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American Journal of Therapeutics 5, 181-187 (1998) Subcutaneous infiltrates Induced by Injection of Mistletoe Extracts (Iscador)by Robert W. Gorter (1,2), Madelon van Wely (1), Matthias Stoss (1) and Uwe Wollina (3)
Supported by the Society for Oncological and Immunological Research. Address for correspondence: Institute for Oncological and Immunological Research, Hardenbergstrasse 19, D-10623, Berlin, Germany Iscador, an aqueous extract of Viscum album L., has been widely used as an anti-cancer drug for several decades. Mistletoe lectins have the capacity to activate nonspecific defense mechanisms, and lectin-carbohydrate interactions may be involved in clinically applicable immunomodulation. During treatment with whole-plant mistletoe extract, an inflammatory reaction usually occurs at the site of the injection, early in therapy. These injection sites were examined histologically. Seven subjects received three subcutaneous injections of Iscador QuFrF or Iscador Qu Spezial (twice 0.1 mg and once 2.5 mg) during 9 days. In all subjects, examination of skin biopsies showed a normal epidermis. The dermal and subcutaneous regions contained a dense perivascular lymphocyte infiltrate and increased monocytes. We could not document any increase of plasma cells, eosinophils, mast cells, neutrophils, or granulocytes, as would be the case for a granulomatous infiltrate. In the blood, we observed a significant increase in neutrophils and monocytes 24 hours after administration of 2.5 mg of Iscador. Keywords: lymphomonocytic infiltrate, Iscador, mistletoe, skin, immune histology. INTRODUCTION For more than 80 years, an aqueous extract of Viscum album L., named Iscador, has been used as an anti- cancer drug. It is licensed for mono- and adjuvant anti-cancer therapy in Switzerland, the European Union, and several countries around the world. V. album L., the European mistletoe, is a hemiparasitic plant that grows in various host trees.(1) In 1920, Rudolf Steiner (1861 – 1925), founder of anthroposophical medicine, suggested that V. album L. would improve the defense mechanisms of the immune system and prevent or overcome tumor growth. Usually, Iscador is administered subcutaneously twice a week over several years. Skin and mucous membranes contain many elements of the immune system, designated the skin immune system (3-5) or skin-associated lymphoid tissue, (6,7) in which penetrating antigens sensitize lymphoid cells within the tissue. Their penetration initiates an immune response to for eign antigens or antigen-mimicking substances to destroy infectious organisms and to neutralize and remove potential toxins as well as damaged tissue. Regularly, at the site of the injection early in therapy, Iscador induces an inflammatory reaction. This inflammatory reaction is dosage-dependent. Higher dosages result in more frequent and stronger local reactions.(8) Normally, the reactions disappear after several weeks of therapy. Studies in rodents show that substances in the mistletoe preparation and not a bacterial contamination are responsible for the inflammation.(9,10) These local inflammatory reactions are probably caused by mistletoe lectins.(11) Other histological studies of single patients with metastaged malign melanoma and gonarthrosis observed subcutaneous infiltrates containing lymphocytes, monocytes, and eosinophils after eight subcutaneous injections of a mistletoe preparation (Plenosol).(12,13) These findings suggested that the observed local reaction might partially be of allergic origin. However, our clinical experience points toward a local inflammatory reaction and not an allergic reaction. The immunohistological study described here was initiated to document the subcutaneous response to Iscador with a view to better understanding of the described reactions. We examined the characteristics of the Iscador induced infiltrate in seven healthy subjects. Additionally, we determined levels of peripheral blood cells before and after administration of Iscador. MATERIALS AND METHODS Iscador For the purpose of the study, we used Iscador QuFrF (new investigational drug) and Iscador Qu Spezial (Institut Hiscia, Arlesheim, Switzerland) in 1 mL ampules of 0.1 and 5 mg produced especially for this trial. Both preparations are aqueous extracts of mistletoe plants growing in oak trees.(14) The initial part of the manufacturing is the same, but Iscador Qu Spezial undergoes a controlled fermentation, whereas Iscador QuFrF remains in an unfermented state. The used Iscador batches are standardized on the basis of their contents of mistletoe lectins and viscotoxins. The concentrations of lectins and viscotoxins in these preparations have been determined by enzyme-linked lectin assay and high-performance liquid chromatopaphy(15,16) as shown in Table 1. These mistletoe preparations also contain several other substances, including amino acids, polysaccharides, and lipids.