Aliment Pharmacol Ther 1999; 13 (Suppl. 4), 3±8.
`
`PAPER 024 DISC
`
`Review article: targeting TNFa as a key cytokine in the in¯ammatory
`processes of Crohn's disease Ð the mechanisms of action of in¯iximab
`
`S . J . H . V A N D E V E N T E R
`Afdeling Maag-Darm-Leverziekten, Academisch Medisch Centrum, Amsterdam, The Netherlands
`
`SUMMARY
`
`Crohn's disease is a chronic, debilitating gastrointestinal
`disorder in which a variety of cellular processes and pro-
`inflammatory mediators influence the pathogenesis of
`the disease. Although the potential roles and functions
`of the pro-inflammatory mediators continue to be
`debated, several mediators, specifically tumour necrosis
`factor-alpha, have been clearly identified as having a
`pivotal role in the inflammation of the bowel mucosa of
`these patients. Therapies specifically focusing on the
`inflammatory process underlying Crohn's disease have
`
`the potential for providing disease modification and
`prolonged remission. Infliximab, an antitumour
`necrosis factor-alpha monoclonal antibody, has been
`demonstrated to neutralize tumour necrosis factor-
`alpha and restore and reset the immunological
`dysbalance of the inflamed mucosa. Preliminary studies
`with infliximab suggested that treatment resulted in a
`rapid and almost complete inhibition of multiple
`inflammatory pathways. In clinical studies of
`infliximab, patients with Crohn's disease achieved rapid
`reduction in clinical signs and symptoms, substantiated
`by both endoscopic and microscopic evaluation.
`
`Ahed
`Bhed
`Ched
`Dhed
`Ref marker
`Fig marker
`Table mar-
`ker
`Ref end
`Ref start
`
`IN TRO DUC TIO N
`
`The chronic in¯ammatory state that characterizes
`Crohn's disease is mediated by several
`factors that
`intracellular cascades.1,2 The
`initiate a number of
`number of di€erent mediators, and the redundancy of
`function and action that
`is evident, have made it
`dicult
`to target a single factor
`responsible for
`precipitating the disease state. Consequently, some
`researchers have suggested that therapies for Crohn's
`disease will have to simultaneously inhibit several
`in¯ammatory mediators
`to attain clinical bene®ts.
`However, results of studies involving in¯iximab, an
`factor-alpha (TNFa) chimeric
`antitumour necrosis
`monoclonal antibody, indicated that speci®cally target-
`ing this cytokine alone may interrupt some of the major
`mechanisms responsible for mucosal in¯ammation in
`
`Correspondence to: Dr S. van Deventer, Afdeling Maag-Darm-Leverziekten,
`Academisch Medisch Centrum, Meibergdreef 9, Amsterdam, The
`Netherlands 1105 AZ.
`
`Crohn's disease. When in¯iximab was used in clinical
`trials, patients with Crohn's disease achieved signi®cant
`endoscopic improvement and substantial reductions in
`clinical signs and symptoms.3±5
`This review discusses the laboratory and clinical data
`implicating TNFa as a pivotal mediator of the in¯am-
`matory response in patients with Crohn's disease. Using
`animal models of colitis combined with a growing
`understanding of the in¯ammatory process in the bowel
`mucosa, it has been possible to gain an appreciation of
`the critical role of TNFa in the pathogenesis of Crohn's
`disease. The use of an agent that speci®cally neutralizes
`TNFa has also provided an insight into the pathways
`that are essential for the genesis of this disease. Finally,
`by comparing what is understood about the action of
`TNFa with clinical data derived from studies using
`in¯iximab, it has been possible to gather information
`about the speci®c mechanism of action of this antibody.
`These data, in turn, may prove useful in predicting the
`clinical responsiveness of patients.
`
`# 1999 Blackwell Science Ltd
`
`3
`
`

`

`4 S . J . H . V A N D E V E N T E R
`
`IN FLA MMA TOR Y MEDIATORS IN THE
`PA THO GENESIS OF CR OHN'S D ISEASE
`
`A large number of in¯ammatory mediators are induced
`in the mucosa of patients with in¯ammatory bowel
`disease (IBD), speci®cally Crohn's disease and ulcerative
`colitis. This list of
`in¯ammatory mediators includes
`eicosanoids, chemokines, cytokines, and other factors
`(i.e. nitric oxide, reactive oxygen intermediates) (Table
`1).1,2,6 The role of pro-in¯ammatory and anti-in¯am-
`matory cytokines in this process, while still the subject
`of debate, continues to be de®ned.
