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Tumour necrosis factor alpha down-regulation parallels inflammatory regression in ulcerative colitis patients treated with infliximab
Digestive and Liver Disease 39 (2007) 811–817
Alimentary Tract
Tumour necrosis factor alpha down-regulation parallels inflammatory regression in ulcerative colitis patients treated with infliximab C. Hassan a,∗ , E. Ierardi b , O. Burattini b , V. De Francesco b , A. Zullo a , G. Stoppino b , C. Panella b , S. Morini a a
Gastroenterology and Digestive Endoscopy Unit, “Nuovo Regina Margherita” Hospital, Rome, Italy Section of Gastroenterology, Department of Medical Sciences, University of Foggia, Foggia, Italy
b
Received 4 February 2007; accepted 6 June 2007 Available online 25 July 2007
Abstract Background. Treatment with the anti-tumour necrosis factor ␣ monoclonal antibody infliximab has been shown to be effective in moderateto-severe ulcerative colitis. However, its effect on the mucosal histopathological abnormalities of this disease is largely unknown. This study aimed to assess the immunohistological effect of infliximab in ulcerative colitis. Methods. Nine patients with moderate-to-severe ulcerative colitis received infliximab (5 mg/kg) at weeks 0, 2 and 6, respectively. Colonic biopsies were collected before therapy and at week 10, when the Mayo score (including the endoscopic subscore) was also assessed. Severity of inflammation was evaluated by histologic score and histomorphometry. Immunohistochemical staining with antibody against tumour necrosis factor ␣ was performed on all biopsies and expressed as percentage of positive stromal cells/1000 counted (tumour necrosis factor ␣ score). Results. A profound down-regulation of mucosal tumour necrosis factor ␣ occurred in all the six patients who achieved a clinical response, but not in the three who did not respond. Median tumour necrosis factor ␣ score dropped from 44.8 (range 35–58.3) to 12.8 (range 5.3–15.3) in the responders (p = 0.03), whilst it remained unchanged in the non-responders. Such effect was related with a dramatic regression of the median histologic score, which dropped from 2.7 (range 2–3) to 0.5 (range 0.0–1.5) in responder patients (p = 0.002). This was related to a virtual disappearance of neutrophils in responders (r = 0.72; p = 0.002; Spearman’s test), but not in those who did not improve. Tumour necrosis factor ␣ score appeared to be correlated with the histologic, endoscopic and clinical activity. Conclusions. A profound tumour necrosis factor ␣ down-regulation appears to be strictly associated with a dramatic regression of the inflammation in patients with moderate-to-severe ulcerative colitis treated with infliximab. Such immunohistochemical effect seems to be critical for a clinical and endoscopic response to therapy. © 2007 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: Biologic therapy; Immunohistochemistry; Infliximab; TNF-␣; Ulcerative colitis
1. Introduction Inflammatory bowel diseases have been shown to be the result of the expression of a genetically determined dysregulation of the host immune response toward luminal antigens, resulting in an immunological imbalance with an excess of ∗ Corresponding author at: Ospedale Nuovo Regina Margherita, Gastroenterologia ed Endoscopia Digestiva, Via Morosini 30, 00153 Rome, Italy. Tel.: +39 06 58446541; fax: +39 06 58446533. E-mail address: [email protected] (C. Hassan).
pro-inflammatory cytokines [1]. In detail, a type 1 helper T cell response has been claimed to play a major role in Crohn’s disease (CD), whilst a type 2 helper T cell response has been implicated in the pathogenesis of ulcerative colitis (UC) [2,3]. Tumour necrosis factor ␣ (TNF-␣) – a 17 kDa polypeptide produced by macrophages, lymphocytes and natural killer cells – has been mainly implicated in the pathogenesis of type 1 helper T cell-mediated disorders, such as CD and rheumatoid arthritis [4,5]. However, high levels of TNF-␣ have also been shown in the blood, stool and intestinal tissues
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of patients with UC, suggesting a possible role even in type 2 helper T cell-mediated diseases [6–8]. Infliximab (Remicade® ; Centocor) – the first and most extensively studied biological agent for inflammatory bowel diseases – is a chimeric monoclonal antibody to TNF-␣ composed of a complement-fixing ‘human’ IgG1 constant region (75%) and a murine-derived antigen-binding variable region (25%), able to bind with high affinity to both soluble and membrane-bound TNF-␣ receptors [9]. Two large randomized, double-blind, placebo controlled trials have shown the high efficacy and safety of infliximab in inducing and maintaining a clinical and endoscopic remission in patients with moderate-to-severe active UC, similarly to the success previously reported in moderate-to-severe luminal and fistulizing CD [10–12]. In CD patients, a profound down-regulation of TNF-␣ in the affected mucosa has been associated with a dramatic histological improvement 4 weeks after infliximab therapy, strengthening the importance of such cytokine in CD pathogenesis [13]. Intriguingly, a similar decrease has been also shown in the synovial tissue of patients with rheumatoid arthritis treated with infliximab, suggesting that a profound TNF-␣ down-regulation is critical for the therapeutic success in type 1 helper T cell disorders [14]. On the other hand, it is still unknown whether in patients with UC – and generally in type 2 helper T cell-related diseases – such a profound down-regulation does occur. This evidence would be important not only to better understand the mechanism of action of the drug, but also to unveil the role played by TNF-␣ in UC pathogenesis. To address these issues, we performed a prospective study in order to assess mucosal TNF-␣ levels before and after infliximab treatment in consecutive patients with moderateto-severe UC, and to associate such values with both the clinical response, and the endoscopic and histological activity.
