range of 0.5 to 10 ng/ml ML-1, Lektinol caused a diminished secretion of IL-10 (p < 0.05) compared with LPS control (Fig. 1B). Preincubation of blood in the presence of Lektinol up to 4 h did not alter the effect on TNF- and IL-10; therefore, all further experiments were carried out with concomitant addition of Lektinol and LPS. The secretion of the cytokines IL-1 and IL-12 in LPS-stimulated blood was not influenced by Lektinol (data not shown) in the tested concentration range of 0.01 to 10 ng/ml ML-1. IFN- and IL-6 were significantly reduced only in concentrations above 5 ng/ml ML-1 (data not shown). These modulations of cytokine release were seen in all of the three lots of Lektinol tested, but the proinflammatory effect was not always as pronounced. The doubling of TNF- release shown in Fig. 1 represents the maximum effect observed. The following data are examples from experiments using large amounts of Lektinol, which showed minimal effects.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of Lektinol on LPS-induced TNF- and IL-10 release in human whole blood. Whole blood in RPMI 1640 (20%) was stimulated with 100 ng/ml LPS in the presence of the indicated concentrations of ML-1 (Lektinol). After 24 h of incubation at 37°C, released cytokines were measured in the cell-free supernatants by ELISA. Data represent means ± S.E.M. of eight different donors. *p < 0.05 versus LPS control.

Time course experiments for TNF- release in LPS-stimulated whole blood showed a peak concentration of TNF- in the supernatant at 6 h of incubation time and a slow decrease in the following hours to about 40% of peak level at 24 h. In the presence of Lektinol (2 ng/ml ML-1), the initial formation kinetics and amounts of TNF- were similar, but the decrease in TNF- levels was attenuated (Fig. 2). In contrast, Lektinol had no effect on the kinetics of IL-1, IL-6, IFN-, and IL-12 release (data not shown). Similar effects on the kinetics of TNF formation were seen when a neutralizing antibody against IL-10 was present in LPS-stimulated whole blood. Here, the initial formation of TNF- was not affected, and anti-IL-10 only increased the levels of TNF- from 10 h of incubation onwards (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of Lektinol on the kinetics of LPS-induced TNF- release in human whole blood. Whole blood in RPMI 1640 (20%) was stimulated with 100 ng/ml LPS ± 2 ng/ml ML-1 (Lektinol). After different incubation times at 37°C, released cytokines were measured in the cell-free supernatants by ELISA. Data represent means ± S.E.M. of 13 different donors. **p < 0.01, ***p < 0.001 versus LPS control.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of anti-IL-10 on the kinetics of LPS-induced TNF- release in human whole blood. Whole blood in RPMI 1640 (20%) was stimulated with 100 ng/ml LPS ± 10 µg/ml anti-IL-10. After different incubation times at 37°C, released cytokines were measured in the cell-free supernatants by ELISA. Data represent means ± S.E.M. of four different donors. *p < 0.05, **p < 0.01 versus LPS control.

To test the hypothesis that Lektinol increases TNF- release via inhibition of IL-10 production, we studied the effect of Lektinol on LPS-induced TNF- release in whole blood in the presence of anti-IL-10. TNF- release in samples with Lektinol (1500 ± 330 pg/ml) was not increased compared with the controls with anti-IL-10 alone (2200 ± 650 pg/ml).

In addition, recombinant ML-1 was studied for its immunomodulatory potency. Recombinant rAB increased TNF- release and attenuated IL-10 release in LPS-stimulated whole blood (Fig. 4), similar to the mistletoe preparation Lektinol but less potent. A concentration of 10 ng/ml rAB resulted in an increase of TNF- release by more than 20% and the inhibition of IL-10 release by more than 60%. The recombinant B-chain alone did not modulate LPS-induced cytokine release, indicating that for this process the A-chain is also required.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of recombinant mistletoe lectin-1 on LPS-induced TNF- and IL-10 release in human whole blood. Whole blood in RPMI 1640 (20%) was stimulated with 100 ng/ml LPS in the presence of the indicated concentrations of rAB. After 24 h of incubation at 37°C, released cytokines were measured in the cell-free supernatants by ELISA. Data represent means ± S.E.M. of eight different donors. **p < 0.01 versus LPS control.

	Discussion

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Our results show that LPS-free mistletoe preparations and recombinant ML-1 do not induce cytokines in human whole blood, a system that has been validated as a pyrogen assay for more than 100 pharmaceuticals and biologicals (Hartung and Wendel, 1996; Fennrich et al., 1999; Jahnke et al., 2000). We therefore conclude that mistletoe lectins as such have no pyrogenic activity. In previous studies, however, mistletoe lectins have been described as cytokine-inducing agents (Hajto et al., 1990; Joller et al., 1996; Ribéreau-Gayon et al., 1996; Möckel et al., 1997). This contradictory observation might be explained by pyrogenic contaminations of tested mistletoe preparations or the cell culture system used. In our hands, some other clinically applied mistletoe preparations showed LPS contamination when tested in the limulus amebocyte lysate assay, and these contaminated preparations also induced high levels of IL-1 release in unstimulated whole blood (data not shown). This hypothesis is supported by the finding that the amount of lectin in clinically applied mistletoe preparations did not correlate with the concentrations of induced cytokines and that even preparations without measurable levels of lectins induced high levels of cytokines in peripheral blood mononuclear cells (Elsässer-Beile et al., 1998). Alternatively, a preactivation of leukocytes during the isolation procedure of mononuclear cells could explain this discrepancy.

