Immunomodulation by Mistletoe

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Based on the studies of Koch (1938b) and Seeger (1965a-c), the biological effects of Viscum album are defined not only by direct cytotoxicity, but also indirect immune activation. The first reports that suggest VA-E may modulate the immune response described splenomegaly and increased clearance of colloidal carbon by phagocytes after intravenous or intraperitoneal injection of the fermented VA-E Iscador (Zschiesche, 1966; Bloksma et al., 1982) and increased thymus weight in mice (Nienhaus and Leroi, 1970; Nienhaus et al., 1970). Other studies found augmentation of delayed type hypersensitivity (DTH) of mice immunized with red blood cells from sheep (SRBC); however, the response was present only when SRBC were mixed with Viscum album and the subcutaneous or intracutaneous injections were Iscador (Bloksma et al., 1979, 1982; Khwaja et al., 1980). Another indication that VA-E activated immune function became evident by the augmentation of splenic plaque-forming cells (PFC) three days after i.p. immunization with SRBC mixed with this drug extract (Bloksma et al., 1979). Thus, intraperitoneal immunization of mice with SRBC induced a humoral response (PFC) and sensitized for DTH. Further experiments found that neither fresh plant juice from mistletoe grown on apple trees in the summer nor the polysaccharides from Viscum album alone induced DTH to SRBC; however, the combined extract and polysaccharides stimulated the antibody-formation (PFC) (Bloksma et al., 1982). The fermented drug extract Iscador M, when applied subcutaneously, induced an early nonspecific inflammation with footpad swelling in mice that disappeared at 72 h, but an unfermented VA-E caused minor swelling 6 h after injection and gradually increased to a dose dependent optimum at 72 h. Application of polysaccharides from Viscum album did not result in paw swelling (Bloksma et al., 1982).

Since the fresh plant extract used for the Iscador drug is fermented by Lactobacillus plantarum, it was unclear if the observed immune responses were due to contamination by microorganisms. However, Iscador and non-viable lactobacilli did not stimulate DTH to SRBC but stimulated the humoral response, however, viable lactobacilli stimulated the cellular response but failed to influence the PFC response (Bloksma et al., 1979). Further, Iscador rendered bacterium-free by centrifugation retained its adjuvanticity for DTH, while sterile-filtration of the drug extract reduced inflammatory and adjuvant properties (Bloksma et al., 1979). Thus, it is unclear whether these soluble components are of plant or microbial origin. Subsequent reports from Bloksma and collegues (1982) suggest that the activities of the fermented drug extract Iscador on immune-related reactions also were attributed to the fresh plant extract with polysaccharides and lactobacilli. However, nowadays Iscador extracts are completely free of bacterial contamination.

Recently published findings of Antony and co-workers (1999) indicate that metastatic B16F10 tumour-bearing mice treated with both, spleen cells activated in vitro with VA-E (Iscador M) or from mice treated intravenously with the extract showed a significant inhibition of tumour nodule formation and increased survival. These findings underline the notion that several effects of Viscum album treatment may be based on an activation of immune cells rather than direct cytotoxicty.

Cellular immune system

In breast cancer patients, Hajto (1986) observed an induction of fever, lymphocytopenia, and an increased number of juvenile granulocytes and zymosan-induced oxidative burst of granulocytes 6 h after intravenous application of the VAE Iscador M. Within 24 h, uptake of [3H]-thymidine in the DNA of mitogen-stimulated lymphocytes, natural killer (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) in the peripheral blood, and numbers of large granular lymphocytes (LGL) increased but returned to baseline levels by 48 to 72 h. After further in-vivo experiments with rabbits, the effects were ascribed to ML I since i.v. administration of ML I or its lectin B chain enhanced NK cytotoxicity and the number of LGL from 24 to 72 h (Hajto et al., 1989). A single i.v. injection of ML I or mistletoe extract Iscador resulted in an increased number of LGL, granulocytes, and oxidative burst stimulated with opsonized zymosan within 24 h in 3 rabbits (ML I at 0.8 ng/kg BW). The effect was also observed in 14 breast cancer patients treated with Iscador (0.33 mg/kg BW corresponding to 1.65 ng/kg ML I). Similar, but less impressive results were observed in 17 breast cancer patients who received a s.c. injection of an unfermented VA-E (Iscador Q FrF at 0.17 mg/kg corresponding to 12 ng/kg ML I). From these studies, an "optimal" ML I concentration of 1 ng/kg BW ML I was postulated. In a later study by Hajto and co-workers, however, this "optimal" immunomodulatory reponse was not confirmed in healthy individuals who received the ML I s.c. (Hajto et al., 1996). In a set of experiments in BALB/c mice treated s.c. with purified ML I or whole plant extract Eurixor, Beuth and co-workers observed that the number and activity of peritoneal macrophages increased as did the number of peripheral monocytes, interleukin-2 receptor-positive lymphocytes, thymus weight, and thymocyte numbers (Lyt-2/CD8+ cells, L3T4/CD4+ cells, and Lyt-2/CD8 L3T4/CD4-double positive cells) (Beuth et al., 1991; 1993a, 1994a, 1995, 1996; Stoffel et al., 1997).

