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Cachexia: a therapeutic approach beyond cytokine antagonism
International Journal of Cardiology 85 (2002) 173–183 www.elsevier.com / locate / ijcard
Cachexia: a therapeutic approach beyond cytokine antagonism S. von Haehling a
a,b ,
*, S. Genth-Zotz a,c , S.D. Anker a,b , H.D. Volk d
Department of Clinical Cardiology, National Heart & Lung Institute, Royal Brompton Hospital, Dovehouse Street, London SW3 6 LY, UK b ¨ Centrum for Molecular Medicine, Berlin, Germany Franz Volhard Klinik ( Charite´ , Campus Berlin-Buch) at Max Delbruck c Department of Medicine II, Johannes Gutenberg-University, Mainz, Germany d Department of Medical Immunology, Charite´ Medical School, Berlin, Germany
Abstract Cachexia is seen in a number of chronic diseases, and it is always associated with a poor prognosis. Irrespective of etiology, the development of cachexia appears to share a common pathophysiological pathway. This includes induction of proteasome-dependent myofibril-degradation, which is thought to be secondary to stimulation by enhanced levels of pro-inflammatory cytokines. Elevation of tumor necrosis factor-a (TNFa) and other plasma cytokines has been demonstrated in many conditions associated with cachexia. Despite improved pathophysiological understanding, a specific treatment for cachexia has not yet been established. Whilst direct TNFa antagonism has therapeutic appeal, this review will focus on manipulation of downstream pathways and the potential benefits. For example, nuclear factor-kB (NF-kB) is one of the most important signal transducers of TNFa, and drugs targeting this signalling cascade might be useful in the treatment of cachexia. Although the use of some of these substances, for example glucocorticoids, remains controversial, others may prove beneficial in the treatment of this syndrome. The role of other approaches such as proteasome-inhibitors remains to be elucidated. Alternatively, interleukin-10 and other immunosuppressive cytokines may also be able to counterbalance certain features of cachexia. 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cachexia; Tumor necrosis factor; Immunosuppression; NF-kB
1. Introduction Cachexia is a common feature of a number of different illnesses, such as cancer, sepsis, chronic heart failure, rheumatoid arthritis and AIDS. It is always associated with poor prognosis. Although cachexia was first described more than 2000 years ago and has been subject to increasing research in recent decades, there is still considerable disagreement even on the question of its definition [1,2]. There is no established therapy that can reverse cachexia. The development of successful therapies *Corresponding author. Tel.: 144-207-351-8127; fax: 144-207-3518733. E-mail address: [email protected] (S. von Haehling).
requires a detailed understanding of the pathophysiology of wasting in chronic illnesses. Irrespective of underlying disease etiology the development of cachexia may share final common pathways. In addition, characteristic immunologic and metabolic abnormalities are seen in all forms of this syndrome. These pathways may offer promising targets for therapy aimed at improving the adverse morbidity and mortality associated with cachexia. Characteristic features of cachexia include the activation of the immune system [3] and muscle wasting through the ubiquitin–proteasome pathway [4]. This review will discuss potential therapeutic approaches beyond the direct antagonism of TNFa and other pro-inflammatory cytokines. Targets include downstream cytokine signalling mechanisms
0167-5273 / 02 / $ – see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 02 )00245-0
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through transcription factors such as nuclear factorkB (NF-kB) and activation protein-1 (AP-1) by immunosuppressive agents including immunomodulatory cytokines, e.g. interleukin (IL)-4, IL-10, IL-13, and transforming growth factor-b (TGF-b), and experimental pharmacologic approaches like proteasome inhibitors.
Elevation of circulating pro-inflammatory cytokines is a common feature of cachexia [5–9]. It is currently accepted that tumor necrosis factor-a (TNFa) plays the key role in the development of this condition. TNFa was first described for its ability to induce weight loss and anorexia in mice and thus termed cachectin [10]. The syndrome was reversed when the injection of TNFa was discontinued. In chronic heart failure, increased levels of TNFa relate to reduced peripheral blood flow [11], apoptosis [12] and lower skeletal muscle mass [13]. In addition, pro-inflammatory cytokines relate to prognosis in chronic heart failure independently of whether cachexia is present or not [14]. In recent years it became clear that other pro-inflammatory mediators, particularly IL-1b and IL-6, might also play an important part in the development of the syndrome [15]. The major sources of these pro-inflammatory cytokines are monocytes and macrophages, although other cell types, such as T lymphocytes or even non-immunological cells like endothelial cells are also capable of their production. The precise mechanism that leads to the secretion of these mediators and ultimately to cachexia is still under investigation and so far only partly understood. Whilst chronic inflammation seems to play a major role in the development of cachexia, abnormalities in other systems may also be important. These include mediators such as catecholamines, steroid hormones and leptin which act either directly or through interaction with pro-inflammatory cytokines [16].
potently inducing this reaction [17]. Maintaining the acute phase response requires an excess of essential amino acids which yields loss of body proteins [18]. Since skeletal muscle accounts for almost half of the body protein mass, this compartment is intensively affected (Fig. 1). Thus, muscle wasting can be initiated by the acute phase response. The predominant pathway of protein turnover in all eukaryotic cell types is the ubiquitin–proteasome pathway, which requires adenosine triphosphate (Fig. 2). Ubiquitin serves as a cofactor that is covalently linked to proteins to be degraded in the 26S proteasome complex. This multisubunit protease specifically degrades ubiquitin-conjugates, and it is the main mediator of muscle wasting in man (Fig. 1) [4]. The impact of protein degradation through the ubiquitin– proteasome pathway has been demonstrated in vivo for cachexia in AIDS [19], sepsis [20], cancer [21], and renal failure [22]. The ubiquitin–proteasome system is present in both the nucleus and the cytosol. Alterations in the activity of this pathway are probably due to changes in the rate of ubiquitin conjugations, and several hormonal and immunological factors seem to be involved in a regulatory fashion. The proteasome is responsible for degradation of proteins from the intracellular compartment that are linked to ubiquitin, while lysosomes degrade proteins from the extracellular compartment. It is not fully understood how ubiquitin-binding to intracellular proteins is regulated, although Varshavsky reported that the half-life of a protein correlates with certain features of its N-terminal sequence [23]. Cytokine signals including TNFa, IL-1 and IL-6 [24–26] stimulate the ubiquitin–proteasome pathway in muscle. However, there is some evidence that these signals may be secondary to the influence of cytokine-induced glucocorticoids [27]. The finding that protein degradation by the proteasome does not yield free amino acids but peptides, suggests that a number of other factors may also be important in the regulation of muscle protein breakdown [28].
2.1. From acute phase response to tissue wasting
2.2. Proteasome inhibitors
Inflammation as well as tissue injury induce a specific reaction of the body known as the acute phase response, and IL-6 is one of the mediators most
The first proteasome inhibitors became available in 1994. All experiments using these agents have thus far been performed in vitro and in animal models.