Immunomodulation by pharmaceuticals can be studied by observing pro- or anti-inflammatory changes in the cytokine response of LPS-stimulated whole blood. In previous reports, the value of this experimental setting to monitor immunomodulation was shown by the concordance of in vitro with ex vivo and in vivo findings in human volunteers (Chernoff et al., 1995; Hartung et al., 1998; Boneberg et al., 2000). This experimental setting, therefore, appeared to be suitable to test whether mistletoe lectins have any immunomodulatory properties. Pure mistletoe lectins alone thus do not stimulate cytokine release, but surprisingly they can modulate LPS-induced cytokine release in a proinflammatory manner by increasing TNF- release and inhibiting the release of anti-inflammatory IL-10. The clinical relevance of the extent of immunomodulation observed requires further investigations. The reduction of IL-10 formation is likely to effect further immune responses besides TNF- formation in a proinflammatory manner. As expected of a plant preparation, we observed differences in the potency of individual lots of Lektinol tested. All preparations increased TNF- release and attenuated IL-10 release in LPS-stimulated whole blood. However, the increase in TNF- release in the presence of 2 ng/ml ML-1 varied between 45 and 200% in the different lots of Lektinol. These variations might be explained by the fluctuations in the amounts of ML-3, which also inhibited IL-10 and increased TNF- release in a similar concentration range as ML-1 (data not shown). Additionally, other substances in the mistletoe preparation might interfere with cytokine release.

After LPS stimulation of whole blood, monocytes release measurable amounts of TNF- within a few hours. TNF- release is then down-regulated by the secretion of its endogenous antagonist IL-10 (de Waal Malefyt et al., 1991). In this feedback loop, IL-10 inhibits the gene transcription of inflammatory cytokines like TNF-, probably via inhibition of the nuclear factor kappa B (Wang et al., 1995). We could show that Lektinol already inhibited the formation of IL-10 at concentrations of ML-1 where the release of other cytokines was not impaired. To exclude a toxic effect of mistletoe lectins on monocytes or lymphocytes during the incubation time, we measured the release of the late cytokines, IL-12 and IFN-, which are produced with comparable kinetics to IL-10. Since Lektinol did not influence the release of these two cytokines, we can conclude that monocytes and lymphocytes were viable and not impaired in their ability to produce proteins in general. The mechanism by which Lektinol exerts its IL-10-inhibiting effects remains to be clarified. The lack of inhibitory IL-10 could result in the observed increase in TNF- release. The experiment with neutralizing antibodies against IL-10 showed that without endogenous IL-10, the late TNF- release is augmented, probably due to a prolonged synthesis phase. This concept might also explain the effects of Lektinol on TNF- release. At 2 ng/ml ML-1, IL-10 release was inhibited, and consequently TNF- release was increased in the late phase (from 14 h after LPS stimulus onwards) of production. This hypothesis is supported by the finding that, after blocking endogenously formed IL-10, Lektinol could no longer increase TNF- release.

Comparison of effects of the plant-derived mistletoe preparation Lektinol with the recombinant heterodimeric ML-1 showed similar effects. The natural, glycosylated lectin and the recombinant, unglycosylated lectin both increased TNF- release and attenuated IL-10 release. The recombinant mistletoe lectin induced significant changes in cytokine release only at higher concentrations than observed for the mistletoe preparation Lektinol. This might be explained by the additional presence of ML-3 in Lektinol or an impaired capacity of the unglycosylated recombinant protein to modulate cytokine release. These data indicate that the modulation of cytokine release by Lektinol can be attributed primarily to its content of ML-1.

The recombinant B-chain alone was not effective in modulating cytokine release. This indicates that the carbohydrate binding B-chain alone does not modulate cytokine release, and for this immunomodulation, the A-chain is also required.

In conclusion, our results provide evidence that mistletoe lectin-1 alone does not induce cytokine release. Surprisingly, however, Lektinol and the recombinant mistletoe lectin showed some immuno-stimulatory effects. Lektinol interferes with the cytokine network of LPS-stimulated whole blood in a proinflammatory manner. Whether this immunomodulation might have any antitumoral effects or prolong the survival time of cancer patients by improving protection against opportunistic infections remains uncertain. The presented data encourage further investigations to clarify possible beneficial immunostimulatory effects of a clinical application of mistletoe lectins in oncology.