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New cytokine therapeutics for inflammatory bowel disease
www.elsevier.com/locate/issn/10434666 Cytokine 28 (2004) 167e173
New cytokine therapeutics for inflammatory bowel disease P.C.F. Stokkers*, D.W. Hommes Department of Gastroenterology and Hepatology, C2-111, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands Received 24 February 2004; accepted 21 July 2004
Abstract Conventional therapy for inflammatory bowel diseases rely on corticosteroids and 5-aminosalicylates combined with immunosuppressive agents for maintenance. These drugs are not always effective and may inflict serious side effects. Other therapies are therefore awaited. Infliximab, a monoclonal antibody against the pro-inflammatory cytokine TNF-alpha has been successfully applied as a treatment for Crohn’s disease. The mechanism of action of this drug extends beyond the level of TNF-alpha scavenging and includes induction of apoptosis of effector cells. Numerous anti-TNF antibodies have been developed and are currently evaluated in clinical trials. Other targets for monoclonal antibodies include integrins and cytokines involved in T-cell differentiation and activation. Likewise recombinant proteins that moderate TNF bioactivity and lymphocyte function have been developed. The therapeutic effect of recombinant interleukin-10 seems to be dependent on local delivery of the protein. Antisense therapy targeting lymphocyte migration has also been tested in IBD. Finally, the conventional drug thalidomide and possibly MAPkinase inhibitors may become novel treatment entities for IBD. Ó 2004 Published by Elsevier Ltd. Keywords: IBO; Crohn’s disease; Ulcerative colitis; Treatment; Biologicals
1. Introduction Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract, which clinically present as one of two disorders, Crohn’s disease (CD) or ulcerative colitis (UC). The inflammation of the intestinal mucosa in IBD is characterized by an influx of neutrophils and macrophages that produce cytokines, eicosanoids, proteolytic enzymes and free radicals that lead to inflammation and ulceration. Hence, IBD patients suffer from diarrhea, rectal blood loss, abdominal pain and weight loss. Complications of disease occur as a toxic megacolon, perforation, malignant transformation and in the case of CD strictures and fistulas [19,43]. The conventional therapies including corticosteroids and 5-aminosalicylates target the inflammatory process * Corresponding author. E-mail address: [email protected] (P.C.F. Stokkers). 1043-4666/$ - see front matter Ó 2004 Published by Elsevier Ltd. doi:10.1016/j.cyto.2004.07.012
on different levels. 5-Aminosalicylates are known to influence the production of prostaglandins and leukotrienes, neutrophil chemotaxis and the scavenging of reactive oxidative radicals. The effects of corticosteroids are even more pleiotropic and only partially understood. Both therapies are effective for induction of disease remission, corticosteroids being the most potent [19,43]. However, 30% of the patients fail to respond on corticosteroids and the side effects of the drug prohibit long term or maintenance treatment [36]. Immunosuppressives such as azathioprine and methotrexate are used for patients dependent on steroids or with steroid refractory disease [12,20]. New therapeutic strategies are therefore awaited and subject of this overview. 2. Immune response and inflammatory pathways The interplay between the immune apparatus and the gut lumen defines mucosal inflammation in IBD. A genetic predisposition for developing an inadequate
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immune response towards some luminal factor seems the underlying cause of the disease [26,74]. This paradigm has been enforced by the identification of Crohn’s disease related mutations in the NOD2 gene [25,39]. NOD2 is a receptor for a pattern associated with microbial pathogens (PAMP) and a family member of several other PAMP-receptors. These receptors bind specific microbial component such as lipopolysaccharide and are mainly expressed in antigen presenting cells (APC). Thus, PAMP-receptors are part of the innate immune system by mediating the initial recognition of pathogens [28,40]. The question is how NOD2 variants lead to the defective peripheral tolerance that is observed in CD. Enterocytes, macrophages, dendritic cells and T-cells all express NOD2 and may have a role in disease development. Dendritic cells (DCs) have an important function at the interface between the innate and adaptive immune systems. We have recently identified two mutually exclusive mucosal DC subpopulations one of which is located directly below the mucosal surface [60]. Although the role of this so-called DC-SIGN-positive subpopulation remains illusive, we speculate that these cells sample the intestinal flora by protruding extensions between the epithelial monolayer [66]. Thus, a first line of defence is warranted by a continuous presentation of antigens via HLA-class 2 molecules to the T-cell receptor. Normally, in the case of indigenous flora, this event should lead to tolerance e.g. induction of anergy of the specific T-cell. Only in case of a pathogenic intruder the DCs become activated expressing co-stimulatory molecules on the membrane that, together with the T-cell receptor complex and the HLA-class 2 molecule, form the immunological synapses. Depending on the type of co-stimulation and cytokine release by the DCs T-cells are activated and differentiate towards a T-helper 1 or T-helper 2 phenotype [4,29,46,66]. A defect in this pathway was recently shown in Stat3 deficient mice, which suffer from chronic enterocolitis and serve as an animal model for IBD. Stat3 deficient macrophages do not produce interleukin-10, which leads to a skewed Th1 immune response upon stimulation with bacterial components [59]. The enterocolitis seems to be induced via TLR-4, a PAMP-receptor and familiar to NOD2, and is mediated by an exaggerated IL-12p40 production by myeloid cells [30]. It is widely acknowledged that the immune response in UC is different from the one seen in CD. In CD the inflammatory reaction is mediated via T-helper 1 lymphocytes expressing interleukin-2 and interferon-g. In UC the mucosal inflammation seems to be T-helper 2 mediated [19,43]. The gut epithelium acts as a structural barrier against pathogens. It has been noted that the epithelium of IBD patients harbours more adherent and intra-epithelial bacteria than healthy controls [53,58]. Enterocytes can