2. Cytokines and the proteasome complex
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Fig. 1. Mechanism of muscle wasting in man: an unknown stimulus, possibly TNFa binding to TNFa-receptors, causes covalent binding of ubiquitin to certain myofibrils. Following ubiquitin-labelling, these proteins are directed to the proteasome-complex. The proteasome releases peptides, which are further broken down into free amino acids by several mechanisms that have not yet been clearly identified. Afterwards, these amino acids are, for example, available for protein synthesis in acute phase response in the liver. Certain pro-inflammatory cytokines such as IL-1, IL-6 and TNFa are known to induce proteasome activity while proteasome inhibitors block it.
Very recently, phase I trials with proteasome inhibitors were started. Four classes of proteasome inhibitors have been described [29,30]: (i) peptide aldehydes primarily inhibit the chymotrypsin-like activity of the proteasome, which is one of its specific proteolytic sites. Removing the peptide aldehyde restores the proteolytic activity. In a model using rat skeletal muscle, these agents had up to 52% greater effect on proteolysis in atrophying muscle than on controls [31]. The overall protein synthesis remained unchanged. Unfortunately, these agents usually require 10–20 h until onset of action [32]. (ii) Lactacystin and its active derivative b-lactone are more specific, but irreversible inhibitors of the proteasome. They act as pseudosubstrates that are covalently bound to one of the subunits of the proteasome [33]. (iii) Vinyl sulfone has been shown to inhibit the proteasome complex irreversibly in a similar manner to lactacystin [29]. In a human lymphoma cell line prolonged inhibition of the proteasome by vinyl sulfone led to the appearance of cell variants with a distinct proteolytic system [34]. (iv) Finally, dipep-
tide boronic acid analogs have been shown to block proteasome activity via reversible binding to its active sites. Nevertheless, nonspecific drugs like b 2 agonists also have the potential to suppress ubiquitin– proteasome-dependent proteolysis during tumor growth. This was recently demonstrated in tumorbearing rats using clenbuterol at a dose of 1 mg / kg body weight daily [35]. Peptide aldehydes, lactacystin and b-lactone have been shown to block up to 90% of the degradation of abnormal proteins and short-lived proteins in the cell [29]. A substance that can specifically block myofibril degradation in skeletal muscle is still waiting to be discovered.
3. Intracellular suppression of TNFa signal transduction The transcription factors nuclear factor-kB (NFkB) and activation protein-1 (AP-1) are found in several cell types. Currently, these proteins are seen
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Fig. 2. Mechanism of protein degradation by the proteasome. It represents the proteolytic mechanism which selectively degrades most proteins of intracellular origin while proteins from the extracellular compartment are degraded by lysosomes. (A) The activation of NF-kB involves degradation of its inhibitory protein, IkB, by the proteasome. Therefore, IkB is marked for degradation by a poly-ubiquitin chain. This ubiquitinylation process requires enzymatic activity of three different enzymes, termed E1, E2 and E3. These enzymes transfer activated ubiquitin to the selected protein. (B) IkB is then rapidly unfolded and degraded in the proteasome, a process requiring adenosine triphosphate (ATP). NF-kB can therefore signal into the nucleus, and transcription of the affiliated genes is induced.
as the most important signal transducers for proinflammatory stimuli. Both are activated following TNFa trimer binding to the TNFa receptor complex (particularly the type I p55 receptor). The complex interaction of these and other transcription factors is responsible for the inhibition or enhancement of certain genes. In general, a coincident activation of several transcription factors is necessary for maximal gene expression [36].
3.1. NF-k B-dependent signalling NF-kB was first described in 1986 as being necessary for immunoglobulin kappa light chain transcription in B cells [37]. It is a heterodimer
consisting of two subunits. In unstimulated cells, cytoplasmatic NF-kB is bound to its inhibitory protein, IkB (Fig. 2). Therefore, it is kept in an inactivated form in the cytoplasm with its nuclear localization signal being masked [38]. Different stimuli such as TNFa, IL-1, bacterial lipopolysaccharide (LPS) or UV light lead to NF-kB activation [39]. Upon activation, IkB is phosphorylated and rapidly degraded in the ubiquitin–proteasome pathway (Fig. 2). The free NF-kB complex can translocate into the nucleus, where NF-kB binds to kB DNA sequences (NF-kB responding elements) in non-coding regions of several ‘acute response’ genes thus promoting the transcription of the affiliated genes (Fig. 3). Interestingly, NF-kB binding sites are found
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Fig. 3. Interactions of different intracellular signalling mechanisms for TNFa. One of the major components activated upon TNFa binding to its receptor is NF-kB, which is kept in an inactive form masked by IkB in the cytoplasm. Binding of other substances like bacterial lipopolysaccharide (LPS) to CD14 / Toll-like receptor (TLR4) or IL-6 to its receptor are also known to stimulate NF-kB activation. IkB is rapidly degraded in the proteasome, and NF-kB binds to promotors of several genes. For example, TNFa and IkB transcription are induced with the latter finally shutting off the signal of NF-kB. Glucocorticoids (GC) can prevent NF-kB from inducing the transcription of certain genes. Therefore, they bind to glucocorticoid-receptors (GR) in the cytoplasm, which serve as transcription factors. IL-10 signalling through STAT1 and STAT3 induces, for example, TNFa receptor release (sTNF-R) which bind TNFa in the plasma and thus inactivate it. IL-10 can also suppress NF-kB signalling.
in a multitude of genes, coding for cytokines, acute phase response proteins, and cell adhesion molecules [39]. In addition, NF-kB up-regulates the transcription of its inhibitory protein IkB which finally shuts off the signal [40]. In principle, NF-kB-mediated signals can be suppressed by targeting IkB degradation, NF-kB translocation, or NF-kB DNA binding as well as by IkB overexpression. In fact, genetic overexpression of IkB blocks NF-kB dependent processes. Drugs targeting the translocation of activated NF-kB such as fumar acid have a high anti-inflammatory potency [41]. Most recently, activation of NF-kB by overexpression of an IkB phosphorylating kinase has been shown to sufficiently block myogenesis, illustrating
the link between NF-kB and cachexia development [42]. As IkB is degraded in the proteasome, the use of proteasome inhibitors is thought to suspend signal transduction via NF-kB into the nucleus. In that context it has to be kept in mind that complete inhibition of NF-kB has proven detrimental. Knockout mice models where the major subunits of NF-kB are targeted show severe immunodeficiency which was lethal in some cases [39]. Whilst the NF-kB pathway seems to be important for up-regulating TNFa and IL-1, the latter are also the most powerful endogenous inducers of NF-kB. In T cells, TNFa production is also controlled by T-cell receptor-mediated NF-AT (nuclear factor of activated T cells) activation. In particular, the p38 mitogen-activated
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protein kinase (MAPK) kinase pathway controls the translation of TNFa.