`Under normal conditions, the gut immune response is
`characterized by a balance between pro-in¯ammatory and
`anti-in¯ammatory factors.
`In patients with Crohn's
`disease, the balance between these two systems in the
`mucosa is dysregulated, thereby shifting the normal
`equilibrium towards a chronic in¯ammatory state. The
`role of speci®c cytokines in this mechanism is complicated
`and, depending on the in¯ammatory content, cytokines
`may act either as pro- or anti-in¯ammatory. For example,
`interferon-gamma (IFNg) is normally pro-in¯ammatory;
`however, in certain systems and disease stages, IFNg may
`have anti-in¯ammatory actions. In addition, when admin-
`istered in high doses, anti-in¯ammatory cytokines such as
`interleukin (IL)-10 may have pro-in¯ammatory properties
`by inducing certain CD+ T lymphocytes to proliferate7
`and by inducing IFNg production by natural killer cells.8
`To further complicate attempts to resolve which cytokines
`have the greatest impact on the development of Crohn's
`disease, the mucosal concentrations of these cytokines do
`not necessarily re¯ect their relative importance or activity
`in the in¯ammatory process. Although IL-1 and IL-8 are
`present in greater concentrations in the intestinal mucosa
`than is TNFa, this latter cytokine appears to have a greater
`importance in the disease process.9,10
`
`Table 1. Inflammatory mediators in inflammatory bowel disease
`
`PAPER 024 DISC
`
`A number of studies have supported a predominant role
`for TNFa in the pathogenesis of Crohn's disease. Mice
`with chronic intestinal in¯ammation induced by 2,4,6,-
`trinitrobenzene sulphonic acid (TNBS) mimic some of the
`characteristics of Crohn's disease in humans.11 Lamina
`propria mononuclear cells from these mice secrete 10- to
`30-fold higher levels of mRNA for TNFa, compared with
`control mice.11 Interestingly, levels of mRNA for IL-1 and
`IL-6 were not elevated to the same degree (Figure 1).
`Similar results have been found in human studies. Levels
`of mRNA and immunoreactivity for TNFa are increased
`in the bowel mucosa of patients with Crohn's dis-
`ease;12,13 TNFa concentrations in stool specimens have
`been shown to parallel disease activity.14 Furthermore,
`TNFa production is increased in organ and cell cultures
`of mucosal biopsy specimens from patients with Crohn's
`disease.6 Nevertheless, despite this evidence pointing to a
`key role of TNFa in the in¯ammatory process, it would be
`naive to conclude that Crohn's disease results from the
`simple overabundance or de®ciency of a single in¯am-
`matory mediator.
`
`MODI FYIN G IMMUNE FUNCTI ON IN CROHN 'S
`DISEASE: THE CRITICAL ROLE OF INFLAMMATORY
`MEDIA TOR S
`
`To fully explore the role of in¯ammatory mediators in
`Crohn's disease, particularly TNFa, it is necessary to have
`a solid understanding of T-cell biology. A number of
`laboratory ®ndings have veri®ed the importance of T lym-
`phocytes in the normal regulation of intestinal responses.