also act as antigen presenting cells. Especially in intestinal infection enterocytes have been shown to express chemokines such as interleukin-8 via an NFkB regulated innate immune response [10,42]. Aberrant function of NOD2 in the enterocytes may very well be important for the pathogenesis of CD since NOD2 expression has been found in several epithelial cell lines and epithelial cells of healthy subject and IBD patients [22,44]. Recently several PAMP-receptors were shown to be expressed by T-regulatory (Treg) cells in mice [7]. Although no data are available for the expression of NOD2 this finding has important implications for future research; Treg cells induce anergy by down regulating T-cell effector function, inhibition of migration of DCs towards mesenteric lymph nodes, and inhibition of T-cell and DC activation. Treg cells responsive to selfantigens and bacterial antigens exert these actions by interleukin-10 and TGF-b [54]. LPS, for which TLR-4 serves as a receptor, promotes Treg cell survival and proliferation, providing a negative inhibition of the immune response [7]. The finding that Treg cells respond to inflammatory chemokines supports this notion [6,27]. Thus, Treg cells may prevent sustained response towards self or indigenous intestinal flora, a process which is hampered in Crohn’s disease [66]. However, preliminary data from our group show that PAMP-receptors including NOD2 are not expressed in human Treg cells (K. Kok et al. data on file). This difference with the murine situation is puzzling and should be further explored. Innate and adaptive immunity meet at different levels in the mucosal immune system. It has become clear that IBD results from a defect somewhere in the pathways between the two. Therapeutic interventions at the level of these pathways may prove more effective and safe than the conventional therapies for IBD.
3. New therapeutic compounds for IBD Fundamental research on the inflammatory pathways underlying IBD has boosted therapeutic interventions with compounds acting on different levels targeting. New therapies include both biological agents as chemically engineered agents. Both classed will be addressed hereafter.
3.1. Biologicals Biological compounds that are used for the treatment of IBD or are under current investigation in clinical trials are either monoclonal antibodies, or recombinant proteins or antisense nucleotides [50].
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3.1.1. Monoclonal antibodies The most prominent advocate of the new biological treatments is the monoclonal antibody infliximab. This chimeric humanemurine antibody has a human IgG1 Fc domain and a murine TNF recognition site [48]. In 1993 the first patient with steroid refractory Crohn’s disease was treated with this antibody showing a remarkable clinical and endoscopical response [8]. Many clinical trials followed and the treatment has now been registered for the treatment of steroid refractory disease and peri-anal fistulizing disease. However, the safety of the drug and the formation of antibodies against chimeric antibodies are issues of concern. Among the concern about safety was the finding of non-Hodgkin lymphomas (NHL) in 3 CD patients and 4 patients with rheumatoid arthritis treated with anti-TNF. For definite conclusions the power of the number of treated patients is too low and the follow up period too short. Furthermore, other immunosuppressive drugs that may confer risk on NHL were used by these patients. The immune suppressive action of the infliximab results in more serious infections [3] and patients should be checked for TBC by means of tuberculin skin testing before infusion of the drug [65]. The formation of human anti-chimeric antibodies (HACAs) has been reported in 13e68% of treated patients and is associated with a poor clinical response and infusion reactions [72]. Concurrent treatment with azathioprine or methotrexate may reduce the chance on HACA formation. The mechanism of action of infliximab remains illusive. Neutralization of TNF-alpha in the inflamed mucosa is unlikely to be a sufficient explanation since many other pro-inflammatory cytokines can do the job instead. Antibody dependent cytotoxicity, lysis of TNF producing cells by complement fixation or apoptosis induction of T-cells by the Fc portion of the antibody seem a more appropriate explanation [41,65]. Our group has focused on the latter possibility showing the effects of infliximab on apoptosis in CD3/CD28 stimulated Jurkat T-lymphocytes. We observed a significant increase of the ratio of Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic). As expected, this resulted in increased apoptosis of infliximab-treated Jurkat cells, but only when the cells were previously activated. Furthermore, we showed that the number of apoptotic T-cells in the mucosa of CD patient increase following treatment with infliximab and that the binding of infliximab to membrane bound TNF induces caspase3 regulated apoptosis of T-cells as well as monocytes [61,64]. CPD571 is an anti-TNF antibody which is supposed to be less immunogenic than infliximab, due to the fact that it is 95% human protein and 5% murine protein [50]. However, the Fc part of the antibody is of the IgG4 type, which does not posses the complement binding action of the Fc part of infliximab. Therefore, CPD571 may lack the apoptotic and cytotoxic properties of