3.2. AP-1 -dependent signalling In comparison to NF-kB, the AP-1-dependent signalling pathway is less well understood. AP-1 forms a family of transcription factors binding to a specific palindromic DNA sequence [43]. Its activity is found in nearly all cell types. Some evidence has accumulated in the past few years that AP-1 functions as an effector of growth factor signalling, although cytokines, particularly TNFa and IL-1, are also potent activators of AP-1. A possible role of AP-1 inhibition for the treatment of cachexia has as yet not been investigated.
4. Immunosuppressive drugs
4.1. Glucocorticoids Glucocorticoids are widely used in the treatment of inflammatory and immune diseases. In cancer cachexia, they have been used to stimulate food intake. Indeed, they seem to mitigate anorexia and asthenia [44]. The anti-inflammatory action of these substances, however, is mainly due to inhibition of NF-kB and AP-1 activation (Fig. 3) [45]. Glucocorticoids freely penetrate cell membranes to bind to the cytosolic glucocorticoid receptor, which serves as a transcription factor. Upon translocation to the nucleus, anti-inflammatory properties of glucocorticoids are initiated due to negative regulation of genes encoding pro-inflammatory cytokines. However, antiinflammatory properties are not strictly dependent on DNA binding of the hormone–receptor complex. In contrast, side effects are mainly due to the transactivating characteristics of glucocorticoid receptors that require DNA binding. This discrepancy opens new possibilities for developing novel glucocorticoids with less side effects. Recently, two interesting studies have suggested that a major mechanism of glucocorticoid action might be the induction of IkB synthesis [46,47]. Moreover, IkB might even be able to remove NF-kB
actively from its DNA binding site. Another mode of glucocorticoid action involves direct protein–protein interaction [48]. Certain transcription factors like NFkB, AP-1 and STAT5 from the signal transducers and activators of transcription (STAT) family have been found to be bound by glucocorticoid-receptors [45]. Therefore, pro-inflammatory genes might be suppressed due to this interaction by masking of transactivating domains. For dexamethasone, a highly potent synthetic glucocorticoid, blocking of the TNFa and IL-1-dependent induction and translocation of NF-kB could be demonstrated [49]. Dexamethasone also inhibits the LPS-induced activation of NF-kB [47]. Whilst glucocorticoid therapy might theoretically be of benefit for the treatment of cachexia, prolonged use of these substances may lead to weakness, osteoporosis, diabetes and even delirium. Therefore, the routine use of these drugs remains controversial, and their role in the treatment of cachexia is far from being established. Novel glucocorticoids without transactivating properties increase the benefit / side effect ratio despite some reduction in their antiinflammatory properties (e.g. no IkB induction, unpublished data). This novel class of glucocorticoids may open new therapeutic opportunities in long-term treatment of cachectic patients.
4.2. Calcineurin inhibitors Cyclosporin A and tacrolimus share similar modes of action, although their chemical structure is highly different. Both are immunosuppressive agents used in the treatment of transplantation and certain autoimmune disorders, and both pass freely through cell membranes. Tacrolimus is more potent than cyclosporin A. Unfortunately, both cyclosporin A and tacrolimus share a narrow range between subtherapeutic and toxic plasma concentration, and therefore frequent drug monitoring is required [50]. Both drugs are nephrotoxic. Another substance with similar immunosuppressive attributes is sirolimus, although it is not a member of the calcineurin inhibitor family per se. The immunosuppressive properties of cyclosporin A were first recognised in 1976 [51]. Cyclosporin A is a small peptide from the fungus Hypocladium inflatum gams widely used in the suppression of
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allograft rejection. Its major targets are T cells [52]. In 1984 one of its modes of action became apparent when its ability to inhibit T cell activation was demonstrated. In these cells, cyclosporin A strongly binds cyclophilins that are involved in protein folding. Cyclosporine–cyclophilin complexes bind to and inhibit calcineurin. Thus, the activation and nuclear translocation of NF-AT, which is involved in IL-2, IL-4 and TNFa transcription, is inhibited [53]. The effects of cyclosporin A on other cell types are less obvious. In a model using a murine macrophage cell line, cyclosporin A acts as an uncompetitive inhibitor of the proteasome, and it suppresses the LPS-induced IkB degradation [54]. Therefore, inhibition of proteasome activity seems to be the mechanism by which cyclosporin A prevents NF-kB activation. Other workers have confirmed the inhibition of NF-kB activation [55]. This mechanism may explain the suppression of pro-inflammatory cytokine production reported in several studies: in an animal model comparing euthymic and athymic mice, cyclosporin A dose-dependently inhibited the production of IL-1b and TNF [56]. The production of IL-6 was blocked only in euthymic mice suggesting that the suppression of its production requires an effective T-cell response. Other workers have at least partly confirmed these data. In U937 monocyte cells, cyclosporin A reduced the secretion of IL-1b, IL-6, IL-8 and TNFa [57], and in human alveolar macrophages cyclosporin A was able to inhibit the production of IL-8 and TNFa as well [58]. In contrast to glucocorticoids, however, it is less efficient in blocking TNFa-mediated effects. A further study demonstrated that cyclosporin A injected into rats with inflammation-induced cachexia prevented the sustained loss of body weight and adipose tissue [59]. Tacrolimus (FK506) is a macrolide antibiotic extracted from Streptomyces tsukubaensis. Like cyclosporin A, tacrolimus mainly inhibits T cell activation via association with calcineurin. It binds to FK506 binding protein (FKBP), and the FKBP– calcineurin complex inhibits NF-AT activation [60]. Tacrolimus has, like cyclosporin A, been shown to reduce TNFa production by certain cell types such as proximal tubular epithelial cells [61]. It is not clear whether the suppression of pro-inflammatory cytokines is due to the inhibition of calcineurin or if other modes of action are involved.
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4.3. Sirolimus (rapamycin) Sirolimus was approved by the US Food and Drug Administration (FDA) in autumn 1999. Like tacrolimus, sirolimus is a macrolide antibiotic; the latter is derived from Streptomyces hygroscopicus [62]. Sirolimus is much less selective in its modes of action than cyclosporin A or tacrolimus, and it acts at a much later stage in the inhibition of T-cell proliferation than tacrolimus [63]. Like tacrolimus, sirolimus binds to FKBP, although the sirolimus–FKBP complex does not inhibit calcineurin but another intracellular protein termed mammalian target of rapamycin (mTOR). This protein takes part in cell cycle regulation. In a whole blood model, LPS-stimulated TNFa production was left unaffected by sirolimus at a concentration of 10 and 500 ng / ml, respectively [64]. Furthermore, the secretion of IL-10 was significantly reduced by 70% in the presence of sirolimus. These findings were in complete contrast to those for tacrolimus and cyclosporin A from the same study, which were able to inhibit TNFa secretion but did not alter IL-10 secretion [64].
