ORIGINAL ARTICLE

Infliximab therapy decreases the levels of TNF-a and IFN-gmRNA in colonic mucosa of ulcerative colitis

TRINE OLSEN1, GUANGLIN CUI1, RASMUS GOLL1, ANNE HUSEBEKK2 &

JON FLORHOLMEN3

1Laboratory of Gastroenterology, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway, 2Department

of Immunology, and 3Department of Medical Gastroenterology, University Hospital North Norway, Tromsø, Norway

AbstractObjective. The mechanisms of action of infliximab (IFX) in the treatment of ulcerative colitis (UC) are poorly understood.The aim of the study was to investigate the changes in tissue expression of tumor necrosis factor-alpha (TNF-a) and othercytokines in UC patients receiving IFX treatment. Material and methods. The levels of TNF-a, interleukin (IL)-10, IL-4,and interferon-gamma (IFN-g) mRNA in colonic biopsies from 32 UC patients during IFX treatment were measured byreal-time polymerase chain reaction (PCR) and compared with those of 19 controls. Immunohistochemistry was performedto characterize the changes of inflammatory cells during treatment. Results. IFX reduced the expression of TNF-a andIFN-g mRNA, but not that of IL-10 and IL-4 mRNA. Reductions in TNF-a mRNA were correlated to clinical andendoscopic improvements, and normalization of TNF-a mRNA was obtained in patients with healed mucosa. The numbersof T lymphocytes and macrophages were significantly decreased in patients with healed mucosa after IFX treatment,although compared to normal controls, there were still increased levels of TNF-a-positive cells after treatment.Conclusions. IFX induced down-regulation of the mucosal TNF-a and IFN-g mRNA expression in UC patients. Thenumbers of T lymphocytes and macrophages were significantly decreased in patients with endoscopically healed mucosaafter IFX treatment.

Key Words: Cytokines, immunohistochemistry, inflammatory bowel disease, quantitative real-time PCR

Introduction

The roles of tumor necrosis factor-alpha (TNF-a)

and the T-cell profile in the pathogenesis of active

ulcerative colitis (UC) are controversial issues [1�5].

UC has been considered a T-helper-2 (Th-2) cell

driven disease with a pathogenesis suggested to be

influenced by other cytokines than TNF-a [6�8].

However, we have recently demonstrated an in-

creased TNF-a mRNA level in inflamed colonic

tissues and its impact on the inflammatory process

[9]. In support of this view is the observed sub-

stantial effect of infliximab (IFX), an anti-TNF-aantibody, in moderate to severe UC [10,11].

The mechanisms of action underlying the ther-

apeutic effects of anti-TNF antibodies used in

inflammatory bowel disease (IBD) are not fully

understood [12]. IFX binds with high affinity to

both soluble and transmembrane TNF-a receptors

[13]. Recently, a possible interaction between IFX

and transmembrane TNF-a mechanisms has been

reported [13,14]. Proposed mechanisms are antago-

nist action by blocking membrane TNF interactions

and agonist actions by initiating reverse signaling,

leading to apoptosis, cell activation, or cytokine

suppression [15�18].

Several groups have focused on the effects of IFX

in Crohn’s disease (CD) [16,19,20]. As far as we

know, only one study has been published on the

effect of IFX in UC [21]. In this small study based

on histological data, IFX reduced the tissue levels of

TNF-a according to the clinical responses [21].

Recently, we have demonstrated increased levels of

TNF-a, IL-4, IL-10, IL-18, and IFN- g mRNA in

mucosa of patients with untreated UC [9]. The aims

Correspondence: Trine Olsen, MD, Laboratory of Gastroenterology, Institute of Clinical Medicine, University of Tromsø, NO-9037, Tromsø, Norway.

Tel: �47 77 644 847. Fax: �47 77 626 670. E-mail: Trine.Olsen@fagmed.uit.no

Scandinavian Journal of Gastroenterology, 2009; 44: 727�735

(Received 16 December 2008; accepted 2 February 2009)

ISSN 0036-5521 print/ISSN 1502-7708 online # 2009 Informa UK Ltd.

