TLR4-mediated activation of mouse macrophages by Korean mistletoe lectin-C (KML-C).

Korean mistletoe lectin (KML-C) is an adjuvant that activates systemic and mucosal immune cells to release cytokines including TNF-alpha, which induces immunity against viruses and cancer cells. Although the immunomodulatory activity of KML-C has been well established, the underlying mechanism of action of KML-C has yet to be explored. When mouse peritoneal macrophages were treated with KML-C, both transcription and translation of TLR4 were upregulated. KML-C-induced TLR4 downstream events were similar to those activated by LPS: the upregulation of interleukin-1 receptor-associated kinase-1 (IRAK1); resulting in macrophage activation and TNF-alpha production. When TLR4 was blocked using a TLR4-specific neutralizing antibody, TNF-alpha production from the macrophages was significantly inhibited. Moreover, TLR4-deficient mouse macrophages treated with KML-C also secreted greatly reduced level of TNF-alpha secretion. Finally, TLR4 molecules were co-precipitated with KML-C, to which agarose beads were conjugated, indicating that those molecules are associated. These data indicate that KML-C activates mouse macrophages to secrete TNF-alpha by interacting with the TLR4 molecule and activating its signaling pathways.

Active Chinese mistletoe lectin-55 enhances colon cancer surveillance through regulating innate and adaptive immune responses.

AIM: To investigate the potential role of active Chinese mistletoe lectin-55 (ACML-55) in tumor immune surveillance. METHODS: In this study, an experimental model was established by hypodermic inoculating the colon cancer cell line CT26 (5 x 10(5) cells) into BALB/c mice. The experimental treatment was orally administered with ACML-55 or PBS, followed by the inoculation of colon cancer cell line CT26. Intracellular cytokine staining was used to detect IFN-gamma production by tumor antigen specific CD8+ T cells. FACS analysis was employed to profile composition and activation of CD4+, CD8+, gammadelta T and NK cells. RESULTS: Our results showed, compared to PBS treated mice, ACML-55 treatment significantly delayed colon cancer development in colon cancer-bearing Balb/c mice in vivo. Treatment with ACML-55 enhanced both Ag specific activation and proliferation of CD4+ and CD8+ T cells, and increased the number of tumor Ag specific CD8+ T cells. It was more important to increase the frequency of tumor Ag specific IFN-gamma producing-CD8+ T cells. Interestingly, ACML-55 treatment also showed increased cell number of NK, and gammadeltaT cells, indicating the role of ACML-55 in activation of innate lymphocytes. CONCLUSION: Our results demonstrate that ACML-55 therapy can enhance function in immune surveillance in colon cancer-bearing mice through regulating both innate and adaptive immune responses.

A lectin from Chinese mistletoe increases gammadelta T cell-mediated cytotoxicity through induction of caspase-dependent apoptosis.

In this study, a mistletoe lectin (ML) was purified from Chinese mistletoe and the effect of this 60 kDa Chinese ML on human gammadelta T cell cytotoxicity, apoptosis and modulation of the cytokine network was studied. The cytotoxic properties of delta T cells was evaluated by using a (51)Cr release test and employed fluorescence-activated cell sorting and enzyme-linked immunosorbent assay analysis to quantify translocation of the cell membrane phospholipid, phosphatidylserine and nuclear DNA fragmentation during apoptosis. It was found that: (i) ML effectively stimulated gammadelta T cell proliferation in a dose- and time-dependent manner; (ii) ML increased gammadelta T cell cytotoxicity; (iii) ML could modulate lipopolysaccharide-induced cytokine release in a pro-inflammatory manner by increasing tumor necrosis factor (TNF)-alpha release and inhibiting the release of anti-inflammatory interleukin (IL)-10; (iv) ML induced apoptosis in caspase-dependent and CD95-independent manner. The results indicated that ML is a potent immunomodulator to human gammadelta T cell cytotoxicity, apoptos is and cytokine production.