5. Immunomodulatory cytokines
5.1. Interleukin-10 Some cytokines possess immunosuppressive properties. The most important one is probably IL-10, which was first described in 1989 as a cytokine synthesis inhibitory factor [65]. IL-10 is produced relatively late following activation of monocytes / macrophages or T cells, and therefore IL-10 is assumed to be a natural dampener of the immune response [66]. Binding of IL-10 to its receptor activates at least two different tyrosine kinases, which in turn activate the transcription factors STAT1 and STAT3, and in some cell types STAT5 [67]. IL-10 is highly potent at the suppression of TNFa, IL-1b and IL-6 expression by monocytes stimulated with LPS. Therefore, it can effectively inhibit the acute phase response [68]. Its transcription is, on the other hand, strongly induced by TNFa by a mechanism which seems to be independent of NF-kB [69], although some workers found an inhibition of NF-kB by IL-10 as well. IL-10 blocks TNFa actions by down-regula-
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tion of TNFa surface receptors and by increasing the secretion of soluble TNFa receptors (Fig. 3) [70]. Infection of mice with Toxoplasma gondii led to an induction of acute phase response and weight loss together with increased serum levels of TNFa and IL-10 [71]. IL-10 gene transfer into tumor-bearing mice prevents the occurrence of cachexia and inhibits the production of IL-6 at the tumor site [72]. In human cachexia a potential benefit of IL-10 treatment has not been evaluated, although it has proven clinically beneficial in several immunologic disorders [73,74].
5.2. Transforming growth factor-b Transforming growth factor-b (TGF-b) is another cytokine with immunosuppressive properties. It has a crucial role in the development, homeostasis and repair of virtually all tissues with its effects highly dependent on the actual responsiveness of the target cells. The principal action of TGF-b is the inhibition of lymphocyte and other leukocyte activation, but it also stimulates synthesis and secretion of extracellular matrix proteins. TGF-b receptor activity has been shown to phosphorylate SMAD proteins, which signal into the nucleus [75]. Some evidence points to the fact that SMAD proteins are degraded in the proteasome [76]. TGF-b is able to block TNFa and IL-6 production in a model using LPS-stimulated whole blood [77]. In contrast, TGF-b injection into mice yielded progressive suppression of erythropoiesis, which could be prevented by erythropoietin [78]. In this model the suppression of erythropoiesis was associated with increased plasma levels of TNFa along with progressive cachexia. In another study in nude mice, the injection of more than 2 mg / day TGF-b led to generalized intestinal fibrosis and cachexia, which was not accompanied by elevated TNFa levels [79]. Therefore, the impact of TGF-b in the treatment of cachexia may be limited.
5.3. Interleukin-4 and interleukin-13 IL-4 and IL-13 are closely related. IL-4 mainly stimulates the development of type 2 helper T cells ¨ T cells. Nevertheless, both IL-4 and IL-13 from naıve
have profound effects on morphology, surface receptor expression and cytokine synthesis of monocytes / macrophages. In LPS-stimulated monocytes, they inhibit the production of IL-1b, IL-6, IL-8 and other pro-inflammatory cytokines [68]. Signal transduction of both IL-4 and IL-13 involves activation of STAT6 [80]. A role for IL-4 in the development of cachexia has been highlighted using a model with IL-4 knock-out mice [81]. Following infection with Schistosoma mansoni these mice demonstrated with systemic illness characterized by cachexia and lethargy, and in severe cases also by anorexia, alopecia, and periorbital edema. The extent of weight loss was significantly increased compared to uninfected IL-4 knock-out mice and wild-type mice. The mortality of the IL-4 knock-out mice was also increased. Studies on IL-4 or IL-13 in humans with cachexia have not been reported yet.
6. Thalidomide Thalidomide is a drug unfortunately associated with tragedy. It was first synthesized by Kunz in 1954 [82]. Thalidomide is used as a racemate, and the enantiomers are subject to fast chiral interconversion [83]. Thalidomide is eliminated almost exclusively by spontaneous hydrolysis in vivo. Malformation of the unborn, however, can be induced by a single dose of the substance [83]. Thalidomide is now known to inhibit selectively the production of TNFa by LPSstimulated human monocytes in vitro leaving other pro-inflammatory cytokines like IL-1b and IL-6 unaltered [84]. Therefore, thalidomide is frequently used in the treatment of erythema nodosum leprosum, a serious complication of leprosy thought to be mainly mediated by TNFa [85]. The inhibitory effect of thalidomide on TNFa production is attributed to an enhanced degradation of TNFa mRNA [86]. Thalidomide has other modes of action which include the upregulation of the secretion of IL-2, IL-4 and IL-5, the inhibition of mitogen-stimulated peripheral mononuclear cells and the enhancement of T-cell responses. Some amino-substituted thalidomide analogues may have fewer side effects and an even greater TNFa suppression potential than the unsubsti-
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tuted drug. Most recently, thalidomide has been proposed as a therapy for chronic heart failure, which frequently is accompanied by cachexia [87]. In a placebo-controlled study in patients with acute pulmonary tuberculosis, either human immunodeficiency virus positive or negative, thalidomide led to significant weight gain [88].
7. Conclusion The complex pathophysiology leading to cachexia is poorly understood. A specific mediator of cachexia has so far not been described. A 24 kDa glycoprotein has recently been identified which seems to trigger weight loss in an animal model of cancer [89,90]. This factor shares physical characteristics with a human molecule of the same molecular weight, which induces skeletal muscle proteolysis. It has therefore been termed proteolysis-inducing factor (PIF). In patients with pancreatic cancer the rate of weight loss was greatest in those whose urine contained the protein [91]. Whether or not this substance shares certain characteristics with or is induced by TNFa remains to be determined. Evidence suggests that TNFa is a key mediator in the development of cachexia, and in cardiac cachexia TNFa is the strongest predictor of the degree of previous weight loss [92]. Suppression of TNFa production, biological activity and downstream signalling processes may therefore be beneficial in the treatment of cachexia. Conventional immunosuppressive drugs and some immunomodulatory cytokines primarily target the production of TNFa. Neutralizing anti-TNFa antibodies and soluble receptor constructs specifically block its biological activity. The suppression of the intracellular signal transduction of TNFa may, however, serve as a new therapeutic intervention to stop the progression of cachexia. Therefore, the role of certain drugs remains to be elucidated in the treatment of this syndrome: whereas proteasome inhibitors not only inhibit NF-kB signalling but also block the wasting process, other drugs like glucocorticoids mainly interfere with NF-kB and AP-1 signalling. Thalidomide finally may serve as a specific TNFa-blocking drug.