DOI: 10.1080/00365520902803507

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of our study were: 1) to correlate the changes in

tissue expression of TNF-a in UC patients to the

clinical effect of IFX treatment, 2) to assess the

effects of IFX on IFN-g, IL-4, and IL-10 levels, and

3) to characterize the IFX response on colorectal

inflammatory cells.

Material and methods

Patients

Thirty-two patients with moderate to severe UC,

defined as Ulcerative Colitis Disease Activity Index

(UCDAI) 6�12, were included in the study [22].

Eighteen patients had pancolitis, 9 had left-sided

colitis, and 5 had proctocolitis. The duration of

disease ranged from 0 to 16 years (mean 4 years).

Nineteen normal controls were included in the

study. The indication for colonoscopy in this group

was irritable bowel syndrome (IBS) without diar-

rhea. Subjects with no diarrhea, normal colono-

scopy, and a normal colon histology examination

served as the normal control group. The patients in

the control group had no history of depression in

their hospital journals. IFX (5 mg/kg) (Remicade;

Centocor Inc., Horsham, Pa., USA) was given as

repeated intravenous infusions at 0, 2, and 6 weeks.

The demographical and clinical characteristics of

patients and controls and the ongoing medication

before and after infusions with IFX are listed in

Table I. The diagnoses were based on established

clinical, endoscopic, and histological criteria [23].

All participants were informed about the procedures

and signed a written consent form. The Regional

Committee of Medical Ethics of North Norway

and the Norwegian Social Science Data Services

approved the storage of the biological material.

Clinical grading and definitions of response to treatment

Patients were enrolled consecutively following a

medical evaluation of the initial clinical and endo-

scopic grading of activity, using colonoscopy at the

start and 4 weeks after the last infusion of IFX. The

degree of illness was evaluated with the UCDAI

scoring system which is based on clinical signs (score

0�12) and on endoscopic evaluation of the distal

colon during colonoscopy (grade 0�3) [24].

Complete response to therapy (clinical remission)

was defined as a reduction of the UCDAI score to

less than 3 together with an endoscopic subscore of 0

or 1 [25]. Clinical response to therapy was defined as

a decrease from baseline in the total UCDAI score of

at least 3 points [25]. Endoscopic remission was

defined as a subscore of 0 or 1 for endoscopy [11].

Tissue samples

Biopsies were taken from the most severely inflamed

mucosa of the colon in UC patients before and 4

weeks after the last infusion of IFX. The biopsies

from healthy controls were taken from the rectum.

Biopsy specimens for histological examination were

fixed in 10% formalin until analysis in hematoxylin &

eosin (H&?E)-stained sections. Biopsy specimens for

RNA extraction were immediately immersed in RNA

later (Applied Biosystems, Ambion Inc., Austin,

Tex., USA) and stored at 48C overnight. The super-

natants were then removed and stored at �208C.

Biopsy specimens for immunohistological examina-

tion were fixed in 10% formalin and embedded in

paraffin routinely.

Cytokine measurements

Pre-PCR and real-time PCR procedures. Real-time

polymerase chain reaction (PCR) procedures have

Table I. Demographic and clinical characteristics in patients with ulcerative colitis before and after treatment with infliximab and in

controls.

Gender Age Endoscopic score

Clinical score;

UCDAI

Biopsy

position Right

colonNumber Female/male Median/range Median/range Median/range Rectum Left colon

UC (32) 12 F/20 M 41 (18�70) 3(2�3) 12 (8�12) 18 14 0

Controls (19) 11 F/8 M 54 (30�89) 1 7 11

Medication in UC patients 5ASA Steroids 5ASA�steroids 5ASA�steroids

�AZA

AZA AZA�5ASA None

Before IFX 5 8 6 9 0 0 4

After IFX 0 0 2 3 11 16 0

Medication in controls Aspirin Beta-

blockers

Insulin Statins Diuretics Warfarin Thyroxine None

19 3 4 2 1 1 1 2 14

Abbreviations: UC�ulcerative colitis; UCDAI�ulcerative colitis disease activity index; IFX� infliximab; 5ASA�5-aminosalicylates, oral

or rectal; AZA�azathioprine, steroids � oral or rectal.