Suppressive effect of a standardized mistletoe extract on the expression of activatory NK receptors and function of human NK cells.

Despite long-term use of mistletoe extracts for cancer treatment, their mode of action remains elusive. In this study, it was studied in vitro if mistletoe extract is able to modulate the expression of natural cytotoxic receptors (NCRs) and NKG2D receptor, which stimulate natural killer cell-mediated cytotoxicity. Unexpectedly, a mistletoe extract, ABNOBA viscum Fraxini, inhibited the expression level of NKp46 and NKG2D receptors in dose- and time-dependent manners. The levels of NKp30 and NKG2D receptors were remarkably induced and NKp44 was slightly induced after 48 h treatment with IL-2 and IL-15 in both mRNA and surface expression. The activatory NK receptors were not induced significantly after treatment with IL-12, IL-18, and IL-21 for 48 h. Induction of activatory NK receptors by IL-2 and IL-15 was suppressed almost to the untreated levels by treatment with mistletoe extract, which appeared to induce apoptosis of NK cells in a dose-dependent manner. However, the treatment with IL-2 and IL-15 did not prevent the mistletoe-induced NK-cell death. Mistletoe extract inhibited significantly the cytotoxic activity of resting and IL-2- or IL-15-stimulated NK cells. These results suggest that inhibition of survival and function of NK cells by mistletoe extract may curtail in part the therapeutic effects of mistletoe.

Immunologic effector mechanisms of a standardized mistletoe extract on the function of human monocytes and lymphocytes in vitro, ex vivo, and in vivo.

Even though mistletoe extracts have been in clinical use for centuries their exact mode of action is still unknown. Currently, the application scheme for registered preparations is a dose-escalating scheme to thus reduce side effects. In this study, healthy controls and patients were evaluated for their immunologic response to treatment with a standardized mistletoe extract (Iscador). It shows a strong effect as adjuvant that induces TNF-alpha and IL-12, which was partly mediated via CD14. Desensitization of the TNF-alpha response could be shown after repeated application in vitro and in vivo. Furthermore, Iscador induces a specific lymphocyte sensitization upon multiple injections and production of IgG1- and IgG3 -mistletoe antibodies. Remarkably, a systemic bystander effect (heterologous immunity against other recall antigens) was observed after long-term treatment. In conclusion, dose-escalation reduces the monocyte-related clinical side effects. A T-lymphocyte sensitization stimulates mainly a specific Th1 response. The most interesting clinical long-term effect is the bystander stimulation of various memory T cells that might mediate in vivo antitumor and antiinfectious T-cell response under mistletoe-extract immunization.

Anaphylaxis to viscotoxins of mistletoe (Viscum album) extracts.

BACKGROUND: Extracts of mistletoe (Viscum album) are used in many countries for adjuvant cancer therapy. These extracts contain mistletoe lectins and viscotoxins that are supposed to have immunostimulating and cytotoxic effects, respectively. The treatment is usually well tolerated. OBJECTIVE: To report a case of severe anaphylaxis secondary to mistletoe extract administration with demonstrable anti-IgE antibodies to mistletoe. METHODS: Skin prick tests, basophil histamine release, basophil activation test, and immunoblotting were performed to characterize the pathophysiology of this reaction. RESULTS: The patient had immediate-type skin prick test reactions to the whole commercial preparation and to its mistletoe extract component. A histamine release test and a flow cytometric basophil activation test performed with the patient's peripheral blood leukocytes by incubation with the mistletoe extract yielded a concentration-dependent histamine release and expression of the activation marker gp53 in up to 98% of anti-IgE-positive cells. Immunoblotting revealed IgE binding to 5-kDa proteins of mistletoe in the patient's serum, which corresponds to the molecular weight of viscotoxins. The results of all these tests were negative in controls. CONCLUSIONS: Until now, anaphylaxis to mistletoe extracts has been only rarely reported. In our patient, viscotoxin specific IgE evidently had induced an anaphylactic reaction.