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Acknowledgements We are indebted to Dr. Paul Kalra for assistance in correcting the English syntax.
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Cachexia: a therapeutic approach beyond cytokine antagonism S. von Haehling a
a,b ,
*, S. Genth-Zotz a,c , S.D. Anker a,b , H.D. Volk d
Department of Clinical Cardiology, National Heart & Lung Institute, Royal Brompton Hospital, Dovehouse Street, London SW3 6 LY, UK b ¨ Centrum for Molecular Medicine, Berlin, Germany Franz Volhard Klinik ( Charite´ , Campus Berlin-Buch) at Max Delbruck c Department of Medicine II, Johannes Gutenberg-University, Mainz, Germany d Department of Medical Immunology, Charite´ Medical School, Berlin, Germany
Abstract Cachexia is seen in a number of chronic diseases, and it is always associated with a poor prognosis. Irrespective of etiology, the development of cachexia appears to share a common pathophysiological pathway. This includes induction of proteasome-dependent myofibril-degradation, which is thought to be secondary to stimulation by enhanced levels of pro-inflammatory cytokines. Elevation of tumor necrosis factor-a (TNFa) and other plasma cytokines has been demonstrated in many conditions associated with cachexia. Despite improved pathophysiological understanding, a specific treatment for cachexia has not yet been established. Whilst direct TNFa antagonism has therapeutic appeal, this review will focus on manipulation of downstream pathways and the potential benefits. For example, nuclear factor-kB (NF-kB) is one of the most important signal transducers of TNFa, and drugs targeting this signalling cascade might be useful in the treatment of cachexia. Although the use of some of these substances, for example glucocorticoids, remains controversial, others may prove beneficial in the treatment of this syndrome. The role of other approaches such as proteasome-inhibitors remains to be elucidated. Alternatively, interleukin-10 and other immunosuppressive cytokines may also be able to counterbalance certain features of cachexia. 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cachexia; Tumor necrosis factor; Immunosuppression; NF-kB
1. Introduction Cachexia is a common feature of a number of different illnesses, such as cancer, sepsis, chronic heart failure, rheumatoid arthritis and AIDS. It is always associated with poor prognosis. Although cachexia was first described more than 2000 years ago and has been subject to increasing research in recent decades, there is still considerable disagreement even on the question of its definition [1,2]. There is no established therapy that can reverse cachexia. The development of successful therapies *Corresponding author. Tel.: 144-207-351-8127; fax: 144-207-3518733. E-mail address: [email protected] (S. von Haehling).
requires a detailed understanding of the pathophysiology of wasting in chronic illnesses. Irrespective of underlying disease etiology the development of cachexia may share final common pathways. In addition, characteristic immunologic and metabolic abnormalities are seen in all forms of this syndrome. These pathways may offer promising targets for therapy aimed at improving the adverse morbidity and mortality associated with cachexia. Characteristic features of cachexia include the activation of the immune system [3] and muscle wasting through the ubiquitin–proteasome pathway [4]. This review will discuss potential therapeutic approaches beyond the direct antagonism of TNFa and other pro-inflammatory cytokines. Targets include downstream cytokine signalling mechanisms
0167-5273 / 02 / $ – see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 02 )00245-0
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through transcription factors such as nuclear factorkB (NF-kB) and activation protein-1 (AP-1) by immunosuppressive agents including immunomodulatory cytokines, e.g. interleukin (IL)-4, IL-10, IL-13, and transforming growth factor-b (TGF-b), and experimental pharmacologic approaches like proteasome inhibitors.
Elevation of circulating pro-inflammatory cytokines is a common feature of cachexia [5–9]. It is currently accepted that tumor necrosis factor-a (TNFa) plays the key role in the development of this condition. TNFa was first described for its ability to induce weight loss and anorexia in mice and thus termed cachectin [10]. The syndrome was reversed when the injection of TNFa was discontinued. In chronic heart failure, increased levels of TNFa relate to reduced peripheral blood flow [11], apoptosis [12] and lower skeletal muscle mass [13]. In addition, pro-inflammatory cytokines relate to prognosis in chronic heart failure independently of whether cachexia is present or not [14]. In recent years it became clear that other pro-inflammatory mediators, particularly IL-1b and IL-6, might also play an important part in the development of the syndrome [15]. The major sources of these pro-inflammatory cytokines are monocytes and macrophages, although other cell types, such as T lymphocytes or even non-immunological cells like endothelial cells are also capable of their production. The precise mechanism that leads to the secretion of these mediators and ultimately to cachexia is still under investigation and so far only partly understood. Whilst chronic inflammation seems to play a major role in the development of cachexia, abnormalities in other systems may also be important. These include mediators such as catecholamines, steroid hormones and leptin which act either directly or through interaction with pro-inflammatory cytokines [16].
potently inducing this reaction [17]. Maintaining the acute phase response requires an excess of essential amino acids which yields loss of body proteins [18]. Since skeletal muscle accounts for almost half of the body protein mass, this compartment is intensively affected (Fig. 1). Thus, muscle wasting can be initiated by the acute phase response. The predominant pathway of protein turnover in all eukaryotic cell types is the ubiquitin–proteasome pathway, which requires adenosine triphosphate (Fig. 2). Ubiquitin serves as a cofactor that is covalently linked to proteins to be degraded in the 26S proteasome complex. This multisubunit protease specifically degrades ubiquitin-conjugates, and it is the main mediator of muscle wasting in man (Fig. 1) [4]. The impact of protein degradation through the ubiquitin– proteasome pathway has been demonstrated in vivo for cachexia in AIDS [19], sepsis [20], cancer [21], and renal failure [22]. The ubiquitin–proteasome system is present in both the nucleus and the cytosol. Alterations in the activity of this pathway are probably due to changes in the rate of ubiquitin conjugations, and several hormonal and immunological factors seem to be involved in a regulatory fashion. The proteasome is responsible for degradation of proteins from the intracellular compartment that are linked to ubiquitin, while lysosomes degrade proteins from the extracellular compartment. It is not fully understood how ubiquitin-binding to intracellular proteins is regulated, although Varshavsky reported that the half-life of a protein correlates with certain features of its N-terminal sequence [23]. Cytokine signals including TNFa, IL-1 and IL-6 [24–26] stimulate the ubiquitin–proteasome pathway in muscle. However, there is some evidence that these signals may be secondary to the influence of cytokine-induced glucocorticoids [27]. The finding that protein degradation by the proteasome does not yield free amino acids but peptides, suggests that a number of other factors may also be important in the regulation of muscle protein breakdown [28].
2.1. From acute phase response to tissue wasting
2.2. Proteasome inhibitors
Inflammation as well as tissue injury induce a specific reaction of the body known as the acute phase response, and IL-6 is one of the mediators most
The first proteasome inhibitors became available in 1994. All experiments using these agents have thus far been performed in vitro and in animal models.
