Probing the cons and pros of lectin-induced immunomodulation: Case studies for the mistletoe lectin and galectin-1

Biochimie 83 (2001) 659−666 © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908401013116/REV

Probing the cons and pros of lectin-induced immunomodulation: Case studies for the mistletoe lectin and galectin-1 Hans-Joachim Gabius* Lehrstuhl für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität München, 80539 Munich, Germany (Received 13 February 2001; accepted 22 June 2001) Abstract — When imagining to monitor animal cells through a microscope with resolution at the molecular level, a salient attribute of their surfaces will be the abundance of glycan chains. They present galactosides at their termini widely extending like tentacles into the extracellular space. Their spatial accessibility and their potential for structural variability endow especially these glycan parts with capacity to act as docking points for molecular sensors (sugar receptors such as lectins). Binding and ligand clustering account for transmission of post-binding signals into the cell interior. The range of triggered activities has turned plant lectins into popular tools in cell biology and immunology. Potential for clinical application has been investigated rigorously only in recent years. As documented in vitro and in vivo for the galactoside-specific mistletoe lectin, its apparent immunomodulatory capacity reflected in upregulation of production of proinflammatory cytokines will not necessarily be clinically favorable but a double-edged sword. In fact, lectin application has been shown to stimulate tumor growth in cell lines, histocultures of human tumors and in two animal models using chemical carcinogenesis or tumor transplantation. When testing immunological effects of the endogenous lectin galectin-1, protection against disorders mediated by activated T cells came up for consideration. Elimination of these cells via CD7-dependent induction of apoptosis, and a shift to the Th2 response by the galectin, are factors to ameliorate disease states. This result encourages further efforts with other galectins. Functional redundancy, synergism, diversity or antagonism among galectins are being explored to understand the actual role of this class of endogenous lectins in inflammation. Regardless of the results of further preclinical testing for galectin-1, these two case studies break new ground in our understanding how glycans as ligands for lectins convey reactivity to immune cells, with impact on the course of a tumor or autoimmune disease. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. apoptosis / cytokines / galectin / immunomodulation / lectin / mistletoe / T cell disease

1. Introduction The classical view of the linear flow of genetic information from DNA to RNA to proteins has turned out to be no longer tenable. The gradual appreciation of the versatility of glycans to form elaborate three-dimensional structures complementary to binding sites of receptors (lectins), which is reflected in the steady growth of literature on this topic [1–4], has translated into the concept of the sugar code, adding a new dimension to our knowledge of biological information transfer [5, 6]. Coining terms such as glycomics [7] attests to the notion that oligosaccharides, in the molecular interplay with lectins [8, 9], harbor capacities that rival those of other classes of biomolecules established to be active in biorecognition. Consequently, it is reasonable to assume that basic research in this area will not be without perspective for potential clinical applications. *Correspondence and reprints. E-mail address: [email protected] (H.J. Gabius).

One topic of interest concerns the activity of lectins to modulate immunological responses, with concanavalin A and phytohemagglutinin as potent mitogens serving as classical role models [10–12]. The successful application of these laboratory tools lends credit to the long-range view that it would have meant missing manifold opportunities not to recruit lectin-glycan interactions for immune regulation. Indeed, endogenous receptors for major classes of mammalian lectins, i.e., C- and I-type lectins, galectins and pentraxins, are intimately involved in cell adhesion and cell activity modulation [13–17]. Selecting a representative from the two main groups currently tested, namely a plant and an endogenous lectin, the lessons that emerged from these studies of lectin-mediated immune responses and how they may relate to practical considerations will be outlined. In detail, the first example documents that augmentation of immune functions, in this instance enhanced production of proinflammatory cytokines, is a double-edged sword in tumor biology. The second case gives reason for guarded optimism to be on track for step-by-step evaluation of downregulating inflammatory and autoimmune processes therapeutically.