a total Mayo score of two points or lower, with no individual subscore exceeding one point [10]. For the purpose of the study, patients who achieved a clinical response or remission were grouped altogether as responder patients. A written informed consent was obtained from all participants in this study. 2.2. Endoscopy and histology A flexible sigmoidoscopy, unless a colonoscopy was clinically indicated, was performed before the start of the therapy and at week 10. Mayo endoscopic subscore was used to assess endoscopic activity. Mucosal healing was defined as an absolute Mayo subscore for endoscopy of 0 or 1. In each patient, at least four biopsy specimens per endoscopy were collected in the sigmoid tract. Biopsy specimens were always taken in the vicinity of lesions or ulcerations, if present. Two specimens were routinely processed and stained with H&E; the remaining specimens were used for immunohistochemistry. In detail, the H&E-stained specimens were semiquantitavely scored according to the histological disease activity. This score represents the mean value of the single scores of the following histological characteristics: polymorphonuclear infiltration of the epithelium and lamina propria, crypt abscesses, loss of glandular parallelism, crypt shortening and/or ramification, mucus epithelial depletion, involvement of muscularis mucosae and/or submucosa [16]. Each histology score ranged from 0 (normal) to 3 (1 mild, 2 moderate and 3 severe). In addition, the total number of neutrophils, lymphocytes, and plasma cells in the lamina propria was counted in five high-power fields (original magnification 400×), selected on the basis of high cellular density, and it was expressed as number of cells/mm2 through a dedicated software (Leica Q Win, Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). 2.3. Immunohistochemistry
2. Methods 2.1. Patients Consecutive patients with an indication to infliximab therapy because of steroid-dependant or steroid-refractory moderate-to-severe UC (Mayo score ≥6) [15] were considered for enrolment. Exclusion criteria were a positive tuberculin skin test, chest X-ray alterations, a previous use of anti-TNF agents, or a diagnosis of indeterminate colitis or Crohn’s disease at the entry endoscopy. Infliximab was administered at weeks 0, 2 and 6, respectively at a standard dose of 5 mg/kg. Mayo score was assessed at enrolment and at week 10, when endoscopy was also performed. Clinical response at week 10 was defined as a decrease from baseline in the total Mayo score of at least three points and at least 30%, with an accompanying decrease in the subscore for rectal bleeding of at least one point or an absolute subscore for rectal bleeding of 0 or 1. Clinical remission was defined as
TNF-␣ expression was assessed by immunohistochemistry, as we previously reported [17]. The cytokine was stained using a polyclonal rabbit antibody (PromoCell, Heidelberg, Germany). The reaction was revealed using a peroxidase/anti-peroxidase (PAP) technique with goat antirabbit immunoglobulins (Dako, Copenhagen, Denmark) and a complex of rabbit antibodies and horseradish peroxidase (Dako, Copenhagen, Denmark). A positive reaction was revealed by aminoethylcarbazole and counterstained by aqueous haematoxylin. For each staining procedure we used positive (Helicobacter pylori chronic gastritis with severe activity) and negative (normal stomach) controls. In the immunoperoxidase-stained biopsy specimens, TNF-␣ score was defined as the percentage of positive stained stromal cells compared with the total number of lamina propria mononuclear cells, counted in five high-power fields (original magnification 400×, at least 1000 total counted cells), randomly selected. Samples from an equal number of sub-
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Fig. 1. Histological changes in colonic biopsies before and after infliximab therapy (H&E; original magnification 400×). (A) Active UC characterized by a dense inflammatory infiltrate of lymphocytes, plasmacells and polymorfonuclear granulocytes, disturbed crypt architecture and epithelial damage with mucus depletion (pointed) and a crypt abscess (pointed) which dramatically improve after therapy (B) with regression of the inflammatory infiltrate and restoring of the epithelial damage with a quite normal goblet cell population.
jects (matched for sex and age) with irritable bowel syndrome (IBS), in whom colonoscopy had been performed for abdominal pain, were used as control. In IBS biopsy samples showed histologically normal epithelium and mucosa. For the purpose of the study, all biopsy specimens were assessed by a single expert gastrointestinal pathologist (E.I.) without prior knowledge of the time of treatment. Biopsy specimens from the same patient before and after treatment, both taken from the most severely inflamed areas, were compared. The results were also compared with those obtained from IBS patients.
3. Statistical analysis Data analysis was performed by using Student’s t-test, Wilcoxon’s test and Mann–Whitney U test as appropriate. Sample size was calculated, as suggested by Sokal and Rohlf [18]. A possible correlation among different findings was evaluated according to the Spearman’s test. A p value <0.05 was considered as indicative of statistical significance.