2. Cytokines and the proteasome complex
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Fig. 1. Mechanism of muscle wasting in man: an unknown stimulus, possibly TNFa binding to TNFa-receptors, causes covalent binding of ubiquitin to certain myofibrils. Following ubiquitin-labelling, these proteins are directed to the proteasome-complex. The proteasome releases peptides, which are further broken down into free amino acids by several mechanisms that have not yet been clearly identified. Afterwards, these amino acids are, for example, available for protein synthesis in acute phase response in the liver. Certain pro-inflammatory cytokines such as IL-1, IL-6 and TNFa are known to induce proteasome activity while proteasome inhibitors block it.
Very recently, phase I trials with proteasome inhibitors were started. Four classes of proteasome inhibitors have been described [29,30]: (i) peptide aldehydes primarily inhibit the chymotrypsin-like activity of the proteasome, which is one of its specific proteolytic sites. Removing the peptide aldehyde restores the proteolytic activity. In a model using rat skeletal muscle, these agents had up to 52% greater effect on proteolysis in atrophying muscle than on controls [31]. The overall protein synthesis remained unchanged. Unfortunately, these agents usually require 10–20 h until onset of action [32]. (ii) Lactacystin and its active derivative b-lactone are more specific, but irreversible inhibitors of the proteasome. They act as pseudosubstrates that are covalently bound to one of the subunits of the proteasome [33]. (iii) Vinyl sulfone has been shown to inhibit the proteasome complex irreversibly in a similar manner to lactacystin [29]. In a human lymphoma cell line prolonged inhibition of the proteasome by vinyl sulfone led to the appearance of cell variants with a distinct proteolytic system [34]. (iv) Finally, dipep-
tide boronic acid analogs have been shown to block proteasome activity via reversible binding to its active sites. Nevertheless, nonspecific drugs like b 2 agonists also have the potential to suppress ubiquitin– proteasome-dependent proteolysis during tumor growth. This was recently demonstrated in tumorbearing rats using clenbuterol at a dose of 1 mg / kg body weight daily [35]. Peptide aldehydes, lactacystin and b-lactone have been shown to block up to 90% of the degradation of abnormal proteins and short-lived proteins in the cell [29]. A substance that can specifically block myofibril degradation in skeletal muscle is still waiting to be discovered.
3. Intracellular suppression of TNFa signal transduction The transcription factors nuclear factor-kB (NFkB) and activation protein-1 (AP-1) are found in several cell types. Currently, these proteins are seen
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Fig. 2. Mechanism of protein degradation by the proteasome. It represents the proteolytic mechanism which selectively degrades most proteins of intracellular origin while proteins from the extracellular compartment are degraded by lysosomes. (A) The activation of NF-kB involves degradation of its inhibitory protein, IkB, by the proteasome. Therefore, IkB is marked for degradation by a poly-ubiquitin chain. This ubiquitinylation process requires enzymatic activity of three different enzymes, termed E1, E2 and E3. These enzymes transfer activated ubiquitin to the selected protein. (B) IkB is then rapidly unfolded and degraded in the proteasome, a process requiring adenosine triphosphate (ATP). NF-kB can therefore signal into the nucleus, and transcription of the affiliated genes is induced.
as the most important signal transducers for proinflammatory stimuli. Both are activated following TNFa trimer binding to the TNFa receptor complex (particularly the type I p55 receptor). The complex interaction of these and other transcription factors is responsible for the inhibition or enhancement of certain genes. In general, a coincident activation of several transcription factors is necessary for maximal gene expression [36].
3.1. NF-k B-dependent signalling NF-kB was first described in 1986 as being necessary for immunoglobulin kappa light chain transcription in B cells [37]. It is a heterodimer
consisting of two subunits. In unstimulated cells, cytoplasmatic NF-kB is bound to its inhibitory protein, IkB (Fig. 2). Therefore, it is kept in an inactivated form in the cytoplasm with its nuclear localization signal being masked [38]. Different stimuli such as TNFa, IL-1, bacterial lipopolysaccharide (LPS) or UV light lead to NF-kB activation [39]. Upon activation, IkB is phosphorylated and rapidly degraded in the ubiquitin–proteasome pathway (Fig. 2). The free NF-kB complex can translocate into the nucleus, where NF-kB binds to kB DNA sequences (NF-kB responding elements) in non-coding regions of several ‘acute response’ genes thus promoting the transcription of the affiliated genes (Fig. 3). Interestingly, NF-kB binding sites are found
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Fig. 3. Interactions of different intracellular signalling mechanisms for TNFa. One of the major components activated upon TNFa binding to its receptor is NF-kB, which is kept in an inactive form masked by IkB in the cytoplasm. Binding of other substances like bacterial lipopolysaccharide (LPS) to CD14 / Toll-like receptor (TLR4) or IL-6 to its receptor are also known to stimulate NF-kB activation. IkB is rapidly degraded in the proteasome, and NF-kB binds to promotors of several genes. For example, TNFa and IkB transcription are induced with the latter finally shutting off the signal of NF-kB. Glucocorticoids (GC) can prevent NF-kB from inducing the transcription of certain genes. Therefore, they bind to glucocorticoid-receptors (GR) in the cytoplasm, which serve as transcription factors. IL-10 signalling through STAT1 and STAT3 induces, for example, TNFa receptor release (sTNF-R) which bind TNFa in the plasma and thus inactivate it. IL-10 can also suppress NF-kB signalling.
in a multitude of genes, coding for cytokines, acute phase response proteins, and cell adhesion molecules [39]. In addition, NF-kB up-regulates the transcription of its inhibitory protein IkB which finally shuts off the signal [40]. In principle, NF-kB-mediated signals can be suppressed by targeting IkB degradation, NF-kB translocation, or NF-kB DNA binding as well as by IkB overexpression. In fact, genetic overexpression of IkB blocks NF-kB dependent processes. Drugs targeting the translocation of activated NF-kB such as fumar acid have a high anti-inflammatory potency [41]. Most recently, activation of NF-kB by overexpression of an IkB phosphorylating kinase has been shown to sufficiently block myogenesis, illustrating
the link between NF-kB and cachexia development [42]. As IkB is degraded in the proteasome, the use of proteasome inhibitors is thought to suspend signal transduction via NF-kB into the nucleus. In that context it has to be kept in mind that complete inhibition of NF-kB has proven detrimental. Knockout mice models where the major subunits of NF-kB are targeted show severe immunodeficiency which was lethal in some cases [39]. Whilst the NF-kB pathway seems to be important for up-regulating TNFa and IL-1, the latter are also the most powerful endogenous inducers of NF-kB. In T cells, TNFa production is also controlled by T-cell receptor-mediated NF-AT (nuclear factor of activated T cells) activation. In particular, the p38 mitogen-activated
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protein kinase (MAPK) kinase pathway controls the translation of TNFa.