Some medicinal plants as immunostimulant for fish.

Immunostimulant effects of the dietary intake of various medicinal plant extracts on fish, rainbow trout (Oncorhynchus mykiss), were investigated. For this purpose fish were fed with diets containing aqueous extracts of mistletoe (Viscum album), nettle (Urtica dioica), and ginger (Zingiber officinale). Food containing lyophilized extracts of these plants as 0.1 and 1% was used at a rate of 2% of body weight per day for three weeks. At the end of the experimental period, various parameters of non-specific defence mechanisms, including extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes and total plasma protein level were examined. Specific growth rates (SGRs) and condition factors (CFs) of the fish were also measured. Plant materials tested for immunostimulatory food additives caused an enhanced extracellular respiratory burst activity (P<0.001) compared to the control group. Especially the rainbow trout fed with a diet containing 1% aqueous extract of powdered ginger roots for three weeks exhibited a significant non-specific immune response. Phagocytosis and extracellular burst activity of blood leukocytes were significantly higher in this group than those in the control group. All plant extracts added to fish diet increased the total protein level in plasma except 0.1% ginger. The highest level of plasma proteins was observed in the group fed with 1% ginger extract containing feed.

Mistletoe lectins enhance immune responses to intranasally co-administered herpes simplex virus glycoprotein D2.

The mucosal adjuvant properties of the three type 2 ribosome-inactivating proteins (RIPs) from the European mistletoe, Viscum album L., were investigated. Mistletoe lectins were compared with cholera toxin (CT) as adjuvants when delivered nasotracheally together with herpes simplex virus glycoprotein D2 (gD2). All three mistletoe lectins (MLI, MLII, MLIII) were potent mucosal adjuvants. Co-administration of MLI, MLII or MLIII with gD2 led to significantly higher levels of gD2-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) antibody than when the antigen was delivered alone. The levels of antibodies induced were similar to those generated in mice immunized with gD2 and the potent mucosal adjuvant CT. Administration of ML1 with gD2 enhanced the antigen-specific splenic T-cell proliferative response. Interleukin-5 (IL-5), but not interferon-gamma (IFN-gamma), was detected in supernatants from splenocytes stimulated in vitro with gD2. This indicates that MLI enhanced type 2 T-helper cell (Th2) responses to the bystander antigen, gD2. Analysis of the gD2- and lectin-specific IgG subclass titres in mice immunized with gD2 and MLI, MLII or MLIII revealed a high ratio of IgG1 : IgG2a, which is compatible with the selective induction of Th2-type immune responses.

In vivo-induction of antibodies to mistletoe lectin-1 and viscotoxin by exposure to aqueous mistletoe extracts: a randomised double-blinded placebo controlled phase I study in healthy individuals.

BACKGROUND: Several studies have been performed in tumour patients to analyse the immunological response to mistletoe extracts. Considering the fact that these extracts are given subcutaneously in most instances, the kind of application resembles a typical immunization schedule. We therefore wanted to see how those extracts act on immunocompetent cells of healthy individuals hoping that this kind of provocation test may give new informations about a more specific application of these extracts in certain diseases. SUBJECTS/METHODS: 47 healthy individuals were exposed for twelve weeks either to Iscador Quercus special (IQ) known to be rich in mistletoe lectin (ML)-1 (n = 16), to Iscador Pini (IP) being poor in ML-1 but enriched in viscotoxins (n = 15), or to placebo (physiological saline) (n = 16) in a randomised, double-blinded placebo-controlled study. Humoral immunoreactivity was analysed by measuring antibodies towards the two compounds ML-1 and viscotoxin VA2 (VA2). Sera were collected in intervals of four weeks up to week 12 and again three months after last exposure. RESULTS: None of the subjects had antibodies to ML-1 or VA2 before exposure. In week 12, anti-ML-1 antibodies of the IgG-type were found in all 16 IQ-treated individuals but only 6 of the 15 probands exposed to IP. In contrast, anti-VA2 IgG-antibodies could be detected in all individuals of both groups. The antibodies were preferentially of the IgG1 and IgG3 type while antibodies of the IgA and IgM type were produced only in a few probands. Antibodies of the IgE-type occurred only in the IQ-exposed individuals and were directed against ML-1 but not VA2. None of the probands receiving placebo developed antibodies to ML-1 or VA2. Severe side effects were not observed in any of the probands. CONCLUSIONS: These data obtained in healthy individuals clearly indicate that IQ and IP-extracts can induce antigen-specific humoral responses. They may, therefore, provide, a solid basic for the evaluation of the humoral immune response in disease states.