728 T. Olsen et al.

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previously been described in detail [9,26]. RNA was

extracted from biopsies in accordance with the

Trizol method (Invitrogen, Paisley, UK). To avoid

contamination of DNA during the extraction proce-

dures, precautions were taken during the RNA

precipitation step. RNA remains exclusively in the

aqueous phase (located in the upper half of the tube)

after the phase-separation step. The aqueous phase

was transferred to a fresh tube. Total RNA concen-

tration was measured at 260 nm using a U-1500 UV/

Vis spectrophotometer (Hitachi Instruments Inc.,

San Jose, Calif., USA). RNA integrity was measured

with an Agilent 2100 Bioanalyzer with RNA 6000

Nano chips (Agilent Technology Inc., Boblingen,

Germany) following the manufacturer’s instructions.

The measurements of RNA quality are scaled from 0

to 10 (RIN values), where a RIN higher than 5 is

considered as good total RNA quality and higher

than 8 is considered as perfect [27]. The mean RIN

values in our samples were above 8 [26].

Reverse transcription of total RNA was done by

iScript (Bio-Rad, Hercules, Calif., USA) according

to the manufacturer’s instructions. Levels of mRNA

for IL-4, IL-10, IFN-g, TNF-a, and b-actin (house-

keeping gene) were determined in duplicate with

real-time quantitative RT-PCR using TaqMan chem-

istry (Applied Biosystems, Foster City, Calif., USA)

and a standardized threshold value. Pre-PCR steps

and assays were evaluated earlier in order to prevent

the methodological pitfalls that have been linked to

this method [26]. In our hands, the quantitative

PCR method can discriminate a difference between

two samples with a variation of 24% or more with a

possibility of 95% over a 5log10 range [27]. Primer

sequences have been published previously [9]. Sta-

bility of b-actin (ACTB) as the housekeeping gene in

the present context has been ascertained earlier [9].

For each cytokine, a specific standard was prepared

and expressed according to our previously published

studies [9,26]. The finally corrected cytokine values

were expressed as copy/mg RNA extracts. The

laboratory investigators were blinded to the clinical

data.

Detection of TNF-a-expressing cells and immune cells by

immunohistochemistry. Detection by immunohisto-

chemistry (IHC) was performed according to our

previously published method [9,28]. In short, sec-

tions were boiled for 15 min in 0.01 M citrate buffer,

pH 6.0, to achieve antigen retrieval. The following

primary antibodies were used: TNF-a-expressing

cells were determined by their intracellular cytokine

pattern using rabbit anti-human TNF-a polyclonal

antibody (working dilution 1:100, from Santa Cruz

Inc., Santa Cruz, Calif., USA), rabbit anti-human

CD3 polyclonal antibody and mouse anti-human

CD68 monoclonal antibody (both at 1:100, both

from DAKO, Carpentaria, Calif., USA) to mark

TNF-a immunoreactivity, lymphocytes, and macro-

phages in the tissue. The negative control slides for

IHC were carried out routinely: 1) primary anti-

bodies were replaced with the isotype-matched and

concentration-matched control antibodies; 2) sec-

ondary antibodies were replaced with phosphate

buffered saline (PBS).

Immunoenzymatic staining in serial sections to

detect TNF-a immunoreactivity in macrophages and

lymphocytes. To identify the cellular source and

distribution of TNF-a, triple serial sections were

stained with TNF-a CD3, CD68 antibodies in 8

patients before and after IFX treatment, according

to the method described above. Primary antibodies

were incubated at 48C overnight and developed with

LSAB-2 system-HRP kits (DAKO) as described

above.