660 2. The mistletoe lectin as biological response modifier In principle, the given case discussion has considerable practical relevance, because this galactoside-specific lectin is a major component of mistletoe extracts. Taking advantage of unique, legally benevolent regulations for anthroposophic and phytotherapeutic preparations, they are on the market in Germany despite lack of undisputed scientific evidence excluding risks and proving efficacy [18–22]. Claims for benefit in cancer treatment are often derived from a dogmatic, purely spiritual concept established by R. Steiner in the first quarter of this century [20, 23, 24]. On the pharmaceutical level of the extracts, their variable compositions dependent on diverse factors including climatic conditions at harvest and the nature of the host tree render it difficult to test reproducibly preclinical attributes. Similar to investigations with the mentioned concanavalin A and phytohemagglutinin it was thus essential to work with purified material in addition to working with extracts of known lectin quantity. The galactoside-specific lectin of mistletoe (Viscum album agglutinin, VAA-I) belongs to the group of enormously potent plant AB toxins with its A-chain acting as deadly rRNA N-glycosidase [25, 26]. Since the B-chain binds the spectrum of natural α-/β-galactosides as prerequisite for entry of the hololectin into the cell without notable preferences [27, 28], this plant toxin lacks cell type specificity and capacity to distinguish malignant from normal cells [29, 30]. In fact, this versatility to home in on animal cells might be explained by the need to protect the mistletoe from predators [31]. While antitumoral activity at a dose exploiting the potent cytotoxic activity of the lectin is still a matter of controversy in tumor models [32–34], results of a phase I study examining the application of the related AB-toxin ricin question the perspective for such plant products as chemotherapeutical [35]. However, its activity as biological response modifier in the range of ng/mL × 0.2–1 × 106 cells (mononuclear cells from peripheral blood, monocytes, cells from cultured lines) or at 1–2 ng/kg body weight given biweekly s.c., as shown to be active in initial studies [36–39], has prompted thorough analysis of the elicited responses on the level of cells in vitro, histotypic cultures, and animal tumor models in vivo. Lectin binding to glycans of the cell surface can cluster target molecules, a pivotal step for initiating cellular signaling pathways [40, 41]. Indeed, this mistletoe lectin is a potent cross-linking agent [42], with marked positive cooperativity of cell surface binding apparent in the non-toxic dose range [38]. As response to lectin binding, quantitative aspects of lipid and protein phosphorylation in THP-1 cells change, and, regarding immune effectors, the production and secretion of cytokines, namely interleukins-1, -6, -10 and -12, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor, are enhanced in vitro and in vivo [37–39, 43–47]. On the

Gabius cellular level, immune parameters such as phagocytic activity of granulocytes and tumoricidal activity of NK cells against cultured tumor cells from established lines are increased, the lectin qualifying as decisive factor for the given parameter changes also by extracts [36, 39]. However, these data should not delude one into assuming that these lectin-dependent responses will principally and exclusively be beneficial for the course of a tumor disease. Having realized the wide range of cytokine functions not at all restricted to immune cells, promises in immunomodulation inevitably come with potential for undesired (yet not unexpectable) risks [20, 39]. In fact, cells of the immune system are the source of growth-promoting substances, proinflammatory cytokines among them. They can help responsive malignant cells to thrive even under adverse environmental conditions, an aspect referred to as ‘understudied and insufficiently acknowledged’ [48–50]. Expression of active cytokine receptors, and the integrity of the intracellular signaling, are documented to facilitate stimulation of tumor cell growth by immune factors such as interleukin-6 [48–50]. To underscore the nature of the problem predicting how tumor parameters are affected by enhanced cytokine availability, it is pertinent to go through the compilation of tumor types responsive to those cytokines upregulated by the mistletoe lectin (table I). Since increased serum levels, especially for interleukin-6, have been delineated as a negative prognostic factor in various tumor diseases including plasmacytoma, metastatic melanoma, and renal cell, prostate and ovarian carcinomas [20], it is a questionable practice to propose with confidence clinical benefit for a treatment modality augmenting this parameter. Recent studies in vitro and in vivo in animal models substantiate the notion that this type of immunomodulation can bear risks, with no proven efficacy outweighing them. In monitoring cell growth in the presence of a non-toxic lectin concentration (50 pg/mL × 105 cells) for eight sarcoma and four melanoma lines, five out of 12 cell responded with significant stimulation [30]. In histotypic cultures established from 30 cases of surgically removed human tumors, the lowest tested concentration (1 ng/mL) still exerted some toxicity. Notably, tumor cells of five specimens incorporated significantly increased levels of radioactive thymidine in the 72 h culture period [30]. The presence of para- and autocrine interleukin-6 loops, for example in benign (and also malignant) prostate, is to be reckoned with to pinpoint the molecular basis for this effect [68]. With respect to portraying a perspective for clinical application of the immunomodulatory lectin activity, the results of three studies of tumor development in rats subjected to chemical carcinogenesis and lectin treatment precisely following the established regimen for immune effects, are also of relevance. Lectin treatment failed to influence the incidence of urinary bladder carcinoma, irrespective of shifting the beginning of lectin treatment relative to the application of