(17) Subjects and procedures Seven healthy, nonsmoking volunteers, four males and three females, between 24 to 44 years of age and with Karnofsky performance scores of 100, who had given their informed consent, were randomized to receive either Iscador QuFrF (n = 4) or Iscador Qu Spezial (n = 3). Injections were given subcutaneously with a 26-gauge x 0.5-inch 0.45 x 12 mm needle (Braun Melsungen AG, Melsungen, Germany) in the right upper quadrant of the musculus gluteus maximus area according to the schedule presented in Table 2. Twenty-four to 26 hours after administering the 2.5 mg (0.5 mL 5.0 mg) of Iscador, two elliptical incision biopsy specimens (diameter [W], 5 mm x 10 mm) were taken. Lidocaine (Xylocane) 0.5% with adzenaline 1: 10,000 (Astra Chem, Wedel, Germany) was used as local anesthetic. No control was taken. One tissue sample was preserved in 4.5% formalin solution and the second sample in sterile 0.9% NaC1, stored at 4'C. This study was approved by the responsible ethics committee. Local inflammation On day 10, the local inflammatory reaction was characterized superficially by measuring the size of the area of erythema and by describing the occurrence of swelling and induration, as well as temperature increase (warmth to the touch). The subjects were questioned regarding whether they were bothered by itching or painful sensations at the injection site and about when each of these reactions first appeared. Immunohistochemical procedures For the formalin preserved samples, the following antibodies were used according to the avidin-biotin- peroxidase complex approach(18,19): pan-lymphocyte marker LCA (Dako, Hamburg, Germany; CD45), pan-B-cell marker L26 (Dako; CD20), pan-T cell marker UCHL1 (Dako; CD45R), and macrophage marker CD68 (Dako). For the native (in 0.9% NaC1 preserved) samples, anti-human interleukin-2 (IL-2) receptor CD25 (Dako), CD38 (Becton Dickinson, San Jose, CA), and anti-human transferrin receptor CD71 (Dako) were used. Laboratory investigations Venous blood (vena cubitale) was collected from all subjects at baseline (two draws at day 1 and day 3) and at the day of biopsy (day 10). Baseline values were determined by taking the average values of day 1 and day 3. After lysing the erythrocytes, the peripheral blood mononuclear cells were assessed for the presence of selected surface markers. Total B lymphocytes, T lymphocytes, and CD3+/CD4+, CD3+/CD8+, CD3+/ CD25+, and CD8+/CD38+ cells were counted by direct immunofluorescence, using monoclonal antibodies (BectonDickinson) and flow cytometry (FACScan; Becton Dickinson, Immunocytometry Systems). Total and differential leukocyte counts were obtained from the same blood samples (Sysmex E-5000/ 100; Sysmex, Langenfeld, Germany), and the absolute number of lymphocytes in each subset was calculated. A differential blood count was performed manually, twice. Statistical analysis Student's t-test for matched pairs was used for statistical analysis. p values < 0.05 were considered significant. RESULTS Biopsies The first sign of a local reaction occurred between 1 and 10 hours after the last injection. A mild to moderate erythematous area (W, 2-5 cm) was observed. Both induration and swelling were found in all subjects between 2.5 and 7 hours, and painful sensations occurred between 1 and 7 hours after injection of the Iscador. None of the subjects was bothered by itching. In all cases, there was increased local warmth to the touch at the reaction site. Histologically, all biopsy specimens showed normal surface epithelium. The epidermis was not affected. Corium and subcutaneous fat tissue showed a superficial .and deep, dense, lymphoid cellular infiltrate. This infiltrate consisted of typical lymphocytes with little cytoplasm. No increase of plasma cells, neutrophils, eosinophils, or mast cells was documented. In all tissue samples, the infiltrate consisted of a dense packing of cells. In comparison, the infiltrates of subjects D18 and E18 were slightly less densely colonized by lymphocytes. Immunohistologically, the infiltrate consisted of 60% LCA-positive lymphocytes. All lymphocytes reacted with the pan-T-cell marker UCHL1. Fifty per-cent of the lymphocytes were CD4+, and 50% were CD8+. None of the cells reacted with the pan-B-cell marker L26. The rest of the cells in the infiltrate responded to macrophage marker CD68. In the native tissue samples, almost all lymphoid cells reacted with the IL-2-receptor receptor (CD25). In four samples, 95% of all cells contained the transferrin receptor marker (CD71), whereas in three samples (subjects D16, D18, and E19), only 50% to 80% of