The B-chain of mistletoe lectin I efficiently stimulates calcium signaling in human Jurkat T-cells.

Mistletoe lectin I (ML I), a heterodimeric disulfide-linked type II ribosome inactivating protein, exhibits immunomodulatory potency in stimulating the cytokine release in vitro and in vivo. However, data concerning early activation events in T-cells induced by ML I and its A and B chain preceding cytokine secretion and the receptors involved are of limited availability. Here we show by flow cytometric measurements that human T-lymphoblastoid Jurkat cells express surface glycoprotein receptors for ML I. One of which is shown to be the CD2 antigen involved in a variety of T-cell signaling events. The lectin induces in Jurkat T-cells an increase of the cytosolic calcium concentration ([Ca(2+)](i)) consisting of both, the transient release of Ca(2+) from internal stores and a sustained influx of extracellular Ca(2+). Studies with isolated A- and B-chains provided evidence that the lectin-induced increase in [Ca(2+)](i) is mediated by ML IB. The ML I and ML IB stimulated cellular calcium responses are inhibited by saccharidic competitors. In transiently transfected E6.1 cells ML IB stimulated the expression of the luciferase reporter construct pNFAT-TA-Luc that is activated through the nuclear factor of activated T-cells (NFAT). The ML IB stimulated expression of the reporter luciferase (Luc) is completely inhibited by cyclosporin A (0.2 microM) and by FK 506 at 0.05 microM. Pretreatment of Jurkat E6.1 cells with 1-deoxymannojirimycin (dMJ), an inhibitor of cis-Golgi alpha-mannosidase I, strongly reduced cell binding of ML IB-FITC and the ML IB induced calcium response. Benzyl-alpha-GalNAc, an inhibitor of O-linked glycosylation, has slightly decreasing effects in ML IB-FITC binding and was without effects on the lectin stimulated increase in [Ca(2+)](i). Inhibition of the lectin induced calcium responses by cholera toxin and by inhibitors of protein kinases as well as the absence of calcium responses in CD3- and CD45- Jurkat T-cell clones suggest that ML IB has the potency to induce early T-cell activation events.

Application of standardized mistletoe extracts augment immune response and down regulates metastatic organ colonization in murine models.

The immunomodulatory and antimetastatic activity of standardized aqueous mistletoe extracts from plants grown on fir trees (ME-A) and pine trees (ME-P) were evaluated in BALB/c-mice. Regular subcutaneous (s.c.) and intraperitoneal (i.p.) applications (three times per week for 14 consecutive days; 5 and 50 microg per injection and mouse) upregulated thymus weight and peripheral blood leukocyte counts in tumor bearing mice. To check the influence of ME-A and ME-P treatment on growth of experimental metastases, RAW 117 H 10 lymphosarcoma cells and L-1 sarcoma cells were intravenously inoculated into BALB/c-mice to establish liver and lung colonization. ME-A and ME-P were regularly administered starting 24 h after tumor cell challenge. Organ colonization was investigated on day 14 after tumor cell inoculation and demonstrated statistically significant (P<0.05) reductions of experimental liver and lung metastases for ME-A and ME-P treated mice.