4. Results Nine patients were enrolled between March 2005 and February 2006. The median Mayo score at entry was 8
(range: 7–11). All patients had a moderate or severe disease activity at flexible sigmoidoscopy (median endoscopic Mayo subscore: 3, range 2–3). Similarly, a moderate to severe inflammatory activity was detected in all biopsies (median histologic score: 3, range 2–3) (Fig. 1). This was associated with a high number of neutrophils (median: 17.2/mm2 ; range 7.8–20), and a moderate number of lymphocytes (median: 7.5/mm2 ; range 4.5–14) and plasma cells (median: 6.3/mm2 ; range 5.4–12.4). Such values were statistically significantly higher as compared to IBS controls (median neutrophils: 1.6/mm2 ; range: 0–3; p < 0.01; median lymphocytes: 4.1/mm2 ; range: 3–5.5; p = 0.04; median plasma cells: 4.3/mm2 : 2.7–4.8; p < 0.01). The increased histologic activity in UC patients was also related with a high expression of TNF-␣ in the colonica mucosa. Immunohistochemical staining showed a TNF-␣ score of 40% (range: 29–58.3%) (Fig. 2). All patients received the scheduled doses of infliximab at weeks 0, 2 and 6, respectively. At week 10, a clinical remission was achieved in two (22.2%) patients, a clinical response in further four (44.5%), whilst no response was observed in the remaining three (33.3%) cases. Characteristics of responder (remission or response) and non-responder patients are given in Table 1. As shown in Fig. 3, Mayo score significantly improved in the responders as compared to baseline values (median: 3.5; range 1–5; p = 0.002), but not in the patients who did not respond (median: 7; range: 6–9; p = 0.6). Flexible sigmoidoscopy at week 10 showed a significant improvement
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Fig. 2. Immunohistochemical changes in colonic TNF-␣ expression before and after infliximab therapy (immunohistochemical stains; original magnification 400×). A high TNF-␣ level may be depicted before therapy with positive cells stained red (pointed) and negative ones stained blue (haematoxylin) (A), with a profound down-regulation after treatment (B).
Fig. 3. Changes of clinical (A), endoscopic (B) and histological scores (C) after infliximab treatment in responder () and non-responder () patients.
Table 1 Demographic and clinical data at entry Age (year), mean ± S.D. Sex (M/F) Duration of disease, mean ± S.D. Mayo score, median (range) Colonic area involved Left-side Extensive Endoscopic Mayo subscore, median (range) Histologic score, median (range) Concomitant medications Sulfasalazine/mesalamine Corticosteroids Azathioprine
Responder (n = 6)
Non-responder (n = 3)
48 ± 17 4/2 7.5 ± 2.2
46 ± 15 2/1 5.3 ± 1.5
9 (7–11)
8 (7–8)
2 4
1 2
3 (2–3)
2 (2–3)
2.75 (2–3)
3 (2–3)
4
2
5 5
3 2
of the Mayo subscore in the responder patients (median: 1; range: 0–2; p = 0.004), whilst no change was observed in the non-responder group (median: 3; range: 2–3; p = 0.6). In detail, mucosal healing occurred in five responder patients and in none of the failure patients. After therapy, a dramatic regression of the inflammatory activity was observed. As shown in Fig. 3, the histological score significantly decreased in the responder group (median: 0.5; range: 0–1.5; p = 0.002), whilst no change was observed in those non-responders (median: 3; range: 2–3). Among responders, mucosal architecture returned to normal in two cases, whilst an incomplete recovery was observed in the remaining cases. As shown in Table 2, the histological improvement was mainly due to an impressive regression of the number of neutrophils associated to the restoration of a normal crypt architecture and mucus
C. Hassan et al. / Digestive and Liver Disease 39 (2007) 811–817
Fig. 4. Changes of TNF-␣ score (A) and absolute number of neutrophils (B) after infliximab treatment in responders () and non-responders ().
content in epithelial cells (see also Fig. 4). However, the number of neutrophils after therapy (median: 4.9/mm2 ; range: 1.9–7.1) was still significantly higher than that observed in IBS controls (median: 1.6/mm2 ; range: 0–3; p = 0.04). On the other hand, no significant reduction of mononuclear cell infiltration was observed both in responders and non-responders. The overall number of TNF-␣ positive cells dramatically decreased after infliximab therapy in responders, with no significant change observed in non-responders (Table 2). TNF-␣ score at week 10 was not only significantly reduced in the responder patients, but it was also similar to that observed in the IBS controls (median: 8%; range 4–12%; p = 0.2). Interestingly, the number of positive cells was more than halved as compared to pre-treatment values in all responder patients (Fig. 4). Grouping altogether pre- and post-treatment values, TNF-␣ score appeared to be correlated with the Mayo score (r = 0.78; p = 0.0004), the endoscopic Mayo subscore (r = 0.80; p = 0.0003), the histologic score (r = 0.59; p = 0.001), and with the number of neutrophils (r = 0.72; p = 0.002).