`Several di€erent animal models of in¯ammatory bowel
`disease have been described, including mice with altera-
`tions in T-cell subpopulations and selection, mice with
`targeted disruption of speci®c cytokine genes, and mice
`
`Eicosanoids
`
`Chemokines
`
`Cytokines
`
`Other factors
`
`Prostaglandin E2
`Leukotriene B4
`Platelet-activating factor
`IL-8
`gro-a
`RANTES
`TNFa
`IL-1
`IL-10
`IL-12
`IFNg
`Nitric oxide
`Reactive oxygen intermediates
`
`Figure 1. Anti-TNF antibody treatment reduces cytokine production
`by lamina propria macrophages in TNBS colitis.11
`
`# 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13 (Suppl. 4), 3±8
`
`

`

`lacking necessary signalling proteins.15±17 All of these
`alterations disrupt normal T-cell regulatory mechanisms,
`which in turn exposes the gut to cell-mediated immune
`attack. In humans, it has been noted that Crohn's disease
`often goes into remission when the immune system is
`weakened (e.g. people with acquired immune de®ciency
`syndrome [AIDS]); similarly, remission is also observed
`when T-cell populations have been replenished following
`bone marrow transplantation. Both these observations
`suggest a role for the T cell in the pathogenesis of Crohn's
`disease. Moreover, in a small open-label clinical study,
`patients with therapy-refractory Crohn's disease re-
`sponded to infusion of a depleting anti-CD4 antibody; in
`some patients long-term remission was observed.18,19
`Activation of T cells occurs upon the presentation of an
`antigen by either major histocompatibility complex
`(MHC) class I or II molecules.2 When the antigen is
`endogenous, it is expressed by MHC class I molecules,
`and when the antigen is exogenous, it is expressed by
`MHC class II molecules. In the latter instance, MHC
`class II molecules are expressed by antigen-presenting
`cells and lead to the activation of CD4+ T lymphocytes
`(Figure 2).20 The CD4+ T cells regulate key aspects of
`the immune response. Based on their function and
`cytokine secretory pro®le, these cells are classi®ed as
`either T-helper 1 (Th1) cells or T-helper 2 (Th2) cells.21
`Th1 cells regulate cell-mediated immunity and secrete
`IL-2, IL-12, IFNg, and TNFa. Th2 cells mediate humoral
`immunity and secrete IL-4, IL-5, IL-6, IL-10, and IL-13.
`The development of each of these subsets is suppressed
`by key cytokines produced by the other subset: the
`development of Th1 cells is suppressed by the action of
`IL-4 produced by Th2 cells, and the development of Th2
`cells is suppressed by IFNg secreted by Th1 cells.
`
`PAPER 024 DISC
`
`R E V I E W : I N F L I X I M A B M E C H A N I S M S O F A C T I O N 5
`
`These polarized populations of Th1 and Th2 cells
`appear to be associated with di€erent disease.22,23
`Crohn's disease is considered a `Th1' disease, while
`ulcerative colitis has characteristics of a `Th2' diseases.
`A number of animal models of
`in¯ammatory bowel
`disease have established that ulcerative colitis and
`Crohn's disease may be distinguished on the basis of
`their immunological pro®le as well.2,24±26
`Under normal conditions,
`luminal antigens derived
`from food and bacteria are continually in contact with
`epithelial cells in the intestinal mucosa and can also be
`found in the lamina propria. However, the action of
`cytokines secreted by Th2 cells (e.g. IL-10, transforming
`growth factor-beta [TGFb]) prevents cell-mediated im-
`mune responses to these antigens. In the case of Crohn's
`disease, where there is a shift to production of Th1-
`secreted cytokines, antigen presence may produce a
`perpetual hyperstimulation of
`the bowel mucosa,
`causing chronic in¯ammation (Figure 3).23,26
`It should be noted that in normal immune responses,
`peripheral T-lymphocyte tolerance is essential to pre-
`vent severe in¯ammation and autoimmune reactions.
`In fact, at least 90% of antigen-activated T lymphocytes
`are deactivated before provoking an in¯ammatory
`response. Deactivation of T lymphocytes can be
`accomplished in a variety of ways. T lymphocytes
`stimulated without costimulation of antigen-presenting
`cells results in anergy (lack of energy) and functional
`unresponsiveness of the T cell. A second mechanism is
`cytokine-mediated regulation,
`in which there
`is
`reciprocal suppression of T-helper subsets by cyto-
`kines secreted by the other subset, as previously
`
`Figure 2. Role of cytokines in the immune response. Adapted from
`Rinco n.20
`
`Figure 3. CD4+-dependent regulation of immune responses in the
`intestine. Adapted from Powrie.26
`
`# 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13 (Suppl. 4), 3±8
`
`

`

`6 S . J . H . V A N D E V E N T E R
`
`described. Finally, programmed cell death of activated
`T lymphocytes through activation of Fas receptors (part
`of the TNF receptor family) also helps regulate T-cell
`number and maintains homeostasis by mediating the
`deletion of mature T cells.27,28 Binding of Fas ligand to
`its receptor on activated T cells results in the formation
`of a signalling complex that activates a family of
`proteases called caspases, which are responsible for
`inducing apoptosis.