Cellular and humoral adjuvant activity of lectins isolated from Korean mistletoe (Viscum album colaratum).

The adjuvant effect of lectins (KML-C) isolated from Korean mistletoe (Viscum album coloratum) on induction of humoral and cellular immune responses against keyhole limpet hemocyanine (KLH) was examined. When mice were immunized subcutaneously (s.c.) with KLH (20 micrograms/mouse) admixed with or without 50 ng/mouse of KML-C (KLH + KML-C), mice immunized with KLH + KML-C showed significantly higher antibody titers against KLH than those immunized with KLH alone, showing the highest titer 5 weeks after immunization. Furthermore, boost immunization with KLH + KML-C at 2-week interval elicited much higher activity than single immunization to enhance antibody responses against KLH. The assay for determining isotypes of antibodies revealed that KML-C augmented KLH-specific antibody titers of IgG1, IgG2a and IgG2b. The culture supernatants obtained from the splenocytes of mice treated with KLH + KML-C also showed a higher level of both KLH-specific Th-1 (IL-2 and IFN-gamma) and Th-2 type cytokine (IL-4). In an in vitro analysis of T lymphocyte proliferation to KLH on week 4, the splenocytes of mice treated with KLH + KML-C showed a significantly higher proliferating activity than those treated with KLH alone. In addition, mice immunized twice with KLH + KML-C and followed by intrafootpad (i.f.) injection of KLH (50 micrograms/site) 14 weeks after the primary immunization induced a higher delayed-type hypersensitivity (DTH) reaction than mice treated with KLH alone. These results suggest that KML-C is a potent immunoadjuvant to enhance cellular and humoral immune responses.

Mistletoe lectin A-chain unfolds during the intracellular transport.

Protein conformation during intracellular routing and translocation of the ribosome-inactivating proteins was investigated on hybridomas producing monoclonal antibodies (monAbs) against mistletoe lectin (ML). Decrease in the toxin activity towards these hybridomas is accounted for by the intracellular interaction of monAbs and the toxin resulting in the interruption of enzymatic subunit translocation into the cytosol. Obtained monAbs interacted with denatured ML A-chain (MLA) and a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. Enzyme-linked immunosorbent assay (ELISA) shows that monAbs recognize five epitopes in denatured MLA. Treatment of MLA by 3 M of guanidine hydrochloride leads to appearance of the epitopes. Hybridoma TA7 has been shown to be insensitive to cytotoxic action of ML. TA7 monAb as we have shown recognizes epitope 101-105, FTGTT, and inhibits the liposome aggregation induced by MLA. A study of the cytotoxicity of ML and ricin for the hybridomas revealed that the unfolding of A-chain is probably required for intracellular transport and cytotoxic activity of ML.

Characterisation of immunological reactivity of patients with adverse effects during therapy with an aqueous mistletoe extract.

Drugs, which are effective are also bond to exert adverse effects. This is also true for mistletoe extracts. Extracts from European mistletoe (Viscum album, VAL) belong to the complementary therapeutic regimens and are used for adjuvant cancer therapy. This study was performed to characterise immunological reactivity of patients with adverse effects during treatment with an aqueous VAL extract (Helixor). VAL-stimulated proliferation and cytokine release of peripheral blood mononuclear cells (PBMC) and anti-mistletoe lectin (ML)-1 antibody production were investigated. 34 patients with proven adverse effects due to VAL therapy (group 1) and 9 patients with unproven relation (group 2) were studied and compared to 14 tumour patients treated with VAL for more than 2 years without side effects (TTP). VAL-stimulated proliferation of PBMC of group 1 was significantly enhanced as compared to group 2 patients and TTP. PBMC from patients with local manifestations proliferated significantly stronger than those from patients with systemic symptoms. Anti-ML-1 antibodies of the IgE type were produced in patients with proven adverse effects but not in patients without adverse effects. Production of Th1 and Th2 specific cytokines varied considerably, indicating that different mechanisms were involved in the induction of adverse effects. In conclusion, our study provide evidence that adverse effects towards VAL (Helixor) are seldom and are dominated by an application site reaction suggesting the involvement of delayed type hypersensitivity (DTH) reactions.