5. Discussion Infliximab seems to trigger a long-lasting profound down-regulation of mucosal TNF-␣ expression in patients
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with moderate-to-severe UC. Indeed, a dramatic decline of mucosal TNF-␣ levels was detected after 10 weeks after the first infusion in our study. Moreover, such down-regulation appeared to be intimately associated with an impressive regression of the active inflammatory process. In particular, an almost complete disappearance of neutrophils occurred in our population. Importantly, such an immunohistochemical effect was strongly associated with both clinical and endoscopic improvement. Notably, TNF-␣ down-regulation, histological regression, and endoscopic improvement were purely confined to those patients achieving a clinical response/remission. In fact, we did not detect TNF-␣ down-regulation or significant reduction of the histological activity in patients who did not respond to infliximab therapy. It has been recently suggested that therapeutic failure in rheumatoid arthritis may be related to the polymorphism at position -308 in the promoter of TNF-␣ gene [19]. Whether a poor response in UC is also associated with an A/A genotype in this locus needs to be assessed by further studies. UC pathogenesis has been widely related with a type 2 helper T cell response, contrary to CD, which has been considered a type 1 helper T cell-related disease [20]. Our data heavily questions the validity and the clinical usefulness of such a classification. TNF-␣, a type 1 cytokine, appeared to play a major role in UC pathogenesis in our study. Not only it was over-expressed in patients with an active disease but also its down-regulation following a blocking antibody was necessary to achieve a histological regression and a clinical and endoscopic response. Such evidence tightly mirrors the high efficacy of infliximab in inducing and maintaining remission in moderate-to-severe UC patients in two large controlled trials [10]. To address this discrepancy, it should be pointed out that the identification in vitro of two types of murine helper T cell clones – type 1 and 2 – has never been supported by their detection in human diseases, whilst the pattern of cytokine produced – either type 1 or 2 – has been used to classify a human disorder as a type 1 or 2 T helper mediated [21]. However, the production of only one type of cytokines has not been described in any human disease. For example, IL-4 – a type 2 cytokine – has been described in CD (reported as being a Th-1 disease), whilst TNF-␣, a type 1 cytokine, has been reported in UC as well, so far described as a Th-2 disease [13,6–8]. Similarly, the efficacy of the biological drugs does not seem to be necessarily associated with such a classification. For example, a therapeutic attempt with IL-10 – an inhibitory type 2 cytokine – has failed in UC [22]. To further confound our confidence, infliximab treatment has been associated with a down-regulation of type 2 cytokines in CD and rheumatoid arthritis, such as IL-4 [13]. Taking all these findings into account, it seems advisable not to prevent a potentially effective and lifesaving biologic therapy only on the basis of our knowledge of the pathogenesis of immune-mediated disorders, which should, on the other hand, be reserved mainly for research purposes.
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Table 2 TNF-␣ scores and number of inflammatory cells per mm2 at baseline (week 0) and after infliximab treatment (week 10) in mucosal biopsies of responders and not responders Respondersa
TNF-␣ score (%) Neutrophils (mm2 ) Lymphocytes (mm2 ) Plasmacells (mm2 ) a b
Non-respondersa
p
Week 0
Week 10
Week 0
Week 10
44.8 (35–58.3) 18.9 (7.8–20) 6 (4.5–14) 8.6 (6–12.4)
12.8 (5.3–15.3) 4.9 (1.9–7.1) 8 (0.4–12.6) 5.9 (3.3–7.9)
32 (29–42) 15 (10–17.2) 9 (7.2–11) 5.8 (5.4–7)
30.2 (30–39.8) 13.7 (11.3–16) 10 (7.2–10.1) 6.8 (6-8)
0.002b 0.04b NS NS
All values are expressed as median (range). Only for responder cases.
The immunohistological response to infliximab observed in our UC patients closely resembled the same effect previously described in CD by Baert et al. [13], who detected a profound down-regulation of TNF-␣ associated with a dramatic regression of the inflammatory activity by 4 weeks after a single infusion of infliximab. Our study reported practically the same scenario in a different disease after a longer time. Suggestively, we also described a similar down-regulation 1 year after the first infliximab infusion in a case of segmental colitis associated with diverticula [23]. All these observations seem to indicate that the long-term effect of infliximab in inflammatory bowel diseases is mainly related to the persistence of the TNF-␣ down-regulation and to the related inhibition of the inflammatory process. However, the reasons of this long-lasting effect are poorly understood. It has been recently suggested that one of the possible mechanisms is related to the apoptosis of TNF-␣ expressing cells responsible for mucosal inflammation, such as monocytes and lymphocytes, via antibody-dependent cell-mediated and complement-dependent cytotoxicity [24,25]. Nevertheless, we failed to detect possible signs of cellular lysis, such as nuclear dust or an increase of pigment-loaded macrophages, similarly to what previously reported by Baert [13]. Alternatively, it could be speculated that the therapeutic effect is related to an inhibition of the migration of inflammatory cells from the systemic circulation through the bowel wall into the lamina propria of the intestinal mucosa. Such cell trafficking is mainly regulated by a variety of adhesion molecules among which ICAM-1 and its ligand LFA-1, as well as syndecan-1, have been shown to play an important role in inflammatory bowel diseases [26,27]. However, a reduction of these molecules has not been described neither in rheumatoid arthritis nor in CD following infliximab infusions [13,28]. In conclusion, our study has shown a profound downregulation of TNF-␣ in moderate-to-severe UC after infliximab, providing a compelling rationale for such a treatment. Infliximab appeared to be able to trigger a “reset of the immunostat” in a type 2 helper T cell-related disorder, contrary to what previously believed [29]. A further evidence that the more we struggle to unravel the inflammatory puzzle, the more puzzling the pathogenetic enigma of inflammatory bowel diseases appears to be.
Practice points • A profound down-regulation of TNF-alpha occurs after infliximab therapy in ulcerative colitis. • TNF-alpha down-regulation is confined only to those patients who achieved a clinical response. • Such down-regulation is associated with a dramatic regression of the inflammatory process, and with an endoscopic and clinical improvement.
Research agenda • Genetic factors responsible for anti-TNF-␣ therapeutic failure in non-responder patients. • Immunological mechanisms responsible for TNF-␣ recurrence in responder patients. • Immunohistochemical factors which may predict therapeutic response, similarly to oestrogen receptors in breast cancer.