3.2. AP-1 -dependent signalling In comparison to NF-kB, the AP-1-dependent signalling pathway is less well understood. AP-1 forms a family of transcription factors binding to a specific palindromic DNA sequence [43]. Its activity is found in nearly all cell types. Some evidence has accumulated in the past few years that AP-1 functions as an effector of growth factor signalling, although cytokines, particularly TNFa and IL-1, are also potent activators of AP-1. A possible role of AP-1 inhibition for the treatment of cachexia has as yet not been investigated.
4. Immunosuppressive drugs
4.1. Glucocorticoids Glucocorticoids are widely used in the treatment of inflammatory and immune diseases. In cancer cachexia, they have been used to stimulate food intake. Indeed, they seem to mitigate anorexia and asthenia [44]. The anti-inflammatory action of these substances, however, is mainly due to inhibition of NF-kB and AP-1 activation (Fig. 3) [45]. Glucocorticoids freely penetrate cell membranes to bind to the cytosolic glucocorticoid receptor, which serves as a transcription factor. Upon translocation to the nucleus, anti-inflammatory properties of glucocorticoids are initiated due to negative regulation of genes encoding pro-inflammatory cytokines. However, antiinflammatory properties are not strictly dependent on DNA binding of the hormone–receptor complex. In contrast, side effects are mainly due to the transactivating characteristics of glucocorticoid receptors that require DNA binding. This discrepancy opens new possibilities for developing novel glucocorticoids with less side effects. Recently, two interesting studies have suggested that a major mechanism of glucocorticoid action might be the induction of IkB synthesis [46,47]. Moreover, IkB might even be able to remove NF-kB
actively from its DNA binding site. Another mode of glucocorticoid action involves direct protein–protein interaction [48]. Certain transcription factors like NFkB, AP-1 and STAT5 from the signal transducers and activators of transcription (STAT) family have been found to be bound by glucocorticoid-receptors [45]. Therefore, pro-inflammatory genes might be suppressed due to this interaction by masking of transactivating domains. For dexamethasone, a highly potent synthetic glucocorticoid, blocking of the TNFa and IL-1-dependent induction and translocation of NF-kB could be demonstrated [49]. Dexamethasone also inhibits the LPS-induced activation of NF-kB [47]. Whilst glucocorticoid therapy might theoretically be of benefit for the treatment of cachexia, prolonged use of these substances may lead to weakness, osteoporosis, diabetes and even delirium. Therefore, the routine use of these drugs remains controversial, and their role in the treatment of cachexia is far from being established. Novel glucocorticoids without transactivating properties increase the benefit / side effect ratio despite some reduction in their antiinflammatory properties (e.g. no IkB induction, unpublished data). This novel class of glucocorticoids may open new therapeutic opportunities in long-term treatment of cachectic patients.
4.2. Calcineurin inhibitors Cyclosporin A and tacrolimus share similar modes of action, although their chemical structure is highly different. Both are immunosuppressive agents used in the treatment of transplantation and certain autoimmune disorders, and both pass freely through cell membranes. Tacrolimus is more potent than cyclosporin A. Unfortunately, both cyclosporin A and tacrolimus share a narrow range between subtherapeutic and toxic plasma concentration, and therefore frequent drug monitoring is required [50]. Both drugs are nephrotoxic. Another substance with similar immunosuppressive attributes is sirolimus, although it is not a member of the calcineurin inhibitor family per se. The immunosuppressive properties of cyclosporin A were first recognised in 1976 [51]. Cyclosporin A is a small peptide from the fungus Hypocladium inflatum gams widely used in the suppression of
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allograft rejection. Its major targets are T cells [52]. In 1984 one of its modes of action became apparent when its ability to inhibit T cell activation was demonstrated. In these cells, cyclosporin A strongly binds cyclophilins that are involved in protein folding. Cyclosporine–cyclophilin complexes bind to and inhibit calcineurin. Thus, the activation and nuclear translocation of NF-AT, which is involved in IL-2, IL-4 and TNFa transcription, is inhibited [53]. The effects of cyclosporin A on other cell types are less obvious. In a model using a murine macrophage cell line, cyclosporin A acts as an uncompetitive inhibitor of the proteasome, and it suppresses the LPS-induced IkB degradation [54]. Therefore, inhibition of proteasome activity seems to be the mechanism by which cyclosporin A prevents NF-kB activation. Other workers have confirmed the inhibition of NF-kB activation [55]. This mechanism may explain the suppression of pro-inflammatory cytokine production reported in several studies: in an animal model comparing euthymic and athymic mice, cyclosporin A dose-dependently inhibited the production of IL-1b and TNF [56]. The production of IL-6 was blocked only in euthymic mice suggesting that the suppression of its production requires an effective T-cell response. Other workers have at least partly confirmed these data. In U937 monocyte cells, cyclosporin A reduced the secretion of IL-1b, IL-6, IL-8 and TNFa [57], and in human alveolar macrophages cyclosporin A was able to inhibit the production of IL-8 and TNFa as well [58]. In contrast to glucocorticoids, however, it is less efficient in blocking TNFa-mediated effects. A further study demonstrated that cyclosporin A injected into rats with inflammation-induced cachexia prevented the sustained loss of body weight and adipose tissue [59]. Tacrolimus (FK506) is a macrolide antibiotic extracted from Streptomyces tsukubaensis. Like cyclosporin A, tacrolimus mainly inhibits T cell activation via association with calcineurin. It binds to FK506 binding protein (FKBP), and the FKBP– calcineurin complex inhibits NF-AT activation [60]. Tacrolimus has, like cyclosporin A, been shown to reduce TNFa production by certain cell types such as proximal tubular epithelial cells [61]. It is not clear whether the suppression of pro-inflammatory cytokines is due to the inhibition of calcineurin or if other modes of action are involved.
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4.3. Sirolimus (rapamycin) Sirolimus was approved by the US Food and Drug Administration (FDA) in autumn 1999. Like tacrolimus, sirolimus is a macrolide antibiotic; the latter is derived from Streptomyces hygroscopicus [62]. Sirolimus is much less selective in its modes of action than cyclosporin A or tacrolimus, and it acts at a much later stage in the inhibition of T-cell proliferation than tacrolimus [63]. Like tacrolimus, sirolimus binds to FKBP, although the sirolimus–FKBP complex does not inhibit calcineurin but another intracellular protein termed mammalian target of rapamycin (mTOR). This protein takes part in cell cycle regulation. In a whole blood model, LPS-stimulated TNFa production was left unaffected by sirolimus at a concentration of 10 and 500 ng / ml, respectively [64]. Furthermore, the secretion of IL-10 was significantly reduced by 70% in the presence of sirolimus. These findings were in complete contrast to those for tacrolimus and cyclosporin A from the same study, which were able to inhibit TNFa secretion but did not alter IL-10 secretion [64].