Using both aqueous (Helixor) and fermented drug extracts (Iscador), Hülsen and co-workers observed increased NK cells activity against leukemia K 562 cells when pre-incubated for 20 h with the drug (Hülsen et al., 1989; Hülsen et al., 1992; Hülsen and Born, 1993). With subcutaneous administration of the whole plant extract with a defined ML I content (Eurixor) to 40 breast cancer patients, an increase of peripheral T helper cells, NK and T cells with expression of interleukin-2-receptor a chains (CD25), and concomitantly acute phase proteins such as complement component C3, C-reactive protein, and haptoglobin was observed within 4 weeks in 10 patients (Beuth et al., 1992, 1993b). In 36 breast cancer patients treated with Eurixor, no significant changes were observed in the lymphocyte subsets; however, plasma levels of ^-endorphin increased at six and twelve weeks after immunization in a subgroup of 25 responders (Heiny and Beuth, 1994).

By the investigation of long-term treated tumour patients (n=23), the amount of lymphocytes and the number of natural killer (NK) cells increased within 7 month of subcutaneous application of VA-E (Helixor), while the number of other lymphocyte subsets (i.e. CD19+ B cells, CD4+ T helper cells, CD8+ CD28- suppressor cells, CD8+ CD28+ cytotoxic cells) and the proportion of CD25+ cells within T cells showed a statistically remarkable trend (Büssing et al., 1999g). For CD19+ B cells, CD4+ T helper cells, CD8+ CD28+ cytotoxic cells and CD16+/CD56+ NK cells, we observed statistically remarkable peaks within the 2nd and the 3rd month of therapy, indicating a sufficient anti-mistletoe antigen response within this time range. Here, no effects of an immunosuppression, as defined by a decrease of defined lymphocyte subsets was observed by an adequate escalation of applied VA-E.

A strong proliferation response of lymphocytes from tumour patients and healthy individuals treated with VA-E was approved several groups (Schultze et al., 1991; Fischer et al., 1997a, b; Stein et al., 1998; Stein and Berg, 1998b). The peaks of [3H] thymidine uptake in the cells were interindividually different, varying from 3 to 10 weeks in tumour patients after the onset of therapy (Stein et al., 1998); later on, this reactivity decreased in 6 out of 8 tested patients, indicating a desensitization of the immune response towards the Viscum album antigens. Few individuals did not showed a proliferation response towards the applied VA-E. However, lymphocytes from tumour patients never exposed to Viscum album antigens showed no specific proliferation response (Schultze et al., 1991).

Another interesting finding came from the group of Kabelitz who observed a stimulation of a defined T cell subset by a heat-treated VA-E as measured by [3H]-thymidine uptake. In response to this extract, T cells with y§ T cell receptor were expanded, i.e. cells with the variable T cell receptor elements Vy9 and V52 (Fischer et al., 1996). A strong stimulation of this small T cell subset which represents about 1 to 10% of peripheral T cells was also observed by heart-treated Mycobacterium tuberculosis (Fischer et al., 1996), indicating that Viscum album-derived ligands share features with mycobacteria-derived ligands for this T cell subset. However, the function of y§ T cells remains unclear.

The in vivo effects of VA-E are highly dependent on the mode of application. Specifically, subcutaneous injection of VA-E from quercus host tree (Iscador) in healthy individuals resulted in a moderate redness, induration, swelling, local warmth, and painfiul sensations within 1 to 10 h (Gorter et al., 1998). Histological analyses of injection sites 10 d after the beginning of the treatment revealed normal surface epithelium and epidermis, while the corium and subcutaneous fat tissue showed a superficial and deep, dense, perivascular infiltrate with 60% T lymphocytes (50% CD4+ T cells and 50% CD8+ cells) and 40% macrophages (Gorter et al., 1998). However, since no controls were investigated, no valid conclusion can bedran from these findings.

Intravenous application of high concentrations of aqueous VA-E did not affect hematopoiesis (Böcher et al., 1996) but increased the number of monocytes and juvenile granulocytes within 24 h after VA-E administration (Büssing et al., 1996f). However, the absolute number of neutrophils, NK, T helper, and T-suppressor/cytotoxic cells did not change significantly (Büssing et al., 1996f). The lack of ML cytotoxicity in a clinical situation might be due to the induction of anti-ML-antibodies during therapy (Stettin et al., 1990; Stein et al., 1997b, 1998) and inhibition of MLs by serum glycoproteins/-lipids (Ribereau-Gayon et al., 1995, 1997). Thus, a clinically relevant ML-mediated cytotoxicity might be induced only in the case of intratumoural or intrapleural injection.