Conflict of interest statement None declared.
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Alimentary Tract
Tumour necrosis factor alpha down-regulation parallels inflammatory regression in ulcerative colitis patients treated with infliximab C. Hassan a,∗ , E. Ierardi b , O. Burattini b , V. De Francesco b , A. Zullo a , G. Stoppino b , C. Panella b , S. Morini a a
Gastroenterology and Digestive Endoscopy Unit, “Nuovo Regina Margherita” Hospital, Rome, Italy Section of Gastroenterology, Department of Medical Sciences, University of Foggia, Foggia, Italy
b
Received 4 February 2007; accepted 6 June 2007 Available online 25 July 2007
Abstract Background. Treatment with the anti-tumour necrosis factor ␣ monoclonal antibody infliximab has been shown to be effective in moderateto-severe ulcerative colitis. However, its effect on the mucosal histopathological abnormalities of this disease is largely unknown. This study aimed to assess the immunohistological effect of infliximab in ulcerative colitis. Methods. Nine patients with moderate-to-severe ulcerative colitis received infliximab (5 mg/kg) at weeks 0, 2 and 6, respectively. Colonic biopsies were collected before therapy and at week 10, when the Mayo score (including the endoscopic subscore) was also assessed. Severity of inflammation was evaluated by histologic score and histomorphometry. Immunohistochemical staining with antibody against tumour necrosis factor ␣ was performed on all biopsies and expressed as percentage of positive stromal cells/1000 counted (tumour necrosis factor ␣ score). Results. A profound down-regulation of mucosal tumour necrosis factor ␣ occurred in all the six patients who achieved a clinical response, but not in the three who did not respond. Median tumour necrosis factor ␣ score dropped from 44.8 (range 35–58.3) to 12.8 (range 5.3–15.3) in the responders (p = 0.03), whilst it remained unchanged in the non-responders. Such effect was related with a dramatic regression of the median histologic score, which dropped from 2.7 (range 2–3) to 0.5 (range 0.0–1.5) in responder patients (p = 0.002). This was related to a virtual disappearance of neutrophils in responders (r = 0.72; p = 0.002; Spearman’s test), but not in those who did not improve. Tumour necrosis factor ␣ score appeared to be correlated with the histologic, endoscopic and clinical activity. Conclusions. A profound tumour necrosis factor ␣ down-regulation appears to be strictly associated with a dramatic regression of the inflammation in patients with moderate-to-severe ulcerative colitis treated with infliximab. Such immunohistochemical effect seems to be critical for a clinical and endoscopic response to therapy. © 2007 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: Biologic therapy; Immunohistochemistry; Infliximab; TNF-␣; Ulcerative colitis
1. Introduction Inflammatory bowel diseases have been shown to be the result of the expression of a genetically determined dysregulation of the host immune response toward luminal antigens, resulting in an immunological imbalance with an excess of ∗ Corresponding author at: Ospedale Nuovo Regina Margherita, Gastroenterologia ed Endoscopia Digestiva, Via Morosini 30, 00153 Rome, Italy. Tel.: +39 06 58446541; fax: +39 06 58446533. E-mail address: [email protected] (C. Hassan).
pro-inflammatory cytokines [1]. In detail, a type 1 helper T cell response has been claimed to play a major role in Crohn’s disease (CD), whilst a type 2 helper T cell response has been implicated in the pathogenesis of ulcerative colitis (UC) [2,3]. Tumour necrosis factor ␣ (TNF-␣) – a 17 kDa polypeptide produced by macrophages, lymphocytes and natural killer cells – has been mainly implicated in the pathogenesis of type 1 helper T cell-mediated disorders, such as CD and rheumatoid arthritis [4,5]. However, high levels of TNF-␣ have also been shown in the blood, stool and intestinal tissues
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of patients with UC, suggesting a possible role even in type 2 helper T cell-mediated diseases [6–8]. Infliximab (Remicade® ; Centocor) – the first and most extensively studied biological agent for inflammatory bowel diseases – is a chimeric monoclonal antibody to TNF-␣ composed of a complement-fixing ‘human’ IgG1 constant region (75%) and a murine-derived antigen-binding variable region (25%), able to bind with high affinity to both soluble and membrane-bound TNF-␣ receptors [9]. Two large randomized, double-blind, placebo controlled trials have shown the high efficacy and safety of infliximab in inducing and maintaining a clinical and endoscopic remission in patients with moderate-to-severe active UC, similarly to the success previously reported in moderate-to-severe luminal and fistulizing CD [10–12]. In CD patients, a profound down-regulation of TNF-␣ in the affected mucosa has been associated with a dramatic histological improvement 4 weeks after infliximab therapy, strengthening the importance of such cytokine in CD pathogenesis [13]. Intriguingly, a similar decrease has been also shown in the synovial tissue of patients with rheumatoid arthritis treated with infliximab, suggesting that a profound TNF-␣ down-regulation is critical for the therapeutic success in type 1 helper T cell disorders [14]. On the other hand, it is still unknown whether in patients with UC – and generally in type 2 helper T cell-related diseases – such a profound down-regulation does occur. This evidence would be important not only to better understand the mechanism of action of the drug, but also to unveil the role played by TNF-␣ in UC pathogenesis. To address these issues, we performed a prospective study in order to assess mucosal TNF-␣ levels before and after infliximab treatment in consecutive patients with moderateto-severe UC, and to associate such values with both the clinical response, and the endoscopic and histological activity.