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infliximab. Three small clinical trials with CPD571, showed encouraging results, and a large placebo controlled phase III trial is currently undertaken to prove efficacy of the drug [11,47,56]. Interfering on leukocyte trafficking natalizumab has been developed [50]. This humanized monoclonal antibody of the IgG4 class recognizes a4 integrin, that dimerizes with b1 unit (a4b1 or VLA-1) or with a b7 unit (a4b7 or MAdCAM-1) VLA-1 is up-regulated on the vascular endothelium at sites of inflammation, and ensures leukocyte rolling and adhesion, the first steps in diapedesis. MAdCAM-1 is also up-regulated on endothelium at sites of chronic inflammation [5,55]. Thus, natalizumab is supposed to block the migration of leukocytes to the inflamed mucosa in IBD. After a promising pilot study, recently the results of a placebo controlled double blind multi-center trial were published [14,17]. The primary end point of this study, remission of disease at the time point of 6 weeks, was not statistically significant compared to placebo. However, it was noted that in the placebo group significantly more patients had used concurrent immunosuppressive drugs when compared to the treatment group [33]. Treatment resulted in higher response rates in all treated groups, and these finding were also reflected in CRP levels and quality of life scores. A larger long term trial is awaited. LDP-2 is another integrin targeted monoclonal antibody. This IgG1 class antibody recognizes MAdCAM-1 and like natalizumab has proven to be an effective treatment for the spontaneous colitis that is seen in cotton top tamarins [21]. Any trials beyond phase II have not been reported so far. Other monoclonal antibodies that have proven efficacy in animal models target interleukin-12 (IL-12), interferon -g (IFN-g), interleukin-18 (IL-18) and the interleukin-2 receptor [50]. IL-12, IL- 18 and IFN-g all play an important role in T-cell differentiation. These cytokines are produced by antigen presenting cells, macrophages and activated lymphocytes and promote Th1 differentiation. In theory interferences on this level could modulate the Th1 biased immune response that is seen in CD. Interleukin-2 is an important proinflammatory cytokine that is produced by Th1 cells. Two antibodies (daclizumab and basiliximab) targeting the interleukin-2 receptor have been created and may be trialed in IBD patients [50]. All new therapeutic strategies based on monoclonal antibodies probably have the same drawbacks as infliximab: formation of antibodies, resulting in diminishing therapeutic efficacy and infusion reactions, high cost in producing the drugs and the necessity of intravenous dosing. In contrast, whilst the therapeutic efficacy of infliximab stretches beyond the scavenging TNF as described above, such mechanisms of action do not necessarily apply to other monoclonal antibodies. In this respect the results of trials with etanercept are
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telling. This genetically engineered fusion protein consists of two p75 TNF receptors mounted on an IgG1 Fc tail [48]. In theory this protein would induce less antibody formation since the protein is fully human. However, in a clinical trial the compound was as effective as placebo [49]. 3.1.2. Recombinant proteins Whilst the etanercept compound was based on recombinant proteins with antibody properties, other TNF scavengers that lack antibody structure have also been investigated. Onercept is a genetically engineered recombinant human p55 TNF receptor. Soluble TNF receptors have been shown to regulate TNF trafficking in chronic inflammatory conditions [48]. After a promising pilot study the compound is now being tested in a phase II clinical trial [45]. Interleukin-10 is a cytokine that has been shown to profoundly inhibit effector functions of activated macrophages, monocytes and dendritic cells [35]. Proinflammatory cytokines production by Th1 cells such as IL-2 and IFN-g is down-regulated by IL-10. In animal models, an important role for IL-10 was suggested by the finding that IL-10 knockout mice develop chronic intestinal inflammation [31] and the observation that IL-10 can prevent colitis that is induced by activated T-cells in a transfer model in mice that lack T-cells [2,18]. Therefore, IL-10 was suggested as a potential therapy for IBD. Encouraging results from pilot studies in IBD patients were tempered by subsequent larger trials [67]. IL-10 given subcutaneously did not prove better than placebo in disease remission induction and only showed a modest clinical response [13,51]. In addition, other studies have linked headache, fever and anemia with high systemic concentrations of IL-10 and suggested that this may be due to IL-10 induced elevation of IFN-g by whole blood cells [32,62,63]. IL-10 also seems to have immunostimulatory effects by up regulating HLA-class 2 molecules on B lymphocytes and by inducing cytotoxic T-cell differentiation [16,34]. Thus, the biology of this cytokine is complex, and the in vivo effects comprise both anti and pro-inflammatory actions. It could very well be that IL10 in higher systemic doses exerts an immunostimulatory effect. This may explain that patients receiving higher doses rhIL10 did worse in the trials. These data, together with the fact that the mucosal bioavailability of rhIL10 upon intravenous administration is probably low due to a rapid clearance, have prompted research towards other methods of more local administration. Van Montfrans et al. have shown that IL-10 delivery to intestinal mucosa in mice is feasible by the using Tlymphocytes that were transduced with a retrovirus construct expressing the cytokine [68,69]. Another genetically engineered vehicle for IL-10 delivery is the bacterium Lactococcus lactis. Daily inoculations with