Morphometric evaluation of the immunostained sections

Single immunostained, well-oriented sections were

examined by light microscope (Reichert Inc., New

York, USA). Semi-quantification of immunoreactive

cells was done in mucosa from 8 UC patients in

endoscopic remission (subscore 0 or 1) before and

after IFX treatment and in 10 normal controls.

In brief, TNF-a expressing cells (labeled by rela-

tive anti-cytokine immunoreactivities (IRs)) were

counted in at least 5 optical fields with abundant

distribution from each slide in �400 high-power

magnification. CD3�and CD68�expressing cells

(labeled by relative anti-cytokine IRs) in each slide

were graded as [29]: nil (0), 1�19 cells/field (1�),

20�49 cells/field (2�) and�50 cells/field (3�) in 5

optical fields (�400) with abundant distribution.

The average values per slide were used for statistical

analysis. The investigator examining the slides was

blinded to the clinical data.

Statistics

All data were checked for frequency distribution,

and tests of normality were run. None of the

absolute cytokine values were normally distributed

after log 10-transformation, and non-parametric tests

were used for between-patient analyses (Mann-

Whitney, Kruskal-Wallis) and within-patients ana-

lyses (Wilcoxon rank sum). In order to analyze the

difference of the levels of TNF-a between several

independent subgroups of patients, we used the

Kruskal-Wallis test. Mann-Whitney tests (two-

tailed) for unpaired samples were used as post-hoc

Infliximab. TNF-a and ulcerative colitis 729

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tests between subgroups. Multiple comparisons were

adjusted with the Bonferroni correction. In addition,

to avoid inflating type I errors, we were selective

concerning the numbers of post-hoc comparisons we

made. The correlation analysis was done using the

Spearman correlation (two-tailed). Central measures

are presented as medians and ranges of the final

corrected values expressed as copy/mg RNA extracts

unless otherwise stated; p-values of less than 0.05

were considered significant, unless otherwise stated.

All statistical analyses were carried out using the

software SPSS 15.0 for Windows.

Results

Clinical outcome of infliximab treatment of UC patients

Thirty-two patients with moderate to severe UC were

treated with three intravenous doses (5 mg/kg) of

IFX. Based on the UCDAI response criteria [24], 10

patients obtained remission, 18 patients had a clinical

response, and 4 patients had no response. The overall

median UCDAI was 12 (range 8�12) before treat-

ment and decreased to 4 (0�11) (pB0.01) after

treatment with IFX. In accordance with the clinical

effect of IFX, the median UCDAI scores decreased

to 1 (0�2) in the remission group (n�10), 4 (3�6) in

the response group (n�18), and 10 (10�11) in the

non-responder group (n�4).

Expressions of TNF-a mRNA level in colorectal mucosa

during treatment with infliximab

The levels of TNF-a mRNA in colorectal mucosa

decreased significantly during treatment to levels

slightly higher than those in controls (Wilcoxon,

Z��4.4, pB0.001, Figure 1). There was a positive

correlation between the levels of TNF-a mRNA after

treatment (post-TNF-a and the endoscopic grading

(0�3) for inflammation (r�0.5, pB0.01), Spear-

man, Figure 2). Earlier, we demonstrated a positive

Figure 1. Expressions of TNF-a mRNA in colorectal mucosa

during treatment with infliximab in ulcerative colitis. Box-plot of

gene expressions of TNF-a copies/microgram in biopsies from

human colorectal mucosa. The Wilcoxon signed-rank test was

used to analyze the differences in TNF-a mRNA levels before and

after treatment in patients with ulcerative colitis. The Mann-

Whitney U-test was used to analyze the differences in TNF-amRNA levels after treatment in patients with ulcerative colitis and

in normal controls; n�32 (ulcerative colitis patients before and

after treatment with infliximab), n�19 (controls). All experi-

ments were done in duplicate.

Figure 2. Correlation of post-TNF-a gene expression and endo-

scopic response in patients with ulcerative colitis after treatment

with infliximab (IFX).

Table II. Changes in TNF-a gene expression in groups according

to endoscopic response in patients with ulcerative colitis during

treatment with infliximab.

