Pediatric Graduate School
Helsinki University Central Hospital
University of Helsinki
Studies of experimental biomarkers during
anti-tumor necrosis factor-alpha therapy in
inflammatory bowel disease and
juvenile idiopathic arthritis
To be presented, with the permission of the Faculty of Medicine of the University of
Helsinki, for public examination in the Niilo Hallman Auditorium, Hospital for Children
on 20 th April 2012, at 12 noon.
Supervised by Docent Kaija-Leena Kolho
Helsinki University Central Hospital
Professor Outi Vaarala
Immune Response Unit
National Institute for Health and Welfare
Reviewed by Professor Yrjö T. Konttinen
University of Helsinki
Docent Katri Kaukinen
University of Tampere
Official opponent Professor Eeva Moilanen
University of Tampere
ISBN 978-952-10-7917-7 (pbk.)
ISBN 978-952-10-7918-4 (PDF)
Unigrafia Oy University Printing House
To Matti, Pyry and Otso
Anti-inflammatory medicines, such as glucocorticoids (GCs) and anti-tumor necrosis
factor-α (anti-TNF-α) agents are used for treatment of inflammatory bowel diseases (IBD),
Crohn`s disease (CD) and ulcerative colitis (UC), and also in juvenile idiopathic arthritis
(JIA). GCs are used for treatment of moderate to severe adult and pediatric IBD. The use
of GC is restricted by inadequate response or development of steroid dependency. In
addition, GCs can induce adverse effects such as suppression of the hypothalamicpituitary-adrenal
axis. In JIA, intra-articular corticosteroid therapy (IACS) is used for
treatment of oligoartcular disease or flare-ups of independent joints. Anti-TNF-α agents
are used for treatment of GC refractory IBD. The anti-TNF-α agent infliximab (IFX) is
indicated for use in pediatric IBD. Anti-TNF-α agents can also have severe adverse effects
including infections and malignancies. A laboratory test in clinical use to predict those
patients who would respond to GC or anti-TNF-α therapy would be important to guide the
therapeutic use of these agents. In this thesis, the attenuation of inflammation during GC
and anti-TNF-α therapies was evaluated by using experimental biomarkers in aim to find
new tools to optimize GC and anti-TNF-α therapy in pediatric and adult IBD patients, and
in JIA patients receiving IACS.
The serum samples were prospectively collected from IBD patients before the start of oral
GC (I, IV) or anti-TNF-α therapy (III, IV), and from the pediatric controls (IV). The
second sample was collected from pediatric IBD patients two to four weeks after the start
of GC therapy (I, IV), and at week two after the first IFX infusion (IV). In all, 57 pediatric
IBD patients were recruited from the Children`s Hospital, University of Helsinki. Twentyseven
children had oral prednisolone, and one had budesonide (I, IV). Sixteen pediatric
IBD patients received IFX (IV). Fifteen adult CD patients from the Clinic of
Gastroenterology, Helsinki University Central Hospital had therapy induction with either
IFX or adalimumab (III). Thirteen pediatric IBD patients with quiescent disease and 19
other pediatric patients served as controls (IV). In addition, 21 JIA patients were
prospectively recruited from Heinola Rheumatism Foundation Hospital, and had 2-7 joints
injected at one time with methylpredinisolone (MP) or MP in combination with
triamcinolone hexacetonide (II). The JIA patients provided serum samples on the morning
before the IACS, seven and 24 hours thereafter, and at two months.
A new biological assay for monitoring the patient serum immune-activation potency
was developed (I-III). The assay is based on analysis of the effect of patient serum on
donor derived allogenic peripheral blood mononuclear cells, refered here as target cells.