these bacteria resulted in attenuated mucosal inflammation in two mouse models [57]. A pilot study using the bacteria in Crohn’s disease patient is currently undertaken by our group. 3.1.3. Antisense molecules To date modification of the immune response on the level of translation has not been widely tested. In IBD patients, treatment with the antisense compound Isis 2302 is the only example. Isis 2302 is an oligonucleotide designed to hybridize to the 3#-untranslated region of the ICAM-1 messenger RNA. ICAM-1 is a ligand that is expressed on endothelial cells and recognizes the lymphocytes homing receptor LFA-1 [15]. The interaction between the two occurs after the leucocytes have marginalized and started rolling upon integrineaddressin interaction, and effectuates a firm adhesion to the endothelial cells. Reminiscent of integrin blockade Isis 2302 can prevent ICAM-1 expression thus preventing lymphocytes from migrating to spots of inflammation [15]. However, two phase III trials failed to demonstrate clinical efficacy [52,73]. In mice models antisense therapy targeting the p65 subunit of NFkB had therapeutic benefit [38]. The active form of NFkB is a heterodimer formed by this p65 subunit together with a p55 subunit. Upon activation this heterodimer is proteolytically cleaved from its inhibitory protein IkB. In this way it is able to pass in the nucleus and act as a transcription factor for a vast spectrum of pro-inflammatory cytokines, immune related transcription factors, enzymes, cell surface receptors and adhesion molecules [37]. As a therapeutic target NFkB holds a great promise, but no studies in humans have been reported so far. 3.2. Small molecules Parallel to the development of the biological drugs described above conventional drugs have been subject to renewed interest because of their specific action on the synthesis pathways of pro-inflammatory cytokines. In addition, novel small molecules sharing these properties are now trialed. A conventional drug, thalidomide, and a novel drug, CNI-1493, are briefly addressed below. Analogous to NKkB the mitogen activated protein kinases (MAP-kinase) constitute major pro-inflammatory cascades from the cell surface to the nucleus. For the same reasons MAP-kinases are promising targets for immune modulation. The MAP-kinases are subdivided into three major signaling modules: ERK1/2, JNK and p38 MAPkinases. Each module is distinctly regulated and results in distinct downstream actions [24]. The ERK1/2 pathway is mainly involved in regulation of cell growth, proliferation and survival, but has also been implicated in several inflammatory processes. The JNK module is involved in regulation of cell proliferation and apoptosis, whilst the
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p38 MAP-kinase cascade is associated with cell growth, cell differentiation, cell death and inflammation [24]. A targeted inhibition of the p38 MAP-kinase pathway resulted in reduced levels of LPS induced TNF production in several animal models [1]. We showed that JNK and p38 MAPK are activated in the mucosa of patients with active CD and significantly increased when compared to levels in mucosal biopsies of healthy controls [23]. CNI-1493 is a gyanylhydrazone that inhibits both JNK and p38 kinase in lymphocytic [23,71]. Thus, it is able to suppress macrophage activation and the production of several pro-inflammatory cytokines such as TNF-alpha, IL-1, IL-6, MIP1-alpha and beta. It has been favorably tested in many preclinical settings and animal models on inflammatory conditions including a mouse model for colitis [24]. In a pilot study in CD patients we showed that CNI administration resulted in mucosal healing and a significant decrease of disease activity [23]. A large controlled dose finding study is currently undertaken. Thalidomide inhibits both TNF-alpha biosynthesis as the Th1 polarizing cytokine interleukin-12 [41]. Two uncontrolled pilot studies have been conducted using thalidomide as a treatment for active CD. In one 80% of the patients experienced fistula closure and 50% showed a clinical response [70]. Another trial reported response rates of 67% and remission of rates of 0e30% [9].
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P.C.F. Stokkers, D.W. Hommes / Cytokine 28 (2004) 167e173 [60] te Velde AA, van Kooyk Y, Braat H, Hommes DW, Dellemijn TA, Slors JF, et al. Increased expression of DCSIGN C IL-12 C IL-18C and CD83 C IL-12 ÿ IL-18ÿ dendritic cell populations in the colonic mucosa of patients with Crohn’s disease. Eur J Immunol 2003;33:143e51. [61] ten Hove T, van Montfrans C, Peppelenbosch MP, van Deventer SJ. Infliximab treatment induces apoptosis of lamina propria T lymphocytes in Crohn’s disease. Gut 2002;50:206e11. [62] Tilg H, Ulmer H, Kaser A, Weiss G. Role of IL-10 for induction of anemia during inflammation. J Immunol 2002;169:2204e9. [63] Tilg H, van Montfrans C, van den EA, Kaser A, van Deventer SJ, Schreiber S, et al. Treatment of Crohn’s disease with recombinant human interleukin 10 induces the proinflammatory cytokine interferon gamma. Gut 2002;50:191e5. [64] Van den Brande JM, Braat H, van den Brink GR, Versteeg HH, Bauer CA, Hoedemaeker I, et al. Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease. Gastroenterology 2003;124:1774e85. [65] van Deventer SJ. Anti-tumour necrosis factor therapy in Crohn’s disease: where are we now? Gut 2002;51:362e3. [66] van Deventer SJ. Taming the mucosal immune response in Crohn’s disease. Best Pract Res Clin Gastroenterol 2002;16: 1035e43. [67] van Deventer SJ, Elson CO, Fedorak RN. Multiple doses of intravenous interleukin 10 in steroid-refractory Crohn’s disease Crohn’s Disease Study Group Gastroenterology 1997;113:383e9.