Pre-TNF-a Post-TNF-aCategory Mean, median (range) Mean, median (range)

Healed

mucosa

56,130, 44930

(165,000)

15,000, 12,600 (1,463,903)*

Not healed

mucosa

87,080, 76,550

(149,000)

43,800, 25,200 (145,224)#

Controls 16,240, 14,520

(36,000)

16,240, 14,520 (36,000)

Differences between the clinical subgroups were significantly

different (pB0.05, Kruskal-Wallis test). Post-treatment levels of

TNF-a mRNA were significantly different in patients in endo-

scopic remission compared to those not in remission (pB0.01,

Mann-Whitney test). Post-TNF-a mRNA was significantly differ-

ent in patients who did not reach remission compared to normal

controls (pB0.01, Mann-Whitney test). There were no differences

between post-TNF-a mRNA in the patients in endoscopic

remission and TNF-a mRNA in normal controls (p�0.05,

Mann-Whitney test). There was a non-significant difference

between the levels of pre-TNF-a in the two different mucosal

groups (p�0.09, Mann-Whitney test).*Significant differences between the groups when comparing

patients before and after treatment with infliximab (IFX); #sig-

nificant differences when comparing patients after treatment and

controls.

730 T. Olsen et al.

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correlation between the tissue mRNA level of TNF-

a and the UCDAI score in untreated UC patients (rs

0.39, p�0.004) [9]. When comparing clinical sub-

groups, the patients with endoscopic remission had

significantly lower post-TNF-a mRNA levels than

the subgroup not in endoscopic remission (Mann-

Whitney U-test (M-WU)�28, pB0.01, Table II).

Moreover, the pre-TNF-a mRNA levels tended

to be higher in the group without endoscopic

remission than in the group with endoscopic remis-

sion (p�0.09, Table II).

When comparing the change in TNF-a mRNA

levels during IFX treatment in the three clinical

subcategories defined by the UCDAI score, respon-

ders, non-responders, and those in remission, the

same tendencies were observed. There was a positive

correlation between the levels of post-TNF-a mRNA

and UCDAI scores after treatment (r�0.5, pB0.01,

Spearman). The non-responder group tended to

have high pre-TNF-a mRNA levels and significantly

higher post-TNF-a compared to normal controls

(M-WU�10; pB0.05). The remission group had

the lowest pre-TNF-a mRNA levels and had nor-

malized post-TNF-a mRNA levels (Table III).

Nine out of 30 patients used stable doses of

azathioprine (AZA) before treatment with IFX.

When comparing the changes in TNF-a mRNA

during IFX treatment in the subgroup of patients

using AZA before treatment with IFX with those not

using AZA, there were no differences between the

groups (delta-TNF in the patients using AZA before

IFX: 38,049 copies/mg mRNA versus 43,101 in the

patients not using AZA, p�0.4, M-WU�84). In

the patients not using AZA before IFX treatment,

the levels of pre-TNF-a and post-TNF-a mRNA

tended to be higher, but were not significantly

different from levels of pre- and post-treatment

TNF-a in the patients using AZA (67,539 copies/

mg pre-TNF-a mRNA versus 54,496, p-value�0.2,

M-WU�77, 24,437 copies/mg post-TNF-a mRNA

versus 15,446, p-value�0.4, M-WU�84).

Gene expression in IL-4, IL-10, and IFN-g in colorectal

mucosa during treatment with infliximab in patients

with UC

IL-4, IL-10, and IFN-g mRNA levels were measured

in the colorectal mucosa before and after IFX

treatment in UC patients and in a control group.

The results are presented in Table IV. The numbers

(n) given in Table IV represent the numbers of the

final detectable real-time PCR results in each

cytokine analyzed and differ from the numbers of

detectable TNF-a results (n�32). Neither the level

of IL-4 nor the level of IL-10 decreased during

treatment (Table IV). The levels of IL-4 and IL-10

mRNA did not correlate to TNF-a mRNA, either

before or after treatment.