`Models of
`in¯ammatory bowel disease suggest that
`suppression of activated T lymphocytes may be defective
`in this disease. IL-10-de®cient mice develop an in¯am-
`matory bowel disease very similar to Crohn's disease. By
`3 weeks of age, 70% of these mice exhibit signs of
`intestinal disease, and by 3 months all of the IL-10-
`de®cient mice have signs of disease (Figure 4).29
`Interestingly, 60% of
`these mice exhibit colorectal
`adenocarcinoma as well. Colonic explants from these
`IL-10-de®cient mice have been shown to produce large
`quantities of pro-in¯ammatory mediators such as IL-1,
`TNFa, IL-6, and IFNg. This suggests that the elimina-
`tion of IL-10 production had lifted the suppressive
`e€ects of anti-in¯ammatory cytokines, resulting in the
`production of chronic in¯ammation.
`Another murine model of colitis was induced by TNBS,
`which produced a Th1-dependent in¯ammatory reac-
`tion.11 Lamina propria mononuclear cells from these
`mice produced 10- to 30-fold higher concentrations of
`TNFa than cells from control mice. When these mice
`were treated with anti-TNFa antibodies, there was an
`improvement of the histological and clinical signs of the
`colitis. Treated mice also produced lower concentrations
`of pro-in¯ammatory cytokines (e.g. IL-1, IL-6) than
`untreated mice. These ®ndings suggest that secretion (or
`lack of secretion) of TNFa a€ects the production of pro-
`
`Figure 4. Development of enterocolitis in IL-10-deficient mice
`(C57BL/6 6 129 Ola, pathogen free).29
`
`PAPER 024 DISC
`
`in¯ammatory cytokines and alters the behaviour of cells
`that are intimately involved in the immune response.
`
`DI SEA SE-MODI FY ING THERA PY IN CROH N'S
`DISEASE: THE MECHANISMS OF ACTION OF
`IN FLI XIMA B
`
`Strictly speaking, current available therapies for Crohn's
`disease are not disease modifying, as they do not correct
`the underlying pathology responsible for development of
`the condition and do not alter the natural course of the
`disease. Although the immediate outcome of a ¯are-up
`is a€ected by treatment, Crohn's disease seems to follow
`a predictable course of remission and relapse, regardless
`of treatment. Standard treatments (i.e. 5-aminosalicylic
`acid
`[5-ASA],
`corticosteroids,
`immunosuppressive
`agents), while e€ective as short-term or maintenance
`therapy, do not prevent relapse in the long term.24
`Corticosteroids are ine€ective in maintaining remissions
`and should not be used as long-term therapy to prevent
`relapse.25 Upon cessation of corticosteroid therapy, 36%
`of patients experience a relapse.26 Similarly, surgical
`resection does not prevent the need for future surgery
`and does not alter long-term outcomes.
`Therapies speci®cally targeting the key events in the
`in¯ammatory process underlying Crohn's disease have
`the potential to be truly disease modifying. Because TNFa
`appears to play a key role in this process, antibodies
`directed toward TNFa have received special attention.
`In patients receiving a single injection of in¯iximab,
`C-reactive protein declined rapidly, reached normal
`concen-trations within 2 weeks, and remained low for
`8 weeks.3 Circulating concentrations of IL-6
`at least
`were also diminished. In a study by Targan and asso-
`ciates, 41% of patients with Crohn's disease exhibited a
`clinical response to therapy 12 weeks after administra-
`tion of a single dose of in¯iximab, compared with 12%
`of placebo-treated patients.4 In addition, 33% of the
`patients treated with in¯iximab experienced a remission
`of the disease, while only 4% of placebo-treated patients
`went into remission. In¯iximab-treated patients also
`had signi®cantly greater mean reductions in their
`Crohn's Disease Activity Index (CDAI) scores and
`signi®cantly better scores on their In¯ammatory Bowel
`Disease Questionnaire (IBDQ). The long duration of
`response to a single dose of in¯iximab (exceeding the
`half-life of the antibody) suggests that mechanisms
`other than neutralization of soluble TNFa are respon-
`sible for its clinical activity.3
`
`# 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13 (Suppl. 4), 3±8
`
`

`

`Mechanisms of action of in¯iximab
`
`Several mechanisms of action have been proposed for
`the ability of in¯iximab to reduce mucosal in¯amma-
`tion. First, in¯iximab binds soluble bioactive TNFa in
`the intestinal mucosa and neutralizes its pro-in¯amma-
`tory e€ects.33,34 In vitro, in¯iximab binds membrane-
`bound TNFa,
`facilitating destruction of
`those cells
`(which have an enhanced cytokine response in Crohn's
`disease) by antibody-dependent cellular toxicity or
`complement-dependent cytotoxicity mechanisms.35
`Recognition of membrane-bound TNFa is a key aspect
`of the action of in¯iximab. Transcription of TNFa mRNA
`results in a TNFa precursor molecule that has to be
`cleaved by a speci®c metalloproteinase (TNFa convert-
`ing enzyme [TACE]) in order to be able to be released
`from the cell membrane. Both the soluble and mem-
`brane-associated TNFa are biologically active as homo-
`trimers; transmembrane TNFa has an important role in
`interactions.22 Some agents
`direct cell-to-cell
`(e.g.