As read-outs, the markers of T-cell activation, interferon-gamma (IFNγ), interleukin-5
(IL-5), forkhead transcription factor 3 (FOXP3) and glucocorticoid-induced tumor
necrosis factor receptor (GITR), were analyzed in the culture supernatants with enzymelinked
immunosorbent assay (ELISA) or in the target cells with polymerase chain
reaction. In addition, serum glucocorticoid bioactivity (GBA) was measured from the JIA
patients (II). In the work IV, matrix metalloproteinases (MMP-7-9), their tissue inhibitors
(TIMP-1-2), alpha-2-macroglobulin (α2M), human neutrophil elastase (HNE) and
myeloperoxidase (MPO) were measured from the serum with ELISA.
In the first study, the serum immune-activation potency was studied in pediatric IBD
patients at the start of oral GC therapy and two to four weeks thereafter (I). In comparison
with pre-treatment serum, patient serum after the treatment reduced target cell IFNγ
secretion (p=0.017 for naїve cells and p=0.006 for activated cells) and GITR expression
(p=0.033 and p=0.005 respectively). The decrease on serum induced FOXP3 expression
was seen on naїve target cells (p=0.05). These changes did not correlate with weightrelated
GC dose, thus the observed changes represented the sum effect of individual
inflammatory activity in patient serum, rather than GC concentration.
In the second study with JIA patients (II), IACS increased serum GBA (p=0.001) and
decreased cortisol (p=0.002) within 24 hours of the injection. In the target cell assay, the
patient serum induced IL-5 secretion showed a trend of decrease (p=0.085). These
findings indicate a leakage of biologically active GC from the joint.
In the third study, the serum immune-activation potency was studied in adult CD
patients at the start of anti-TNF-α therapy and its association with endoscopic disease
activity (CDEIS) within three months (III). Before the start of the therapy, the high serum
induced FOXP3 and GITR expression on activated target cells were associated with low
CDEIS (r=-0.621, p=0.013 for FOXP3 and r=-0.625, p=0.013 for GITR) and to low
erythrocyte sedimentation rate (r=-0.548, p=0.034 for FOXP3). Interestingly, the low pretreatment
FOXP3 and GITR expression on target cells were associated with a pronounced
decrease of CDEIS during the therapy (r=0.600, p=0.018 for FOXP3 and r=0.589,
p=0.021 for GITR). Restoring the number and function of regulatory T-cells (Tregs) is one
central therapeutic effect of anti-TNF-α agents. It is possible that there is a group of IBD
patients who do not benefit from additional enhancement of Tregs induced by anti-TNF-α
In the final study, serum MMPs, TIMPs, α2M, HNE and MPO were measured from
pediatric IBD patients during GC and anti-TNF-α therapies (IV). The serum level of
MMP-7, MMP-8, MMP-9, α2M, HNE, MPO in all IBD patients and TIMP-1 in the GC
treatment group was higher than in the controls (all p≤0.022). During the GC therapy, the
attenuation of inflammation was seen as a decrease of MMP-7 and TIMP-1 (both
p≤0.001). During the anti-TNF-α therapy, α2M and HNE increased (both p≤0.026). As the
α2M is the major plasma inhibitor of MMPs, its increase was suggested as an additional
mechanism mediating anti-TNF-α agent’s therapeutic effect.
Taken together, studying the patient serum immune-activation potency and measurement
of serum MMPs, TIMPs, α2M and HNE seemed promising approaches to explore the antiinflammatory
effects of GC and anti-TNF-α therapies in IBD and JIA. It should be noted
that the number of patients was relatively small, thus further studies are needed to evaluate
the clinical usefulness of the proposed means to monitor the treatment response during GC
and anti-TNF-α therapies.