Immunomodulation by lectins: friend or foe?

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Table I. Stimulation of tumor proliferation or aggressiveness in vivo by cytokines inducible by the mistletoe lectin. Tumor type

Cytokine

Study object

Reference

B cell tumors Plasmacytoma Primary effusion lymphoma Melanoma Melanoma Advanced-stage melanoma Glioma Non-small cell lung cancer Hepatocellular carcinoma Sarcoma Renal cell carcinoma Renal cell/ovarian carcinomas Brest carcinoma

IL-6 IL-6 IL-6 IL-1/IL-6 TNF-α IL-6 GM-CSF GM-CSF IL-6 IL-6 IL-6 IL-6 IL-1β

IL-6 overexpression Gene deficiency (KO mutant) SCID mice Metastasis formation and growth of injected tumor cells Carcinogenesis in KO mutants Transfected cells Expression of factor and receptor in advanced tumor stage Xenograft IL-6 overexpression and metastatic spread Growth of transplantable tumors Definition of aggressive subgroup by IL-6R expression Clinical case reports Correlation of expression with aggressiveness

[51–53] [54, 55] [56] [57, 58] [59] [60] [61] [62] [63] [64] [65] [66] [67]

the carcinogen (N-methyl-N-nitrosourea or N-butyl-N-(4hydroxylbutyl)-nitrosamine) [69–71]. When monitoring tumor size in control and treated groups tumors proved significantly larger (P = 0.02) in the latter groups after an experimental period of 15 months [70, 71]. Indications for multifocal tumor development and increased incidence of urothelial carcinomas with an invasive growth [69–71] cannot be reconciled with a paradigm that biological response modifiers from herbal medicine are free of preclinical risk potential. Another unrelated tumor model, namely transplantation of C3L5 murine mammary adenocarcinoma cells (5 × 105 viable cells) subcutaneously into the left axillary region of C3H/HeJ female mice, revealed that the given observations on urothelial carcinoma development under lectin treatment are not exceptional. Growth of the tumors subcutaneously and in metastatic lesions to the lung, as well as their frequency, were significantly increased in the course of the immunomodulatory treatment protocol relative to control animals, the experimental period lasting 21 days [72]. In view of the documented evidence for growth factor activity of proinflammatory cytokines on lymphoma/leukemia cells in vivo (table I), case reports on disease progression in connection with application of mistletoe extract deserve not to be neglected [73, 74]. Similarly, the risk of immunostimulation and immunopathogenesis will have to be examined rigorously, case reports in this respect also giving reason for concern [75, 76]. Although it is tempting to use the readily accessible mistletoe lectin as potent effector for endogenous production of a cytokine set, the evidence from preclinical investigations casts serious doubts on the ethical justification and legitimacy to perform clinical trials in this area. To predict responses for the individual patient in the network of lectin-dependent immune regulation, and to exclude adverse effects of such an experimental treatment compromising the patient’s safety, is currently hardly possible.