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[68] van Montfrans C, Hooijberg E, Rodriguez Pena MS, De Jong EC, Spits H, te Velde AA, et al. Generation of regulatory gut-homing human T lymphocytes using ex vivo interleukin 10 gene transfer. Gastroenterology 2002;123:1877e88. [69] van Montfrans C, Rodriguez Pena MS, Pronk I, Ten Kate FJ, te Velde AA, van Deventer SJ. Prevention of colitis by interleukin 10-transduced T lymphocytes in the SCID mice transfer model. Gastroenterology 2002;123:1865e76. [70] Vasiliauskas EA, Kam LY, Abreu-Martin MT, Hassard PV, Papadakis KA, Yang H, et al. An open-label pilot study of lowdose thalidomide in chronically active, steroid-dependent Crohn’s disease. Gastroenterology 1999;117:1278e87. [71] Waetzig GH, Seegert D, Rosenstiel P, Nikolaus S, Schreiber S. p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease. J Immunol 2002;168:5342e51. [72] Wagner CL, Schantz A, Barnathan E, Olson A, Mascelli MA, Ford J, et al. Consequences of immunogenicity to the therapeutic monoclonal antibodies ReoPro and Remicade. Dev Biol (Basel) 2003;112:37e53. [73] Yacyshyn BR, Bowen-Yacyshyn MB, Jewell L, Tami JA, Bennett CF, Kisner DL, et al. A placebo-controlled trial of ICAM-1 antisense oligonucleotide in the treatment of Crohn’s disease. Gastroenterology 1998;114:1133e42. [74] Yang H, Taylor KD, Rotter JI. Inflammatory bowel disease. I. Genetic epidemiology. Mol Genet Metab 2001;74:1e21.
New cytokine therapeutics for inflammatory bowel disease P.C.F. Stokkers*, D.W. Hommes Department of Gastroenterology and Hepatology, C2-111, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands Received 24 February 2004; accepted 21 July 2004
Abstract Conventional therapy for inflammatory bowel diseases rely on corticosteroids and 5-aminosalicylates combined with immunosuppressive agents for maintenance. These drugs are not always effective and may inflict serious side effects. Other therapies are therefore awaited. Infliximab, a monoclonal antibody against the pro-inflammatory cytokine TNF-alpha has been successfully applied as a treatment for Crohn’s disease. The mechanism of action of this drug extends beyond the level of TNF-alpha scavenging and includes induction of apoptosis of effector cells. Numerous anti-TNF antibodies have been developed and are currently evaluated in clinical trials. Other targets for monoclonal antibodies include integrins and cytokines involved in T-cell differentiation and activation. Likewise recombinant proteins that moderate TNF bioactivity and lymphocyte function have been developed. The therapeutic effect of recombinant interleukin-10 seems to be dependent on local delivery of the protein. Antisense therapy targeting lymphocyte migration has also been tested in IBD. Finally, the conventional drug thalidomide and possibly MAPkinase inhibitors may become novel treatment entities for IBD. Ó 2004 Published by Elsevier Ltd. Keywords: IBO; Crohn’s disease; Ulcerative colitis; Treatment; Biologicals
1. Introduction Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract, which clinically present as one of two disorders, Crohn’s disease (CD) or ulcerative colitis (UC). The inflammation of the intestinal mucosa in IBD is characterized by an influx of neutrophils and macrophages that produce cytokines, eicosanoids, proteolytic enzymes and free radicals that lead to inflammation and ulceration. Hence, IBD patients suffer from diarrhea, rectal blood loss, abdominal pain and weight loss. Complications of disease occur as a toxic megacolon, perforation, malignant transformation and in the case of CD strictures and fistulas [19,43]. The conventional therapies including corticosteroids and 5-aminosalicylates target the inflammatory process * Corresponding author. E-mail address: [email protected] (P.C.F. Stokkers). 1043-4666/$ - see front matter Ó 2004 Published by Elsevier Ltd. doi:10.1016/j.cyto.2004.07.012
on different levels. 5-Aminosalicylates are known to influence the production of prostaglandins and leukotrienes, neutrophil chemotaxis and the scavenging of reactive oxidative radicals. The effects of corticosteroids are even more pleiotropic and only partially understood. Both therapies are effective for induction of disease remission, corticosteroids being the most potent [19,43]. However, 30% of the patients fail to respond on corticosteroids and the side effects of the drug prohibit long term or maintenance treatment [36]. Immunosuppressives such as azathioprine and methotrexate are used for patients dependent on steroids or with steroid refractory disease [12,20]. New therapeutic strategies are therefore awaited and subject of this overview. 2. Immune response and inflammatory pathways The interplay between the immune apparatus and the gut lumen defines mucosal inflammation in IBD. A genetic predisposition for developing an inadequate