5. Immunomodulatory cytokines
5.1. Interleukin-10 Some cytokines possess immunosuppressive properties. The most important one is probably IL-10, which was first described in 1989 as a cytokine synthesis inhibitory factor [65]. IL-10 is produced relatively late following activation of monocytes / macrophages or T cells, and therefore IL-10 is assumed to be a natural dampener of the immune response [66]. Binding of IL-10 to its receptor activates at least two different tyrosine kinases, which in turn activate the transcription factors STAT1 and STAT3, and in some cell types STAT5 [67]. IL-10 is highly potent at the suppression of TNFa, IL-1b and IL-6 expression by monocytes stimulated with LPS. Therefore, it can effectively inhibit the acute phase response [68]. Its transcription is, on the other hand, strongly induced by TNFa by a mechanism which seems to be independent of NF-kB [69], although some workers found an inhibition of NF-kB by IL-10 as well. IL-10 blocks TNFa actions by down-regula-
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tion of TNFa surface receptors and by increasing the secretion of soluble TNFa receptors (Fig. 3) [70]. Infection of mice with Toxoplasma gondii led to an induction of acute phase response and weight loss together with increased serum levels of TNFa and IL-10 [71]. IL-10 gene transfer into tumor-bearing mice prevents the occurrence of cachexia and inhibits the production of IL-6 at the tumor site [72]. In human cachexia a potential benefit of IL-10 treatment has not been evaluated, although it has proven clinically beneficial in several immunologic disorders [73,74].
5.2. Transforming growth factor-b Transforming growth factor-b (TGF-b) is another cytokine with immunosuppressive properties. It has a crucial role in the development, homeostasis and repair of virtually all tissues with its effects highly dependent on the actual responsiveness of the target cells. The principal action of TGF-b is the inhibition of lymphocyte and other leukocyte activation, but it also stimulates synthesis and secretion of extracellular matrix proteins. TGF-b receptor activity has been shown to phosphorylate SMAD proteins, which signal into the nucleus [75]. Some evidence points to the fact that SMAD proteins are degraded in the proteasome [76]. TGF-b is able to block TNFa and IL-6 production in a model using LPS-stimulated whole blood [77]. In contrast, TGF-b injection into mice yielded progressive suppression of erythropoiesis, which could be prevented by erythropoietin [78]. In this model the suppression of erythropoiesis was associated with increased plasma levels of TNFa along with progressive cachexia. In another study in nude mice, the injection of more than 2 mg / day TGF-b led to generalized intestinal fibrosis and cachexia, which was not accompanied by elevated TNFa levels [79]. Therefore, the impact of TGF-b in the treatment of cachexia may be limited.
5.3. Interleukin-4 and interleukin-13 IL-4 and IL-13 are closely related. IL-4 mainly stimulates the development of type 2 helper T cells ¨ T cells. Nevertheless, both IL-4 and IL-13 from naıve
have profound effects on morphology, surface receptor expression and cytokine synthesis of monocytes / macrophages. In LPS-stimulated monocytes, they inhibit the production of IL-1b, IL-6, IL-8 and other pro-inflammatory cytokines [68]. Signal transduction of both IL-4 and IL-13 involves activation of STAT6 [80]. A role for IL-4 in the development of cachexia has been highlighted using a model with IL-4 knock-out mice [81]. Following infection with Schistosoma mansoni these mice demonstrated with systemic illness characterized by cachexia and lethargy, and in severe cases also by anorexia, alopecia, and periorbital edema. The extent of weight loss was significantly increased compared to uninfected IL-4 knock-out mice and wild-type mice. The mortality of the IL-4 knock-out mice was also increased. Studies on IL-4 or IL-13 in humans with cachexia have not been reported yet.
6. Thalidomide Thalidomide is a drug unfortunately associated with tragedy. It was first synthesized by Kunz in 1954 [82]. Thalidomide is used as a racemate, and the enantiomers are subject to fast chiral interconversion [83]. Thalidomide is eliminated almost exclusively by spontaneous hydrolysis in vivo. Malformation of the unborn, however, can be induced by a single dose of the substance [83]. Thalidomide is now known to inhibit selectively the production of TNFa by LPSstimulated human monocytes in vitro leaving other pro-inflammatory cytokines like IL-1b and IL-6 unaltered [84]. Therefore, thalidomide is frequently used in the treatment of erythema nodosum leprosum, a serious complication of leprosy thought to be mainly mediated by TNFa [85]. The inhibitory effect of thalidomide on TNFa production is attributed to an enhanced degradation of TNFa mRNA [86]. Thalidomide has other modes of action which include the upregulation of the secretion of IL-2, IL-4 and IL-5, the inhibition of mitogen-stimulated peripheral mononuclear cells and the enhancement of T-cell responses. Some amino-substituted thalidomide analogues may have fewer side effects and an even greater TNFa suppression potential than the unsubsti-
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tuted drug. Most recently, thalidomide has been proposed as a therapy for chronic heart failure, which frequently is accompanied by cachexia [87]. In a placebo-controlled study in patients with acute pulmonary tuberculosis, either human immunodeficiency virus positive or negative, thalidomide led to significant weight gain [88].
7. Conclusion The complex pathophysiology leading to cachexia is poorly understood. A specific mediator of cachexia has so far not been described. A 24 kDa glycoprotein has recently been identified which seems to trigger weight loss in an animal model of cancer [89,90]. This factor shares physical characteristics with a human molecule of the same molecular weight, which induces skeletal muscle proteolysis. It has therefore been termed proteolysis-inducing factor (PIF). In patients with pancreatic cancer the rate of weight loss was greatest in those whose urine contained the protein [91]. Whether or not this substance shares certain characteristics with or is induced by TNFa remains to be determined. Evidence suggests that TNFa is a key mediator in the development of cachexia, and in cardiac cachexia TNFa is the strongest predictor of the degree of previous weight loss [92]. Suppression of TNFa production, biological activity and downstream signalling processes may therefore be beneficial in the treatment of cachexia. Conventional immunosuppressive drugs and some immunomodulatory cytokines primarily target the production of TNFa. Neutralizing anti-TNFa antibodies and soluble receptor constructs specifically block its biological activity. The suppression of the intracellular signal transduction of TNFa may, however, serve as a new therapeutic intervention to stop the progression of cachexia. Therefore, the role of certain drugs remains to be elucidated in the treatment of this syndrome: whereas proteasome inhibitors not only inhibit NF-kB signalling but also block the wasting process, other drugs like glucocorticoids mainly interfere with NF-kB and AP-1 signalling. Thalidomide finally may serve as a specific TNFa-blocking drug.
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Acknowledgements We are indebted to Dr. Paul Kalra for assistance in correcting the English syntax.
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