a total Mayo score of two points or lower, with no individual subscore exceeding one point [10]. For the purpose of the study, patients who achieved a clinical response or remission were grouped altogether as responder patients. A written informed consent was obtained from all participants in this study. 2.2. Endoscopy and histology A flexible sigmoidoscopy, unless a colonoscopy was clinically indicated, was performed before the start of the therapy and at week 10. Mayo endoscopic subscore was used to assess endoscopic activity. Mucosal healing was defined as an absolute Mayo subscore for endoscopy of 0 or 1. In each patient, at least four biopsy specimens per endoscopy were collected in the sigmoid tract. Biopsy specimens were always taken in the vicinity of lesions or ulcerations, if present. Two specimens were routinely processed and stained with H&E; the remaining specimens were used for immunohistochemistry. In detail, the H&E-stained specimens were semiquantitavely scored according to the histological disease activity. This score represents the mean value of the single scores of the following histological characteristics: polymorphonuclear infiltration of the epithelium and lamina propria, crypt abscesses, loss of glandular parallelism, crypt shortening and/or ramification, mucus epithelial depletion, involvement of muscularis mucosae and/or submucosa [16]. Each histology score ranged from 0 (normal) to 3 (1 mild, 2 moderate and 3 severe). In addition, the total number of neutrophils, lymphocytes, and plasma cells in the lamina propria was counted in five high-power fields (original magnification 400×), selected on the basis of high cellular density, and it was expressed as number of cells/mm2 through a dedicated software (Leica Q Win, Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). 2.3. Immunohistochemistry
2. Methods 2.1. Patients Consecutive patients with an indication to infliximab therapy because of steroid-dependant or steroid-refractory moderate-to-severe UC (Mayo score ≥6) [15] were considered for enrolment. Exclusion criteria were a positive tuberculin skin test, chest X-ray alterations, a previous use of anti-TNF agents, or a diagnosis of indeterminate colitis or Crohn’s disease at the entry endoscopy. Infliximab was administered at weeks 0, 2 and 6, respectively at a standard dose of 5 mg/kg. Mayo score was assessed at enrolment and at week 10, when endoscopy was also performed. Clinical response at week 10 was defined as a decrease from baseline in the total Mayo score of at least three points and at least 30%, with an accompanying decrease in the subscore for rectal bleeding of at least one point or an absolute subscore for rectal bleeding of 0 or 1. Clinical remission was defined as
TNF-␣ expression was assessed by immunohistochemistry, as we previously reported [17]. The cytokine was stained using a polyclonal rabbit antibody (PromoCell, Heidelberg, Germany). The reaction was revealed using a peroxidase/anti-peroxidase (PAP) technique with goat antirabbit immunoglobulins (Dako, Copenhagen, Denmark) and a complex of rabbit antibodies and horseradish peroxidase (Dako, Copenhagen, Denmark). A positive reaction was revealed by aminoethylcarbazole and counterstained by aqueous haematoxylin. For each staining procedure we used positive (Helicobacter pylori chronic gastritis with severe activity) and negative (normal stomach) controls. In the immunoperoxidase-stained biopsy specimens, TNF-␣ score was defined as the percentage of positive stained stromal cells compared with the total number of lamina propria mononuclear cells, counted in five high-power fields (original magnification 400×, at least 1000 total counted cells), randomly selected. Samples from an equal number of sub-
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Fig. 1. Histological changes in colonic biopsies before and after infliximab therapy (H&E; original magnification 400×). (A) Active UC characterized by a dense inflammatory infiltrate of lymphocytes, plasmacells and polymorfonuclear granulocytes, disturbed crypt architecture and epithelial damage with mucus depletion (pointed) and a crypt abscess (pointed) which dramatically improve after therapy (B) with regression of the inflammatory infiltrate and restoring of the epithelial damage with a quite normal goblet cell population.
jects (matched for sex and age) with irritable bowel syndrome (IBS), in whom colonoscopy had been performed for abdominal pain, were used as control. In IBS biopsy samples showed histologically normal epithelium and mucosa. For the purpose of the study, all biopsy specimens were assessed by a single expert gastrointestinal pathologist (E.I.) without prior knowledge of the time of treatment. Biopsy specimens from the same patient before and after treatment, both taken from the most severely inflamed areas, were compared. The results were also compared with those obtained from IBS patients.
3. Statistical analysis Data analysis was performed by using Student’s t-test, Wilcoxon’s test and Mann–Whitney U test as appropriate. Sample size was calculated, as suggested by Sokal and Rohlf [18]. A possible correlation among different findings was evaluated according to the Spearman’s test. A p value <0.05 was considered as indicative of statistical significance.