Pleurodesis and elimination of tumour cells in response to the intrapleural administration of VA-E in patients with malignant pleural effusions (Böck and Salzer, 1980; Böck, 1983a, b; Salzer, 1986; Salzer and Popp, 1990; Stumpf and Schietzel, 1994) was associated with an increase of eosinophils, T helper cells, and NK cells (Salzer, 1986; Salzer and Popp, 1990), while in the investigations of our group no significant changes of lymphocyte subsets (i.e., CD4+ T helper cells, CD8+ T suppressor/ cytotoxic cells or CD16+/CD56+ NK cells) were observed; however the decline of tumour cells was associated with a transient increase of macrophages and eosinophils only in the responder group (Stumpf and Büssing, 1997b).

Intratumoural application of VA-E did not produce significant changes of peripheral immunocompetent cells, but slightly increased granulocytes, modestly decreased monocytes (Scheffler et al., 1996), and slightly increased CD8+ CD28- putative suppressor cells (Stumpf et al., 1997a). Nevertheless, the effects of VA-E on tumour-infiltrating immune cells may be more relevant than on peripheral immune cells, but no investigations have been reported at this time.

Induction of cytokines

Immunostimulation of immunocompetent cells by VA-E was further evidenced by a release of TNF-a nd IL-6 in the supernatants of VA-E-stimulated peripheral mononuclear cells, indicating an activation of monocytes/macrophages, and a release of T cell-associated cytokines INF-y and IL-4 with individual variations (Stein et al., 1996b, 1998a; Stein and Berg, 1998b). Similar responses were observed in cultured cells from healthy individuals treated subcutaneously with VA-E (Stein and Berg, 1998b). At present, however, no valid conclusions can be drawn whether Viscum album treatment drives the immune response towards a "cytotoxic" Th1-associated reaction (IL-2, IL-12, IFN-y) or towards a "humoral" Th2-associated direction (IL-4, IL-5, IL-10). Although especially the VA-E containing micelles from the cell membranes of the plant (Abnoba) predominantly induced IFN-y/IL-2 and of TNF-a/IL-6, while IL-4/IL-5 was induced only in few cases, a large proportion of investigated patients (about 38%) showed a release of both Th-1- and Th2-associated cytokines (Stein et al., 1998a). The simultaneous secretion of Th1- and Th2-associated cytokines was also observed in healtyh individuals treated with an aqueous VA-E, while a dominance of Th2-associated cytokines was observed (Stein and Berg, 1998b). It remains to be clarified (1) whether a Th1 response is favourable in tumour patients, (2) whether a VA-E-directed Th2 response may favour suppression of cellular anti-tumour response or not, and (3) whether the anti-Viscum album antigen response and anti-tumour response are independent effects. Anyway, these results indicate that a modulation of the Th1/Th2 balance is highly dependent on the individual tested and on the stimulating components present in the applied drug.

Humoral immune system

During therapy with VA-E, antibodies are produced against antigens from Viscum album, predominantly ML (Stettin et al., 1990; Stein et al., 1997b, 1998a,b; Stein and Berg, 1998b). In most cases, these anti-ML antibodies are of IgG class, IgA was found in some cases, and IgE only in rare cases, while IgM antibodies were detected only infrequently (Stettin et al., 1990; Stein et al., 1998a, b; Stein and Berg, 1998b).

Human anti-ML antibodies were found to detect different epitopes of ML and other components from VA-E (Stein et al., 1998b), i.e. an epitope of about 45 kD present in at least two VA-E from pine mistletoe (Iscador P and Helixor P). This epitope remains to be characterized.

Subcutaneous application of VA-E obviously results in a presentation of antigens from Viscum album, i.e. ML, to T and B cells in the lymph nodes. In fact, maximal anti-ML antibody responses were observed within 3 to 10 weeks and 4 to 12 weeks, respectively, after the VA-E therapy started (Stein et al., 1998a; Stein and Berg, 1998b); within this time range, the number of B cells and T helper cells showed a peak (Büssing et al., 1999g). Figure 2.

The major task of CD8+ cytotoxic T cells is the lysis of MHC class I+ virally infected cells and tumour cells, while CD4+ cytotoxic T cells may have an immunomodulatory role as they eliminate activated MHC class II+ immune cells by Fas/FasL interaction to prevent overreactions in ongoing immune responses and to remove potentially hazardous immune cells (Hahne et al., 1995). In this regard it is of importance to notice that apoptotic CD4+ and CD8+ cells showed increased level of intracellular IL-4 (Stein et al., submitted for publication). In agreement with this observation, Fasmediated apoptosis of lymphoid cells was reported to result in a rapid production of IL-10 in these cells (Gao et al., 1998). In a physiological context, this may indicate that apoptosis and tolerance are linked through the production of anti-inflammatory Th2-associated cytokines which may inhibit Th-1-associated immune reactions, and thus may prevent deleterious immune responses, such as strong local immune reactions induced by s.c. application of VA-E.

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