In a broader context, work on this lectin epitomizes the capacity of protein-carbohydrate binding and glycan cross-linking to influence immune parameters. Similar to plant agglutinins, animal lectins share the property to form clusters with their ligands [9, 42, 77, 78]. By placing carbohydrate recognition domains at opposing sides of a lectin (for example a homodimeric lectin [79]) an efficient cross-linking molecule is established. Actually, the illustrated structure [79] depicts the folding pattern of a class of animal lectins which like the mistletoe lectin homes in on galactosides, i.e., the galectins [15, 16]. The different binding-site topologies of these two unrelated types of galactose receptor lead to distinct subspecificities of ligand selection. This difference has been determined by a panel of ligand analogues such as chemically engineered ligand derivatives [4, 27, 28]. Moreover, non-uniform ligand selection translates into distinct three-dimensional modes of how to accommodate the same saccharide in binding pockets of different lectins. Given the flexibility of various carbohydrate ligands to oscillate between defined conformations in solution, not every detectable topology version might be bioactive. Indeed, conformer selection abounds, and various lectins can distinguish between bioinert and bioactive conformations following their own way, a process dubbed differential conformer selection [4, 8, 80–82]. These biochemical differences of ligand sequence and conformation intimate that the plant agglutinin and a galectin will elicit disparate profiles of immune reactions. In fact, a prodigious aspect of immunomodulation by the non-toxic proto-type galectin-1 concerns intervention in activated-T-cell-dependent disorders including autoimmune diseases. 3. Galectin-1: inductor of T cell apoptosis In the quest to delineate functions of the rather abundant galectin-1, its prophylactic and therapeutic action on

662 experimental autoimmune myasthenia gravis in rabbits pointed to attenuation of the cellular response against self-antigens [83]. The protective effect of galectin-1 against experimental autoimmune encephalitis, a paralytic T-cell-mediated disease directed against myelin basic protein, furthered the notion for a potential perspective of galectin-1 in treatment of activated-T-cell-dependent diseases [84]. Concanavalin-A-induced hepatitis, a T-celldependent model of liver disease, and collagen-induced arthritis, an experimental model for rheumatoid arthritis, were two further manifestations of immune dysregulation which were subject to successful intervention by galectin-1 [85, 86]. Pivotal for the analysis of mechanisms of action of galectin-1 on the cellular level was the detection of 10-fold upregulation of its gene expression by dexamethasone in the glucocorticoid-sensitive human leukemia line CEM C7 [87]. This enhancement occurred during the induction of apoptosis, and the actual experimental demonstration of galectin-1 as effector of apoptosis of activated but not resting T cells and various T leukemia cell lines was crucial for establishing this paradigm of galectin-1 function in immunosuppression [88]. Corroborating this reasoning, activation of galectin-1 gene expression by demethylation of its promoter region (among other reactive genes) in susceptible MOLT-4 leukemia cells led to reduced proliferation [89]. Since CD7 is active as counterreceptor for galectin-1 to transmit proapoptotic signals [90], regulation of sensitivity to apoptosis can comprise the level of expression of the CD marker. Consequently, loss of CD7 might enhance survival of neoplastic and autoreactive T cells, and CD7negative memory T cells or tumor cells could thereby escape the detrimental effects of the death-signaling pair CD7/galectin-1. Expansion of CD7-negative clones in the course of a Sézary syndrome has been linked to this autoprotective mechanism [91]. Actually, galectin-1 presence in human skin [92] is conceivable to be an important factor in this environment removing CD7-positive malignant cells from the cutaneous T cell neoplasms. Having observed galectin-1 also in most other types of tumor cells [93, 94], the question is intriguing to figure out ways to turn its proapoptotic potential into tumor growth regulation. The observation that its overexpression by transfection with a gene-carrying pR14BA plasmid entails inhibition of proliferation and an increased rate of apoptosis in the prostate cancer LNCaP line illustrates proapoptotic activity beyond T cells [95]. In addition to triggering a cell death pathway in activated T cells, immunosuppression by galectin-1 can come about by preventing synthesis and/or release of proinflammatory Th1-derived cytokines [85, 96], as well as reducing prostaglandin E2 secretion and arachidonic acid release from stimulated peritoneal macrophages [97] and eosinophil migration [98]. Within the arthritogenic process in mice, galectin-1 application skewed the overall balance towards a Th2 profile with reduction of the level