the cells reacted to antibody against transferrin receptor (Table 3). In six of seven samples, in deeper areas of the infiltrate, anti-CD38 reacted with about 80% of all cells. More superficially, a CD38 expression of 80% was found in samples from three subjects. In samples from the other four subjects, CD38 was expressed on only 30% of cells (Table 3). Peripheral blood cell differentiation In all seven subjects, a marked increase in absolute neutrophilic cell number and monocyte count compared with baseline was found after the final Iscador injection on day 10 (Table 4). The mean increases in both neutrophilic cells and monocytes (3225 to 8514 and 390 to 706, respectively) were statistically significant (p < 0.001) (Table 4). The number of lymphocytes decreased in all but one subject, and the mean decrease was statistically significant (p < 0.05) (Table 4). There were no changes in eosinophils or total B-cell counts. Absolute CD4+ cell numbers decreased in six subjects, and absolute CD8+ cell numbers as well as the number of natural killer cells decreased in five subjects. Interestingly, absolute numbers of both the activated T-cell subsets (CD3+/CD25+ and CD8+/ CD38+ lymphocytes) increased slightly in five subjects. DISCUSSION In this study, we demonstrate that, after administration of 2.5 mg of Iscador QuFrF or Iscador Spezial, a specific subepidermal lymphomonocytic infiltrate is induced at the injection site. At the surface of the skin, a local erythema, followed by swelling and induration, occurred in about 4.5 hours. Our histological examinations show that the epidermis was normal in all samples. However, the dermal and subcutaneous regions of the skin contained a dense perivascular lymphocyte infiltrate. This infiltrate was mainly localized in the subcutis and accounted for approximately 60% of the T lymphocytes, with an equal distribution of CD4+ and CD8+ cells (CD4:CD8 ratio = 1). The remaining 40% of the cells in the infiltrate were identified as macrophages. No cells reacted to antibody against L26, so the presence of B cells could be excluded. It should be noted that the epidermis was not involved, as was observed for allergy testing through intracutaneous antigen application(20). Moreover, there was no increase in plasma cells, eosinophils, mast cells, neutrophils, or granulocytes, as would be the case for a granulomatous infiltrate. In normal skin, dermal lymphocytes are known to make up 90% of the total number of T lymphocytes(4). Small numbers of T lymphocytes can be found in the upper reticular dermis, and about 2% of all T lymphocytes dwell in the connective tissue. B cells are not found in normal skin(3,4). It was remarkable that almost all T lymphocytes in each biopsy expressed the IL-2 – receptor marker (CD25). Most of the cells in the infiltrate expressed the transferrin marker (CD71), which is normally present on proliferating cells and on macrophages. Furthermore, 80% of the cells in the subcutaneous layers were CD38+ in all subjects. CD38 is expressed both by activated T lymphocytes and macrophages (antigen- presenting cells). The increase of the activation markers indicates an enhanced cytokine production by the lymphocytes and monocytic cells that express them(21,22). From these observations, it can be concluded that both Iscador QuFrF and Iscador Qu Spezial administration induce an activation of CD4+ and CD8+ lymphocytes. These results confirm earlier observations regarding peripheral blood mononuclear cells from healthy individuals and human immunodeficiency virus-positive individuals on Iscador therapy(14). All blood cell values remained within the normal range. A decrease was observed in CD4+ and CD8+ lymphocytes and in natural killer cells, but this was not statistically significant. We found a clear, significant (p < 0.001) increase in neutrophilic and monocytic cell number. We would like to point out here that the blood of all seven subjects was drawn at the same time of day, with a variation of up to 2 hours. It is possible that the daily intrapersonal variation can partly ex plain the observed change in absolute cell number. However, the threefold increase in cell number of neutrophilic cells was remarkable. This result is consistent with earlier observations in which, after a single intravenous infusion of Iscador, significant increases of 2.7 to 3.2 times baseline values were described(23,24). As far as we know, an increase of monocytic cells during Iscador therapy has not been described before. Our results show that the observed local reaction is not an allergic reaction, but a nonspecific inflammatory reaction like a late-phase reaction. This local inflammation leads to activation of both specific and nonspecific components of the immune system. The specific activation is expressed by a increase of CD25+ and CD71+ lymphocytes at the injection site. Furthermore, an increase of activated cells in peripheral blood was found, which is consistent with earlier observations.