Mistletoe in immunology and the clinic (short review).

The recent meeting of the AGMIF (Working Group for Mistletoe Therapy and Immunological Research) held in Herdecke, Germany, on October, 3rd, 1997, covered recent developments in the field of immunological and biological properties of Viscum album L., the European mistletoe, which is used for adjuvant cancer treatment. So far, one extract component, the mistletoe lectin (ML)-1, was propagated by some researchers to be the only relevant substance within the extracts. However, immunological activities of other extract components such as polysaccharides, vesicles, chitin-binding lectin and their interactions discussed in the first part, underline the significance of the other components as well. In the second part, clinical evidence for the beneficial effects of subcutaneous and intratumoral application of mistletoe therapy was presented by different working groups. However, further research is of great importance to carefully analyse and characterize the involved molecules and exact mechanisms underlying the beneficial effects reported in this meeting.

Modulation of the cellular and humoral immune responses of tumor patients by mistletoe therapy.

There is evidence from recent data that mistletoe extracts exert immunostimulatory properties which could explain their therapeutic effects observed in some tumor patients. Aim of our study was, therefore, to investigate the effect of a subcutaneous 16-weeks therapy with a mistletoe extract (ABNOBAviscum Mali, AM) on the cellular and humoral immune responses in eight breast cancer patients. Mistletoe therapy induced a strong initial proliferation of peripheral blood mononuclear cells (PBMC) in all individuals, which, however, decreased in six patients during the observation period, indicating that not only activating but also inhibitory mechanisms have been induced. In all supernatants of AM-stimulated cell cultures TNF-alpha or IL-6 were found, indicating the activation of cells of the monocyte-/macrophage lineage by mistletoe extracts. Further analyses revealed, that AM induced in vitro also the release of low amounts of IFN-gamma and IL-4 with individual variations. At the end of the therapy, a shift to Th1- related cytokines could be observed in the in vitro cell culture system. All patients produced anti-mistletoe lectin 1 antibodies of the IgG-type during therapy and in four of them additionally antibodies of the IgE-type were found. It, therefore, seems that AM can influence the Th1/Th2 balance and, in case of a Th1 shift, this may favourably influence the tumor growth.

Decrease of activated lymphocytes four and nine hours after a subcutaneous injection of a Viscum album L. extract in healthy volunteers.

The immunomodulatory substance, VaQuFrF (an aqueous extract of Viscum album L. of the oak tree) is used as an adjuvant and as monotherapy in the treatment of cancer and AIDS. After subcutaneous injection, there is a local inflammatory reaction at the injection site and systemic elevation of activated lymphocytes. The immunomodulatory effect of VaQuFrF in the first 24 h after subcutaneous injection on blood leukocyte and lymphocyte subpopulation was investigated. Because a significant natural circadian variation of these cellular parameters exist, the influence was studied in regard to this. In two groups of healthy volunteers, one group receiving VaQuFrF, the following parameters were measured every 2-3 h over a period of 24 h: leukocytes, band form, segmented and eosinophilic granulocytes, monocytes, total lymphocytes and CD4-, CD8-, CD3/25- and CD8/38-positive lymphocytes in count and percentage. In regard to the natural circadian variation 24 h after injection of VaQuFrF, a statistically significant fall in the absolute numbers and percentage of CD3/ 25- and CD8/38-positive lymphocytes was observed. Also, monocytes in percent and absolute numbers show a transient fall 6-9 h, lymphocytes only in absolute and CD4-positive lymphocytes only in percentage 2 h after injection. The results demonstrate that there is increased extravasation of (activated) lymphocytes and monocytes after subcutaneous injection of 1 mg VaQuFrF.