The IFN-g mRNA level was significantly de-

creased during treatment. Moreover, when com-

pared to the control group, the post-IFN-g level

was significantly increased compared with that in the

control group (pB0.05, M-WU). The IFN-gmRNA level tended to be lower in patients in

endoscopic remission (n�7) than that in patients

not in remission (n�7, p�0.05, M-WU). IFN-gmRNA levels did not correlate to either endoscopic

subscore or the UCDAI score (data not shown).

However, the pre-IFN-gamma mRNA correlated

significantly to the level of pre-TNF-a mRNA (r�0.84, pB0.01, Spearman) and the corresponding

post-IFN-g and TNF-a mRNA correlated almost to

significance (r�0.39, p�0.05, Spearman).

Expression of TNF-a-positive cells, T lymphocytes and

macrophages in the lamina propria during treatment

with infliximab

Patients obtaining clinical remission during IFX

treatment are of special clinical interest because

this subgroup has a more favorable long-term

clinical outcome [30]. Previous reports have shown

that endoscopic mucosal healing at 8 weeks in the

active UC trials predicted a long-term remission

Table III. Changes in TNF-a gene expression in groups accord-

ing to clinical response based on UCDAI scores in patients with

ulcerative colitis during treatment with infliximab.

Pre-TNF-a

Category

Mean,

median (range)

Post-TNF-aMean, median (range)

Response 60,240, 51,030

(160,000)

18,500, 18,800 (53,151)

Remission 57,030, 59,950

(10,000)

14,400, 15,500 (33,450)

No response 97,290, 94,840

(16,700)

58,300, 24,900 (162,029)#

Controls 16,240, 14,520

(36,000)

16,240, 14,520 (36,000)

Differences in TNF-a levels between the clinical subgroups

showed a tendency, but were not significantly different (pre-

TNF-a levels: p�0.5, Kruskal-Wallis test, post-TNF-a levels:

p�0.1, Kruskal-Wallis test). There were no differences between

the post-treatment levels of TNF-a mRNA in patients in clinical

remission and the TNF-a mRNA in controls (p�0.05, Mann-

Whitney test). The non-responder group had increased levels of

post-TNF-a compared to normal controls (pB0.05). There was a

non-significant difference between the levels of pre-TNF-a in the

remission- and response group when compared to the non-

responder group (p�0.05, respectively, Mann-Whitney test).

For further details, see Statistics.#Significant difference when comparing the patients after treat-

ment and controls.

Infliximab. TNF-a and ulcerative colitis 731

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during maintenance with IFX [11]. As described

above, in our group that reached endoscopic remis-

sion after IFX treatment, an apparent ‘‘normal-

ization’’ of mucosal TNF-a mRNA levels was

observed. We carried out a serial adjacent well-

oriented section with single staining of TNF-a,

CD3�(T lymphocytes) and CD68 (macrophages)

in slides of lamina propria, before and after IFX

treatment, in 8 of the 24 UC patients in endoscopic

remission (one endoscopic subscore of 0, and 7

endoscopic subscores of 1) and in 10 normal

controls. The post-TNF-a mRNA level was 10,096

copies/mg, slightly below the mean level of normal

controls (16,243 copies/mg mRNA, p�0.05). In this

mucosa with endoscopic remission, TNF-a-positive

cells, T lymphocytes and macrophages were found

predominantly in the stroma. Most of the cells were

present in the upper-half region towards the lumen.

Some intraepithelial TNF-a-positive cells were also

found. There were no changes in the localization or

distribution of examined immune cells after treat-

ment with IFX. The distribution and localization of

the immune cells did not differ in normal controls.

The grading of the lymphocytes (CD3�), macro-

phages (CD68�), and the number of TNF-a-

positive cells in the 8 patients in remission and in

healthy controls are listed in Table V. First, the

numbers of macrophages and lymphocytes in UC

patients were significantly decreased after the IFX

treatment and even significantly lower than those in

normal controls (Table V). The number of TNF-a-

positive cells was slightly and non-significantly

decreased after the IFX treatment (Table V). Com-

pared to the healthy controls, the number of TNF-a-

positive cells was increased in the UC patients after

treatment (Table V).