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immune response towards some luminal factor seems the underlying cause of the disease [26,74]. This paradigm has been enforced by the identification of Crohn’s disease related mutations in the NOD2 gene [25,39]. NOD2 is a receptor for a pattern associated with microbial pathogens (PAMP) and a family member of several other PAMP-receptors. These receptors bind specific microbial component such as lipopolysaccharide and are mainly expressed in antigen presenting cells (APC). Thus, PAMP-receptors are part of the innate immune system by mediating the initial recognition of pathogens [28,40]. The question is how NOD2 variants lead to the defective peripheral tolerance that is observed in CD. Enterocytes, macrophages, dendritic cells and T-cells all express NOD2 and may have a role in disease development. Dendritic cells (DCs) have an important function at the interface between the innate and adaptive immune systems. We have recently identified two mutually exclusive mucosal DC subpopulations one of which is located directly below the mucosal surface [60]. Although the role of this so-called DC-SIGN-positive subpopulation remains illusive, we speculate that these cells sample the intestinal flora by protruding extensions between the epithelial monolayer [66]. Thus, a first line of defence is warranted by a continuous presentation of antigens via HLA-class 2 molecules to the T-cell receptor. Normally, in the case of indigenous flora, this event should lead to tolerance e.g. induction of anergy of the specific T-cell. Only in case of a pathogenic intruder the DCs become activated expressing co-stimulatory molecules on the membrane that, together with the T-cell receptor complex and the HLA-class 2 molecule, form the immunological synapses. Depending on the type of co-stimulation and cytokine release by the DCs T-cells are activated and differentiate towards a T-helper 1 or T-helper 2 phenotype [4,29,46,66]. A defect in this pathway was recently shown in Stat3 deficient mice, which suffer from chronic enterocolitis and serve as an animal model for IBD. Stat3 deficient macrophages do not produce interleukin-10, which leads to a skewed Th1 immune response upon stimulation with bacterial components [59]. The enterocolitis seems to be induced via TLR-4, a PAMP-receptor and familiar to NOD2, and is mediated by an exaggerated IL-12p40 production by myeloid cells [30]. It is widely acknowledged that the immune response in UC is different from the one seen in CD. In CD the inflammatory reaction is mediated via T-helper 1 lymphocytes expressing interleukin-2 and interferon-g. In UC the mucosal inflammation seems to be T-helper 2 mediated [19,43]. The gut epithelium acts as a structural barrier against pathogens. It has been noted that the epithelium of IBD patients harbours more adherent and intra-epithelial bacteria than healthy controls [53,58]. Enterocytes can
also act as antigen presenting cells. Especially in intestinal infection enterocytes have been shown to express chemokines such as interleukin-8 via an NFkB regulated innate immune response [10,42]. Aberrant function of NOD2 in the enterocytes may very well be important for the pathogenesis of CD since NOD2 expression has been found in several epithelial cell lines and epithelial cells of healthy subject and IBD patients [22,44]. Recently several PAMP-receptors were shown to be expressed by T-regulatory (Treg) cells in mice [7]. Although no data are available for the expression of NOD2 this finding has important implications for future research; Treg cells induce anergy by down regulating T-cell effector function, inhibition of migration of DCs towards mesenteric lymph nodes, and inhibition of T-cell and DC activation. Treg cells responsive to selfantigens and bacterial antigens exert these actions by interleukin-10 and TGF-b [54]. LPS, for which TLR-4 serves as a receptor, promotes Treg cell survival and proliferation, providing a negative inhibition of the immune response [7]. The finding that Treg cells respond to inflammatory chemokines supports this notion [6,27]. Thus, Treg cells may prevent sustained response towards self or indigenous intestinal flora, a process which is hampered in Crohn’s disease [66]. However, preliminary data from our group show that PAMP-receptors including NOD2 are not expressed in human Treg cells (K. Kok et al. data on file). This difference with the murine situation is puzzling and should be further explored. Innate and adaptive immunity meet at different levels in the mucosal immune system. It has become clear that IBD results from a defect somewhere in the pathways between the two. Therapeutic interventions at the level of these pathways may prove more effective and safe than the conventional therapies for IBD.
3. New therapeutic compounds for IBD Fundamental research on the inflammatory pathways underlying IBD has boosted therapeutic interventions with compounds acting on different levels targeting. New therapies include both biological agents as chemically engineered agents. Both classed will be addressed hereafter.
3.1. Biologicals Biological compounds that are used for the treatment of IBD or are under current investigation in clinical trials are either monoclonal antibodies, or recombinant proteins or antisense nucleotides [50].