4. Results Nine patients were enrolled between March 2005 and February 2006. The median Mayo score at entry was 8
(range: 7–11). All patients had a moderate or severe disease activity at flexible sigmoidoscopy (median endoscopic Mayo subscore: 3, range 2–3). Similarly, a moderate to severe inflammatory activity was detected in all biopsies (median histologic score: 3, range 2–3) (Fig. 1). This was associated with a high number of neutrophils (median: 17.2/mm2 ; range 7.8–20), and a moderate number of lymphocytes (median: 7.5/mm2 ; range 4.5–14) and plasma cells (median: 6.3/mm2 ; range 5.4–12.4). Such values were statistically significantly higher as compared to IBS controls (median neutrophils: 1.6/mm2 ; range: 0–3; p < 0.01; median lymphocytes: 4.1/mm2 ; range: 3–5.5; p = 0.04; median plasma cells: 4.3/mm2 : 2.7–4.8; p < 0.01). The increased histologic activity in UC patients was also related with a high expression of TNF-␣ in the colonica mucosa. Immunohistochemical staining showed a TNF-␣ score of 40% (range: 29–58.3%) (Fig. 2). All patients received the scheduled doses of infliximab at weeks 0, 2 and 6, respectively. At week 10, a clinical remission was achieved in two (22.2%) patients, a clinical response in further four (44.5%), whilst no response was observed in the remaining three (33.3%) cases. Characteristics of responder (remission or response) and non-responder patients are given in Table 1. As shown in Fig. 3, Mayo score significantly improved in the responders as compared to baseline values (median: 3.5; range 1–5; p = 0.002), but not in the patients who did not respond (median: 7; range: 6–9; p = 0.6). Flexible sigmoidoscopy at week 10 showed a significant improvement
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Fig. 2. Immunohistochemical changes in colonic TNF-␣ expression before and after infliximab therapy (immunohistochemical stains; original magnification 400×). A high TNF-␣ level may be depicted before therapy with positive cells stained red (pointed) and negative ones stained blue (haematoxylin) (A), with a profound down-regulation after treatment (B).
Fig. 3. Changes of clinical (A), endoscopic (B) and histological scores (C) after infliximab treatment in responder () and non-responder () patients.
Table 1 Demographic and clinical data at entry Age (year), mean ± S.D. Sex (M/F) Duration of disease, mean ± S.D. Mayo score, median (range) Colonic area involved Left-side Extensive Endoscopic Mayo subscore, median (range) Histologic score, median (range) Concomitant medications Sulfasalazine/mesalamine Corticosteroids Azathioprine
Responder (n = 6)
Non-responder (n = 3)
48 ± 17 4/2 7.5 ± 2.2
46 ± 15 2/1 5.3 ± 1.5
9 (7–11)
8 (7–8)
2 4
1 2
3 (2–3)
2 (2–3)
2.75 (2–3)
3 (2–3)
4
2
5 5
3 2
of the Mayo subscore in the responder patients (median: 1; range: 0–2; p = 0.004), whilst no change was observed in the non-responder group (median: 3; range: 2–3; p = 0.6). In detail, mucosal healing occurred in five responder patients and in none of the failure patients. After therapy, a dramatic regression of the inflammatory activity was observed. As shown in Fig. 3, the histological score significantly decreased in the responder group (median: 0.5; range: 0–1.5; p = 0.002), whilst no change was observed in those non-responders (median: 3; range: 2–3). Among responders, mucosal architecture returned to normal in two cases, whilst an incomplete recovery was observed in the remaining cases. As shown in Table 2, the histological improvement was mainly due to an impressive regression of the number of neutrophils associated to the restoration of a normal crypt architecture and mucus
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Fig. 4. Changes of TNF-␣ score (A) and absolute number of neutrophils (B) after infliximab treatment in responders () and non-responders ().
content in epithelial cells (see also Fig. 4). However, the number of neutrophils after therapy (median: 4.9/mm2 ; range: 1.9–7.1) was still significantly higher than that observed in IBS controls (median: 1.6/mm2 ; range: 0–3; p = 0.04). On the other hand, no significant reduction of mononuclear cell infiltration was observed both in responders and non-responders. The overall number of TNF-␣ positive cells dramatically decreased after infliximab therapy in responders, with no significant change observed in non-responders (Table 2). TNF-␣ score at week 10 was not only significantly reduced in the responder patients, but it was also similar to that observed in the IBS controls (median: 8%; range 4–12%; p = 0.2). Interestingly, the number of positive cells was more than halved as compared to pre-treatment values in all responder patients (Fig. 4). Grouping altogether pre- and post-treatment values, TNF-␣ score appeared to be correlated with the Mayo score (r = 0.78; p = 0.0004), the endoscopic Mayo subscore (r = 0.80; p = 0.0003), the histologic score (r = 0.59; p = 0.001), and with the number of neutrophils (r = 0.72; p = 0.002).