Discussion

In this study, treatment with IFX in UC patients

strongly reduced the levels of TNF-a and IFN-gmRNA in colorectal mucosa. The expression levels

of TNF-a mRNA correlated well with the clinical

and endoscopic response to the treatment. No

significant changes were observed in the mucosal

levels of IL-4 or IL-10 mRNA. Our results indicate

that the effects of IFX in UC patients are not only a

direct suppression of tissue expression of TNF-a,

but also an indirect suppression of other TH1

cytokines such as IFN-g.

The mechanisms of action underlying the ther-

apeutic effects of anti-TNF antibodies in the treat-

ment of IBD are not fully understood [12]. Possible

mechanisms fall into two categories: blockage of

TNF receptor-mediated mechanisms and induction

of transmembrane TNF-mediated mechanisms [13].

In vitro studies support the theory that these anti-

TNF drugs bind to transmembrane TNF and

initiate reverse signaling back into the cell, leading

to apoptosis, cell activation, or cytokine suppression

[17,18]. According to this theory, the decrease in

TNF-a and IFN-g at transcription level found in our

study may be ascribed to a reverse signaling mechan-

ism of IFX. The mechanisms of IFX in UC patients

may be the same as those in CD, but to the best of

our knowledge no comparable studies have been

done.

In our study the reduction of TNF-a levels was

intimately associated with clinical and endoscopic

improvement of colonic mucosa. There are few data

Table V. TNF-a-positive cells, T lymphocytes (CD3�) and

macrophages (CD68�).

TNF-a�cells CD3�cells CD68�cells

Category Median (range) Median (range) Median (range)

Before 15 (42) 2.4* (2.5) 2.9* (0.7)

After 13# (35) 1.0# (1.0) 1.8# (2.0)

Controls 3 (4) 2 (0.8) 2.2 (0.7)

*Significant differences between the groups when comparing the

patients before and after treatment with infliximab (IFX); #sig-

nificant difference when comparing the patients after treatment

and a normal control group.

TNF-a-expressing cells were counted in at least 5 optical fields

with abundant distribution. CD3�and CD68�expressing cells in

each slide were graded as [29]: nil (0), 1�19 cells/field (1�),

20�49 cells/field (2�) and�50 cells/field (3�) in 5 optical fields

(�400) with abundant distribution. Data are expressed as

medians (range).

Table IV. Gene expression of IFN-g, interleukin-4 and interleukin-10 before and after treatment with infliximab in patients with ulcerative

colitis.

IFN-g IL-10 IL-4

Category Median (range) Median (range) Median (range)

Before 23,947 (262,424) n�12 31,125 (1,463,903) n�19 2565 (8850) n�12

After 6696* (54,048) 15,827 (932,880) 2387 (8195)

Controls 262# (3450) n�11 1580# (4315) n�11 360# (2595) n�18

*Significant differences between the groups when comparing the patients before and after treatment with infliximab (IFX); #significant

difference when comparing the patients after treatment with a normal control group.

All experiments were done in duplicate.

732 T. Olsen et al.

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correlating TNF-a data to clinical response to IFX

in UC. In one UC study with a limited number of

patients, histological data showed reductions of

tissue levels of TNF-a closely correlating to clinical

improvement [21]. This is in agreement with our

data. Furthermore, we observed that the levels of

pretreatment TNF-a mRNA strongly correlated

with the levels of IFN-g mRNA (r�0.8, pB0.01),

and IFN-g mRNA was reduced after the IFX

treatment. Increased levels of IFN-g in UC patients

have not been reported earlier by our group and

others [9,31]. Decreased levels of IFN-g mRNA in

IFX-treated UC patients have also recently been

described in IFX-treated cultures [32].