(14) The decrease in T lymphocytes in peripheral blood might be interpreted as indicating a migration of these cells into the skin. The significant increase in neutrophils is a nonspecific response induced by the inflammatory reaction to Iscador administration. New studies are planned to further study these observations. It is known from in vitro and in vivo experiments that mistletoe lectins stimulate monocytes to secrete tumor necrosis factor-alpha and stimulate peripheral blood mononuclear cells to IL-1, IL-2, and IL-6 secretion(24-28). The release of IL-2 by human mononuclear cells in vivo after Iscador administration has been described in different studies(25,26). In the Skin 2 model, an in vitro human skin bioassay, a clear stimulation of IL-1 and IL-6 release was observed after 24 hours of incubation with mistletoe lectin-1 (ML-1) standardized mistletoe extract(29). Furthermore, studies in cancer patients showed a significant increase of IL-6 in blood serum in the first 6 hours after Iscador injection(27). Hence, it is expected that Iscador injection in the skin induces the release of IL-1, IL-2, and IL-6. If this is true, patients challenged with these interleukins may develop similar local reactions. Indeed, in several studies, analogous reactions have been described. Subcutaneous injection of IL-2 in patients with an atopical condition and in healthy subjects induced an erythematous reaction after 24 to 28 hours. In this study, a clear increase was observed in the expression of the activation markers CD25 and HLA-DR+. The cellular response to this single injection of IL-2 showed an infiltrate consisting of lymphomonocytic cells and neutrophilic granulocytes(28). After subcutaneous injection of IL-1alpha (10 and 60 U), a maximal erythematous reaction was observed after 6 to 9 hours, and this reaction lasted for at least 24 hours. As described for the IL-2-induced infiltrate, the lymphomonocytic infiltrate subsequently contained neutrophilic granulocytes(30). It should be noted that we did not observe neutrophils in our lymphomonocytic infiltrate. In patients with an atopical condition, it was found that, after antigen challenge, IL-6 was produced at the location of swelling. Interestingly, a relationship was found between IL-6 release in the first 6 hours after antigen challenge and the size of the erythematous area, as well as a relationship between IL-6 in the second 6 hours and eosinophil influx in the infiltrate(31). Another study on cytokine release through tuberculin- induced, delayed-type hypersensitivity found, in addition to IL-1 and IL-6, an increase in tumor necrosis factor-alpha release. Here, the observed mononuclear infiltrate was mainly composed of monocytes(32). We interpret our observed local infiltrate as a late- phase reaction. The Iscador-induced reactions appear to be very similar to the described responses to IL-1, IL-2, and IL-6 injections. We did not, however, observe an increased number of neutrophils in our lympho- monocytic infiltrate, which has been described after IL-1 or IL-2 administration, nor did we detect eosinophils in the infiltrate, as has been described after IL-6 administration. It is not clear to us why in the study with Plenosol, after eight subcutaneous injections in one melanoma and one gonarthrosis patient, a strong influx of eosinophils in the infiltrate was observed(12,13). We postulate three possible explanations that should be further examined: (1) the lectin/viscotoxin ratio in the different mistletoe preparations induces distinctive reactions; (2) the local reaction will be different after different numbers of injections or (3) the mistletoe extract-induced reaction of the immune system in healthy subjects is different from that in ill subjects. We could not document obvious differences between the effects of Iscador QuFrF and Iscador Qu Spezial. Clearly, both Iscador QuFrF and Iscador Qu Spezial provoke a characteristic, nonspecific, antigen-induced immune response in healthy humans with a massive activation of T lymphocytes at the injection site. An increase of activated CD4+ and CD8+ cells and of macrophages was observed. Activation of macrophages by mistletoe lectins has been previously reported in melanoma effusions(33). To our knowledge, this is the first study to characterize the skin reaction to Iscador. Table 1
Table 3
Table 4
ACKNOWLEDGEMENT We thank Arno Triebskorn for taking the biopsies, Helmut Laff for performing the histological analyses, and Maria Linder for her critical comments on the manuscript.
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