Induction of anti-mistletoe lectin antibodies in relation to different mistletoe-extracts.

Mistletoe extracts are frequently applied in adjuvant cancer treatment. The mistletoe lectins are especially suggested to mediate an antitumorous effect. During treatment with mistletoe lectin-rich extracts, anti-mistletoe lectin antibodies preferentially of the immunoglobulin G type are produced against mistletoe lectin (ML)-1. Interestingly, after application of mistletoe extracts containing natural micelles, anti-mistletoe lectin antibodies of the immunoglobulin G as well as one of the immunoglobulin E type were induced in parallel, suggesting that the nature and preparation of the antigens within the extract modifies immune responses. Anti-mistletoe lectin antibodies were shown to neutralize the cytotoxic effect of mistletoe lectin on peripheral blood mononuclear cells in vitro. Thus, the mode of application of these extracts seems to be of importance with respect to the therapeutic effect.

A plant lectin derived from Viscum album induces cytokine gene expression and protein production in cultures of human peripheral blood mononuclear cells.

A plant lectin from Viscum album (ML-I) has been shown to increase the number and cytotoxic activity of natural killer cells and to induce antitumor activity in animal models. However, the mechanisms underlying the effects of ML-I on natural host defenses are unknown. After 24 h incubation of peripheral blood mononuclear cells in the presence of 10 and 1 ng/ml of ML-I, mRNA expression and secretion of a panel of cytokines were evaluated by reverse polymerase chain reaction and by ELISA, respectively. The lectin induced expression of interleukin (IL)-1 alpha, IL-1 beta, IL-6, tumor necrosis factor-alpha, interferon-gamma, granulocyte-monocyte colony-stimulating factor and IL-10 genes but no expression of IL-2 and IL-5 genes could be detected. Regarding cytokine secretion, IL-6 and TNF-alpha production was induced by 10 ng/ml ML-I. On the other hand, IL-10 secretion was only stimulated by 1 ng/ml lectin. No production of IFN-gamma or, as expectable, IL-2 could be detected. In addition, ML-I increased the percentage of HLA-DR+ T lymphocytes in vitro. In tests performed on whole blood, monocytes and granulocytes bound the fluorescence-conjugated ML-I molecules to a higher degree than lymphocytes. Expression of IL-1 beta and IFN-gamma genes could also be observed upon ML-I stimulation of nonadherent cells. These results suggest that lectin-sugar interactions on the cell surface of immunocompetent cells can induce cytokine gene expression and protein synthesis.

Hybridoma cells producing antibodies against A-chain of mistletoe lectin I are resistant to this toxin.

The cytotoxic effect of mistletoe lectin I (MLI) on TA5 hybridoma cells which produce monoclonal antibodies (mAb) to MLI A-chain (MLA) was investigated. In vitro cytotoxic tests with colorimetric assay were carried out for LD50 determination. TA5 hybridoma cells were 100 times more resistant to MLI and 30 times to chimeric toxin consisting of MLA and ricin B-chain (MLA/RTB) than control cells. The TA5 mAb (IgG1) recognized MLI A-chain in Western blotting and bound 125I-labeled MLI with Ka of 0.43 x 10(8) M-1. The TA5 and control hybridomas had the same number of 125I-labeled MLI binding sites. Therefore cell-surface TA5 antibodies did not influence MLI binding with the cell. The cytotoxic effect and binding of MLI were completely blocked in the presence of 20 mM lactose. Thus, MLI cytotoxicity was mediated only by cell-surface galactosyl residues; intracellular mAb molecules block MLI toxicity. Our data suggest that MLA molecules mediating cytotoxicity pass through an anti-MLA antibody-containing vesicular compartment and that mAbs inhibit the translocation activity of MLI A-chain from intracellular vesicles into the cytosol.