In this present study, tissue levels of IL-4 and IL-

10 mRNA expression were also measured. These

cytokines are increased in UC [9,33,34]. There was

no effect of IFX on these cytokines. Therefore, the

apparent reverse signaling of IFX in UC seems to be

ascribed to TH1-like cytokines as TNF-a and IFN-gonly. The clinical significance of increased levels of

IL-4, IL-10, and IFN-g mRNA in patients in clinical

remission is intriguing and awaits further studies.

Decreased numbers of T lymphocytes and macro-

phages were verified by IHC examinations in UC

patients in remission after treatment. This could

indicate that IFX induces a reduction of these two

main cell sources of TNF-a production in colon

mucosa [35]. Several studies have shown that IFX

induces apoptosis of monocytes and T lymphocytes

in CD [16,19,20]. One study reported decreased

numbers of T lymphocytes and macrophages after

treatment with IFX by IHC in the mucosa of

patients with CD [36], in line with our results.

Unexpectedly, there was only a slight decrease in

the number of TNF-a-positive cells after treatment.

Our observation may be ascribed to other TNF-a-

producing cell types, such as B lymphocytes and

natural killer cells [35]. These cells may not respond

to IFX treatment. Another important point is that

the amount of TNF-a produced in each cell depends

on the activation of the cell and the inducing

stimulus [35]. In addition, the number of TNF-a-

positive cells does not necessarily correlate to the

exact intracellular amount of TNF-a.

Notably, most of the patients in our study started

with AZA in addition to IFX, to prevent the

development of antibodies to IFX [12,37]. We

cannot out rule that, to some degree, AZA may

have influenced our results. However, when measur-

ing the levels of pre-and post-treatment TNF-a in

these subgroups of patients, there were no significant

differences between the groups. The present study

was, however, not powered for this subgroup analy-

sis. Another limitation of our study was the dis-

crepancy between the biopsy position of the patients

included and the controls, which could influence the

results. Lastly, our controls were IBS patients and

some studies suggest that IBS is associated with

colonic inflammation [38]. Furthermore, IBS is

associated with depression, which is associated with

inflammation [39]. Our controls had no history of

depression and did not use any antidepressant

medication. However, there was no assessment of

mood, and this is a limitation of the study.

Of special interest are the observations that the

patients with healed mucosa evaluated by endo-

scopy had TNF-a expressions in the range of those

of normal healthy controls. The patients with the

best long-term clinical outcome had a normal

endoscopy [40]. There is an ongoing discussion on

whether healing of the mucosa should be the

primary end-point and therapeutic goal in clinical

studies of IBD [30]. It should also be borne in mind

that mucosal healing is suggested as the strongest

predictor of a reduced risk of cancer among UC

patients [40,41].

In the current study, IFX (5 mg/kg) was given as

repeated intravenous infusions, in line with the study

by Rutgeerts et al. [11]. Our results demonstrate that

the patients with high pre-TNF-a mRNA still have

increased post-treatment TNF-a mRNA and do not

achieve remission. Our hypothesis is that this sub-

group of patients may need IFX treatment for a

longer period of time or higher doses of anti-TNF-amedication before they achieve remission.

In conclusion, we have demonstrated a down-

regulation of mucosal TNF-a and IFN-g mRNA

expression in moderate to severe UC treated with

IFX. Post-treatment TNF-a mRNA levels correlated

well with the clinical and endoscopic improvement

in UC patients. Our observation of normalized

TNF-a mRNA levels in patients in endoscopic

remission supports the idea of healed mucosa as

the optimal therapeutic goal in clinical studies in

IBD.

Acknowledgements

We thank Ingrid Christiansen, Marian Remijn, and

Line Wilsgard for superb technical assistance and we

also express our thanks to the clinicians J. M.

Kvamme, K. Johnsen, E. Paulsen, and D. Malm

for supporting the project by providing biopsies

during the endoscopic procedures.

Declaration of interest: The project was funded in

full by the Medical Research Program, Northern

Norway Regional Health Authority; Grant number

SFP-328-05.

Infliximab. TNF-a and ulcerative colitis 733

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