5. Discussion Infliximab seems to trigger a long-lasting profound down-regulation of mucosal TNF-␣ expression in patients
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with moderate-to-severe UC. Indeed, a dramatic decline of mucosal TNF-␣ levels was detected after 10 weeks after the first infusion in our study. Moreover, such down-regulation appeared to be intimately associated with an impressive regression of the active inflammatory process. In particular, an almost complete disappearance of neutrophils occurred in our population. Importantly, such an immunohistochemical effect was strongly associated with both clinical and endoscopic improvement. Notably, TNF-␣ down-regulation, histological regression, and endoscopic improvement were purely confined to those patients achieving a clinical response/remission. In fact, we did not detect TNF-␣ down-regulation or significant reduction of the histological activity in patients who did not respond to infliximab therapy. It has been recently suggested that therapeutic failure in rheumatoid arthritis may be related to the polymorphism at position -308 in the promoter of TNF-␣ gene [19]. Whether a poor response in UC is also associated with an A/A genotype in this locus needs to be assessed by further studies. UC pathogenesis has been widely related with a type 2 helper T cell response, contrary to CD, which has been considered a type 1 helper T cell-related disease [20]. Our data heavily questions the validity and the clinical usefulness of such a classification. TNF-␣, a type 1 cytokine, appeared to play a major role in UC pathogenesis in our study. Not only it was over-expressed in patients with an active disease but also its down-regulation following a blocking antibody was necessary to achieve a histological regression and a clinical and endoscopic response. Such evidence tightly mirrors the high efficacy of infliximab in inducing and maintaining remission in moderate-to-severe UC patients in two large controlled trials [10]. To address this discrepancy, it should be pointed out that the identification in vitro of two types of murine helper T cell clones – type 1 and 2 – has never been supported by their detection in human diseases, whilst the pattern of cytokine produced – either type 1 or 2 – has been used to classify a human disorder as a type 1 or 2 T helper mediated [21]. However, the production of only one type of cytokines has not been described in any human disease. For example, IL-4 – a type 2 cytokine – has been described in CD (reported as being a Th-1 disease), whilst TNF-␣, a type 1 cytokine, has been reported in UC as well, so far described as a Th-2 disease [13,6–8]. Similarly, the efficacy of the biological drugs does not seem to be necessarily associated with such a classification. For example, a therapeutic attempt with IL-10 – an inhibitory type 2 cytokine – has failed in UC [22]. To further confound our confidence, infliximab treatment has been associated with a down-regulation of type 2 cytokines in CD and rheumatoid arthritis, such as IL-4 [13]. Taking all these findings into account, it seems advisable not to prevent a potentially effective and lifesaving biologic therapy only on the basis of our knowledge of the pathogenesis of immune-mediated disorders, which should, on the other hand, be reserved mainly for research purposes.
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Table 2 TNF-␣ scores and number of inflammatory cells per mm2 at baseline (week 0) and after infliximab treatment (week 10) in mucosal biopsies of responders and not responders Respondersa
TNF-␣ score (%) Neutrophils (mm2 ) Lymphocytes (mm2 ) Plasmacells (mm2 ) a b
Non-respondersa
p
Week 0
Week 10
Week 0
Week 10
44.8 (35–58.3) 18.9 (7.8–20) 6 (4.5–14) 8.6 (6–12.4)
12.8 (5.3–15.3) 4.9 (1.9–7.1) 8 (0.4–12.6) 5.9 (3.3–7.9)
32 (29–42) 15 (10–17.2) 9 (7.2–11) 5.8 (5.4–7)
30.2 (30–39.8) 13.7 (11.3–16) 10 (7.2–10.1) 6.8 (6-8)
0.002b 0.04b NS NS
All values are expressed as median (range). Only for responder cases.
The immunohistological response to infliximab observed in our UC patients closely resembled the same effect previously described in CD by Baert et al. [13], who detected a profound down-regulation of TNF-␣ associated with a dramatic regression of the inflammatory activity by 4 weeks after a single infusion of infliximab. Our study reported practically the same scenario in a different disease after a longer time. Suggestively, we also described a similar down-regulation 1 year after the first infliximab infusion in a case of segmental colitis associated with diverticula [23]. All these observations seem to indicate that the long-term effect of infliximab in inflammatory bowel diseases is mainly related to the persistence of the TNF-␣ down-regulation and to the related inhibition of the inflammatory process. However, the reasons of this long-lasting effect are poorly understood. It has been recently suggested that one of the possible mechanisms is related to the apoptosis of TNF-␣ expressing cells responsible for mucosal inflammation, such as monocytes and lymphocytes, via antibody-dependent cell-mediated and complement-dependent cytotoxicity [24,25]. Nevertheless, we failed to detect possible signs of cellular lysis, such as nuclear dust or an increase of pigment-loaded macrophages, similarly to what previously reported by Baert [13]. Alternatively, it could be speculated that the therapeutic effect is related to an inhibition of the migration of inflammatory cells from the systemic circulation through the bowel wall into the lamina propria of the intestinal mucosa. Such cell trafficking is mainly regulated by a variety of adhesion molecules among which ICAM-1 and its ligand LFA-1, as well as syndecan-1, have been shown to play an important role in inflammatory bowel diseases [26,27]. However, a reduction of these molecules has not been described neither in rheumatoid arthritis nor in CD following infliximab infusions [13,28]. In conclusion, our study has shown a profound downregulation of TNF-␣ in moderate-to-severe UC after infliximab, providing a compelling rationale for such a treatment. Infliximab appeared to be able to trigger a “reset of the immunostat” in a type 2 helper T cell-related disorder, contrary to what previously believed [29]. A further evidence that the more we struggle to unravel the inflammatory puzzle, the more puzzling the pathogenetic enigma of inflammatory bowel diseases appears to be.
Practice points • A profound down-regulation of TNF-alpha occurs after infliximab therapy in ulcerative colitis. • TNF-alpha down-regulation is confined only to those patients who achieved a clinical response. • Such down-regulation is associated with a dramatic regression of the inflammatory process, and with an endoscopic and clinical improvement.
Research agenda • Genetic factors responsible for anti-TNF-␣ therapeutic failure in non-responder patients. • Immunological mechanisms responsible for TNF-␣ recurrence in responder patients. • Immunohistochemical factors which may predict therapeutic response, similarly to oestrogen receptors in breast cancer.
Conflict of interest statement None declared.
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