Gabius of interferon-γ and a clear increase in interleukin-5 production [86]. Importantly, this activity is opposite to that of another member of the galectin family. Galectin-3 reduces interleukin-5 production at the level of increased activity of the REIII site, a negative regulatory transcription element [99, 100]. Equally pertinent for a cellular mechanism to hold the proapoptotic activity of galectin-1 at bay, overexpression of galectin-3 in Jurkat cells and BT549/Evsa-T breast cancer cells confers resistance to apoptosis. Acquisition of this attribute probably involves interaction with proteins from the bcl-2 gene family and protects against alteration of the mitochondrial membrane potential and formation of reactive oxygen species [101–103]. It is instructive to note that besides overlapping ligand profiles, the individual galectins most probably also have binding capacity for distinct glycan chains. For example, eosinophil chemoattractive activity of galectin-9 is not seen with galectins-1, -3 and -8 [104]. In an animal model for the crescentic form of glomerulonephritis, galectin-1 failed to induce apoptosis in CD8-positive cells, while this tandem-repeat family member (galectin-9) was active [105]. Interestingly, its linker peptide connecting the two lectin domains can be extended by 31/32 amino acids in murine and human isoforms, and point mutations for sequence truncations have been mapped [106, 107]. These results have implications for the interpretation or prediction of cellular activities based on galectin presence. It is premature to draw conclusions from monitoring only one or two activities. The extent to which human cell types use their genome-encoded galectin diversity has recently been assessed by galectin fingerprinting using RT-PCR for 61 tumor cell lines flanked by biochemical assays in selected cell extracts [108]. In tumor cells in culture, the capacity of galectin expression often goes well beyond the commonly studied galectins-1 and -3 [108]. This conclusion has a pronounced bearing on further attempts to regulate immune functions and on prognostic evaluations in tumor pathology. Having expression vectors, purified galectins and galectin-specific markers available, the extents of functional overlap or even antagonism will have to be defined rigorously on the way to devise rational therapeutic strategies with members of this family of endogenous lectins. 4. Conclusions Being readily accessible at the non-reducing end of glycan chains is a decisive factor to turn β-galactosides and derivatives thereof into important docking points for lectins. Regarding this role, it is not surprising that the synthetic machinery for such epitopes is very elaborate. At least 13 different β1,3(4)-galactosyltransferases are available to add galactose moieties to growing chains [109]. Variations in their activity profile open the way for

Immunomodulation by lectins: friend or foe? versatile modulation of this aspect of glycosylation. Otherwise identical proteins carrying glycan chains with structural alterations, termed glycoforms [110], contribute to establish the typical glycoproteome of the cell. It can be likened to a signature with relevance for cell typing. Besides sequence alterations, the shifting of conformational equilibria and presentation of bioactive or bioinert forms of the same saccharide might provide a further level of regulation in glycocoding [111]. As prophetically supposed in this respect in 1965 by Eylar [112], ‘presumably the specificity’ of the interaction of the glycan with its receptor ‘would reside in the tertiary conformation of the carbohydrate unit’. Insights into the topological details of the molecular rendez-vous will then be crucial to devise custom-made ligands for interfering therapeutically with e.g. bacterial/parasitic or metastatic adhesion [4, 113, 114]. For therapeutical approaches, not only oligosaccharides can find their place to combat disease-causing or -accompanying cell adhesion. The two examples in this review demonstrate that lectins are potent immune regulators. One salient lesson we have learned is the doubleedged nature of the activity of the mistletoe lectin. The respective results caution against the naive expectation that any biological response modifier, this one from herbal medicine, is free of risk potential. Under the given circumstances, further extensive preclinical work is mandatory to foretell with confidence when lectin effects will either be friend, neutral or foe for the individual patient. Reviewing the available evidence, the situation looks different for the non-toxic and non-immunogenic endogenous galectin-1, although, among other parameters, the effects of long-term administration in models will certainly have to be assessed. In gauging possibilities to put endogenous lectins of this family to work clinically, it is fair to conclude at present that immune and growth regulatory effects of galectins should not undeservedly be assigned to take second place relative to selectins. Moreover, the discussed roles of galectins in immunology add another strong argument that being ‘overshadowed by their more glamorous companions, nucleic acids and proteins’ [115], will not do justice to the manifold talents of glycans in their interaction with lectins in biological information transfer.

Acknowledgments I am grateful to R. Ohl for excellent processing of the manuscript, the Dr.-Mildred-Scheel-Stiftung für Krebsforschung, the Volkswagen-Stiftung and the Wilhelm Sander-Stiftung for generous financial support and the exchange programs between the DAAD and Spain (Acciones Integradas) and the NSF (USA) and also the EC program for Euro-Conferences for travel grants.

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