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3.1.1. Monoclonal antibodies The most prominent advocate of the new biological treatments is the monoclonal antibody infliximab. This chimeric humanemurine antibody has a human IgG1 Fc domain and a murine TNF recognition site [48]. In 1993 the first patient with steroid refractory Crohn’s disease was treated with this antibody showing a remarkable clinical and endoscopical response [8]. Many clinical trials followed and the treatment has now been registered for the treatment of steroid refractory disease and peri-anal fistulizing disease. However, the safety of the drug and the formation of antibodies against chimeric antibodies are issues of concern. Among the concern about safety was the finding of non-Hodgkin lymphomas (NHL) in 3 CD patients and 4 patients with rheumatoid arthritis treated with anti-TNF. For definite conclusions the power of the number of treated patients is too low and the follow up period too short. Furthermore, other immunosuppressive drugs that may confer risk on NHL were used by these patients. The immune suppressive action of the infliximab results in more serious infections [3] and patients should be checked for TBC by means of tuberculin skin testing before infusion of the drug [65]. The formation of human anti-chimeric antibodies (HACAs) has been reported in 13e68% of treated patients and is associated with a poor clinical response and infusion reactions [72]. Concurrent treatment with azathioprine or methotrexate may reduce the chance on HACA formation. The mechanism of action of infliximab remains illusive. Neutralization of TNF-alpha in the inflamed mucosa is unlikely to be a sufficient explanation since many other pro-inflammatory cytokines can do the job instead. Antibody dependent cytotoxicity, lysis of TNF producing cells by complement fixation or apoptosis induction of T-cells by the Fc portion of the antibody seem a more appropriate explanation [41,65]. Our group has focused on the latter possibility showing the effects of infliximab on apoptosis in CD3/CD28 stimulated Jurkat T-lymphocytes. We observed a significant increase of the ratio of Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic). As expected, this resulted in increased apoptosis of infliximab-treated Jurkat cells, but only when the cells were previously activated. Furthermore, we showed that the number of apoptotic T-cells in the mucosa of CD patient increase following treatment with infliximab and that the binding of infliximab to membrane bound TNF induces caspase3 regulated apoptosis of T-cells as well as monocytes [61,64]. CPD571 is an anti-TNF antibody which is supposed to be less immunogenic than infliximab, due to the fact that it is 95% human protein and 5% murine protein [50]. However, the Fc part of the antibody is of the IgG4 type, which does not posses the complement binding action of the Fc part of infliximab. Therefore, CPD571 may lack the apoptotic and cytotoxic properties of
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infliximab. Three small clinical trials with CPD571, showed encouraging results, and a large placebo controlled phase III trial is currently undertaken to prove efficacy of the drug [11,47,56]. Interfering on leukocyte trafficking natalizumab has been developed [50]. This humanized monoclonal antibody of the IgG4 class recognizes a4 integrin, that dimerizes with b1 unit (a4b1 or VLA-1) or with a b7 unit (a4b7 or MAdCAM-1) VLA-1 is up-regulated on the vascular endothelium at sites of inflammation, and ensures leukocyte rolling and adhesion, the first steps in diapedesis. MAdCAM-1 is also up-regulated on endothelium at sites of chronic inflammation [5,55]. Thus, natalizumab is supposed to block the migration of leukocytes to the inflamed mucosa in IBD. After a promising pilot study, recently the results of a placebo controlled double blind multi-center trial were published [14,17]. The primary end point of this study, remission of disease at the time point of 6 weeks, was not statistically significant compared to placebo. However, it was noted that in the placebo group significantly more patients had used concurrent immunosuppressive drugs when compared to the treatment group [33]. Treatment resulted in higher response rates in all treated groups, and these finding were also reflected in CRP levels and quality of life scores. A larger long term trial is awaited. LDP-2 is another integrin targeted monoclonal antibody. This IgG1 class antibody recognizes MAdCAM-1 and like natalizumab has proven to be an effective treatment for the spontaneous colitis that is seen in cotton top tamarins [21]. Any trials beyond phase II have not been reported so far. Other monoclonal antibodies that have proven efficacy in animal models target interleukin-12 (IL-12), interferon -g (IFN-g), interleukin-18 (IL-18) and the interleukin-2 receptor [50]. IL-12, IL- 18 and IFN-g all play an important role in T-cell differentiation. These cytokines are produced by antigen presenting cells, macrophages and activated lymphocytes and promote Th1 differentiation. In theory interferences on this level could modulate the Th1 biased immune response that is seen in CD. Interleukin-2 is an important proinflammatory cytokine that is produced by Th1 cells. Two antibodies (daclizumab and basiliximab) targeting the interleukin-2 receptor have been created and may be trialed in IBD patients [50]. All new therapeutic strategies based on monoclonal antibodies probably have the same drawbacks as infliximab: formation of antibodies, resulting in diminishing therapeutic efficacy and infusion reactions, high cost in producing the drugs and the necessity of intravenous dosing. In contrast, whilst the therapeutic efficacy of infliximab stretches beyond the scavenging TNF as described above, such mechanisms of action do not necessarily apply to other monoclonal antibodies. In this respect the results of trials with etanercept are
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telling. This genetically engineered fusion protein consists of two p75 TNF receptors mounted on an IgG1 Fc tail [48]. In theory this protein would induce less antibody formation since the protein is fully human. However, in a clinical trial the compound was as effective as placebo [49]. 