`oxpentifylline) interfere with transcription of TNFa by
`increasing intracellular cyclic adenosine monopho-
`sphate (cAMP) concentrations. Other agents
`(e.g.
`corticosteroids) a€ect
`translation, while still other
`agents target TACE. Although metalloproteinase inhi-
`bitors reduce release of soluble TNFa, they do not
`decrease expression of the membrane-bound cytokine
`and therefore,
`the mechanism of action of
`these
`inhibitors di€ers from monoclonal antibodies that target
`both soluble and transmembrane TNFa.
`Recent studies have indicated that membrane-bound
`TNFa may play a role in cell death. The Bcl family of
`proteins normally regulates apoptosis of T cells. Anti-
`apoptotic members (e.g. Bcl-2) prevent T cells from
`undergoing death when they are deprived of activating
`stimuli or growth factors (passive cell death).28 The
`function of these anti-apoptotic members is regulated by
`pro-apoptotic members (e.g. BAX). Therefore, the balance
`of interaction between BAX and Bcl-2 determines the fate
`of the cell. When quiescent T lymphocytes are exposed to
`in¯iximab, there is no change in the relative concentra-
`tions of BAX and Bcl-2 because there is little expression of
`TNFa on these cells. However, when these T lymphocytes
`are activated and express TNFa on their surface, the
`addition of in¯iximab provokes a sharp increase in the
`ratio of BAX to Bcl-2, and cell death is accelerated.
`Further studies con®rmed that the e€ect of in¯iximab on
`apoptosis appears to be speci®c for activated, but not
`resting, T lymphocytes. This action of in¯iximab may be
`
`PAPER 024 DISC
`
`R E V I E W : I N F L I X I M A B M E C H A N I S M S O F A C T I O N 7
`
`responsible for its prolonged duration of action. (T. Ten
`Hove, S. J. H. van Deventer, unpublished results).
`In¯iximab does not appear to be unique in its apoptotic
`e€ect. Methotrexate has been reported recently to induce
`apoptosis of activated T cells in the S phase and G2 phase
`of the cell cycle.36 This results in the clonal deletion of T
`cells that are activated by antigen at the time of metho-
`trexate administration. The parallel activity of in¯iximab
`and methotrexate on apoptosis may have clinical
`implications as well, because a trial of combination
`therapy using these two agents has demonstrated marked
`synergism in the treatment of rheumatoid arthritis.37
`
`CONCLU SION
`
`Normal immunological homeostasis in the bowel mucosa
`is a consequence of active and ®ne-tuned regulation of T-
`lymphocyte proliferative responses. Even subtle distur-
`bance in this system may result in an uncontrolled and
`prolonged in¯ammatory response that results in tissue
`damage and ®brosis. TNFa is a pivotal pro-in¯ammatory
`cytokine in Crohn's disease that is responsible for the
`activation of a wide range of secondary in¯ammatory
`pathways. Neutralization of TNFa by administration of
`in¯iximab has very rapid e€ects, with a detectable
`decrease of
`in¯ammatory parameters at 24 h after
`from the direct TNFa neutralizing
`infusion. Apart
`activity, in¯iximab is able to target activated T lympho-
`cytes and augment activation-induced programmed cell
`death. This latter mechanism may be immune-modulat-
`ing and may result in a long-term response (about 3
`months) after a single infusion of
`the monoclonal
`antibody. Recently, other immune-modulating agents,
`in particular methotrexate, have shown a similar e€ect
`on T lymphocytes.36 These data provide an outlook for
`the design of novel, potentially disease-modifying ther-
`apeutic strategies in Crohn's disease.
`
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