3.1.2. Recombinant proteins Whilst the etanercept compound was based on recombinant proteins with antibody properties, other TNF scavengers that lack antibody structure have also been investigated. Onercept is a genetically engineered recombinant human p55 TNF receptor. Soluble TNF receptors have been shown to regulate TNF trafficking in chronic inflammatory conditions [48]. After a promising pilot study the compound is now being tested in a phase II clinical trial [45]. Interleukin-10 is a cytokine that has been shown to profoundly inhibit effector functions of activated macrophages, monocytes and dendritic cells [35]. Proinflammatory cytokines production by Th1 cells such as IL-2 and IFN-g is down-regulated by IL-10. In animal models, an important role for IL-10 was suggested by the finding that IL-10 knockout mice develop chronic intestinal inflammation [31] and the observation that IL-10 can prevent colitis that is induced by activated T-cells in a transfer model in mice that lack T-cells [2,18]. Therefore, IL-10 was suggested as a potential therapy for IBD. Encouraging results from pilot studies in IBD patients were tempered by subsequent larger trials [67]. IL-10 given subcutaneously did not prove better than placebo in disease remission induction and only showed a modest clinical response [13,51]. In addition, other studies have linked headache, fever and anemia with high systemic concentrations of IL-10 and suggested that this may be due to IL-10 induced elevation of IFN-g by whole blood cells [32,62,63]. IL-10 also seems to have immunostimulatory effects by up regulating HLA-class 2 molecules on B lymphocytes and by inducing cytotoxic T-cell differentiation [16,34]. Thus, the biology of this cytokine is complex, and the in vivo effects comprise both anti and pro-inflammatory actions. It could very well be that IL10 in higher systemic doses exerts an immunostimulatory effect. This may explain that patients receiving higher doses rhIL10 did worse in the trials. These data, together with the fact that the mucosal bioavailability of rhIL10 upon intravenous administration is probably low due to a rapid clearance, have prompted research towards other methods of more local administration. Van Montfrans et al. have shown that IL-10 delivery to intestinal mucosa in mice is feasible by the using Tlymphocytes that were transduced with a retrovirus construct expressing the cytokine [68,69]. Another genetically engineered vehicle for IL-10 delivery is the bacterium Lactococcus lactis. Daily inoculations with
these bacteria resulted in attenuated mucosal inflammation in two mouse models [57]. A pilot study using the bacteria in Crohn’s disease patient is currently undertaken by our group. 3.1.3. Antisense molecules To date modification of the immune response on the level of translation has not been widely tested. In IBD patients, treatment with the antisense compound Isis 2302 is the only example. Isis 2302 is an oligonucleotide designed to hybridize to the 3#-untranslated region of the ICAM-1 messenger RNA. ICAM-1 is a ligand that is expressed on endothelial cells and recognizes the lymphocytes homing receptor LFA-1 [15]. The interaction between the two occurs after the leucocytes have marginalized and started rolling upon integrineaddressin interaction, and effectuates a firm adhesion to the endothelial cells. Reminiscent of integrin blockade Isis 2302 can prevent ICAM-1 expression thus preventing lymphocytes from migrating to spots of inflammation [15]. However, two phase III trials failed to demonstrate clinical efficacy [52,73]. In mice models antisense therapy targeting the p65 subunit of NFkB had therapeutic benefit [38]. The active form of NFkB is a heterodimer formed by this p65 subunit together with a p55 subunit. Upon activation this heterodimer is proteolytically cleaved from its inhibitory protein IkB. In this way it is able to pass in the nucleus and act as a transcription factor for a vast spectrum of pro-inflammatory cytokines, immune related transcription factors, enzymes, cell surface receptors and adhesion molecules [37]. As a therapeutic target NFkB holds a great promise, but no studies in humans have been reported so far. 3.2. Small molecules Parallel to the development of the biological drugs described above conventional drugs have been subject to renewed interest because of their specific action on the synthesis pathways of pro-inflammatory cytokines. In addition, novel small molecules sharing these properties are now trialed. A conventional drug, thalidomide, and a novel drug, CNI-1493, are briefly addressed below. Analogous to NKkB the mitogen activated protein kinases (MAP-kinase) constitute major pro-inflammatory cascades from the cell surface to the nucleus. For the same reasons MAP-kinases are promising targets for immune modulation. The MAP-kinases are subdivided into three major signaling modules: ERK1/2, JNK and p38 MAPkinases. Each module is distinctly regulated and results in distinct downstream actions [24]. The ERK1/2 pathway is mainly involved in regulation of cell growth, proliferation and survival, but has also been implicated in several inflammatory processes. The JNK module is involved in regulation of cell proliferation and apoptosis, whilst the
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p38 MAP-kinase cascade is associated with cell growth, cell differentiation, cell death and inflammation [24]. A targeted inhibition of the p38 MAP-kinase pathway resulted in reduced levels of LPS induced TNF production in several animal models [1]. We showed that JNK and p38 MAPK are activated in the mucosa of patients with active CD and significantly increased when compared to levels in mucosal biopsies of healthy controls [23]. CNI-1493 is a gyanylhydrazone that inhibits both JNK and p38 kinase in lymphocytic [23,71]. Thus, it is able to suppress macrophage activation and the production of several pro-inflammatory cytokines such as TNF-alpha, IL-1, IL-6, MIP1-alpha and beta. It has been favorably tested in many preclinical settings and animal models on inflammatory conditions including a mouse model for colitis [24]. In a pilot study in CD patients we showed that CNI administration resulted in mucosal healing and a significant decrease of disease activity [23]. A large controlled dose finding study is currently undertaken. Thalidomide inhibits both TNF-alpha biosynthesis as the Th1 polarizing cytokine interleukin-12 [41]. Two uncontrolled pilot studies have been conducted using thalidomide as a treatment for active CD. In one 80% of the patients experienced fistula closure and 50% showed a clinical response [70]. Another trial reported response rates of 67% and remission of rates of 0e30% [9].
4. Conclusion New therapeutic approaches to the treatment of inflammatory bowel disease are based on the knowledge of the immunological mechanisms that characterize the pathogenesis of the diseases. The great success of the anti-TNF antibody infliximab has boosted research towards other compounds that share infliximab’s feature of specific intervention in the immune response. However, it seems that the success of infliximab is rather due to its pleiotropic effects, which are observed in ongoing research to the working mechanism of the drug. The therapeutic effect of recombinant interleukin10 seems to be dependent on local delivery of the protein, since higher systemic doses appear to have and pro-inflammatory effect. Many new approaches using monoclonal antibodies, recombinant proteins or small molecules await to be evaluated. References [1] Badger AM, Bradbeer JN, Votta B, Lee JC, Adams JL, Griswold DE. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J Pharmacol Exp Ther 1996;279:1453e61.
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