List of original publications 10
Review of the literature 15
1 Inflammatory bowel diseases 15
1.1 Classification and clinical picture 15
1.2 Diagnosis 16
1.3 Etiology 17
2 Juvenile idiopathic arthritis 18
2.1 Diagnosis and classification 18
2.2 Etiology 19
3 Immunological aspects in IBD and JIA 20
3.1 Evoking the immune response 20
3.2 T-cells 20
3.3 Regulatory T-cells 21
3.3.1 FOXP3 22
3.3.2 GITR 22
3.4 Immunopathogenesis of IBD 23
3.4.1 Innate immunity mediated IBD pathogenesis 23
3.4.2 T-cell response in IBD 24
3.5 Immunopathogenesis of JIA 25
3.5.1 T-cell response in JIA 25
3.5.2 Autoantibodies 26
4 Matrix metalloproteinases, their inhibitors and neutrophil released enzymes 27
4.1 Basic concepts of MMPs and TIMPs 27
4.1.1 MMPs 27
4.1.2 TIMPs 28
4.2 MMPs, TIMPS, α2M, MPO and HNE in IBD 28
4.2.1 MMPs and TIMPs 28
4.2.2 α2M 28
4.2.3 MPO 30
4.2.4 HNE 30
5 Therapeutic options of IBD and JIA 31
6 Glucocrticoids in IBD and JIA 31
6.1 Intra-articular corticosteroid therapy in JIA 36
6.2 Immunological effects of GCs 36
6.2.1 GC effects on cytokine profile 38
6.2.2 GC effect on Tregs 39
7 Anti-TNF-α agents in IBD 39
7.1 Immunological effect of anti-TNF-α agents 40
7.2 Predicting therapeutic response to anti-TNF-α agents in IBD 40
Aims of the study 43
Study subjects and methods 44
1 Study subjects and samples 44
1.1 Pediatric IBD patients (I, IV) 45
1.2 Adult CD patients (III) 46
1.3 Patients with JIA (II) 46
1.4 Controls (IV) 46
2 Laboratory methods 47
2.1 T-cell stimulation assay for measurement of serum immune-activation
2.2 Enzyme-linked immunoabsorbent assays (ELISAs) 47
2.2.1 Cytokine ELISAs 47
2.2.2 ELISAs for MMPs, TIMPs, α2M, HNE and MPO 48
2.3 Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) 48
2.4 Glucocorticoid bioactivity (GBA) 49
3 Statistics 49
Results and discussion 51
1 Serum immune activation potency on target cells (I-III) 51
1.1 The assay development 51
1.2 The effect of GC treated patient`s serum on target cell cytokine profile (I,II) 52
1.2.2 JIA patients with IACS (II) 54
1.3 The effect of GC treated patient serum on target cell FOXP3 and GITR
expression (I) 55
1.4 Clinical implications of serum immune activation potency (II, III) 56
1.4.1 In JIA patients (II) 56
1.4.2 Serum immune activation potency related to CDEIS and ESR in
adult CD patients (III) 57
2 GBA (II) 58
2.1 The effect of IACS to serum GBA and cortisol 58
2.2 Comparison between GBA and serum immune activation potency 59
2.3 Clinical implications 60
3 MMPs, their inhibitors and neutrophil released enzymes during GC and anti-
TNF-α therapy (IV) 61
3.1 Comparison between patients with active IBD and controls 61
3.2 Changes in measured serum markers during oral GC and IFX therapy 62
3.3 Clinical implications 65
4 General discussion 65
4.1 Strengths and weaknesses of the study 65
4.1.1 Subjects and samples 65
4.1.2 Pitfalls in biological assay for monitoring the treatment effects 66
4.1.3 Drug of choice in JIA 66
4.2 Future prospective 67
List of original publications
This thesis is based on the following publications that are referred to in the text by their
I Rintamäki H, Salo HM, Vaarala O, Kolho KL. New means to monitor the
effect of glucocorticoid therapy in children. World J Gastroenterol
II Rintamäki H, Tamm K, Vaarala O, Sidoroff M, Honkanen V, Raivio T,
Jänne O, Kolho KL. Intra-articular corticoid injection induces circulating
glucocorticoid bioactivity and systemic immune activation in juvenile
idiopathic arthritis. Scand J Rheumatol. 2011;40:347-53.
III Rintamäki H, Sipponen T, Salo HM, Vaarala O, Kolho K-L. Serum immuneactivation
potency and response to anti-tnf-alpha therapy in Crohn´s disease.
World J Gastroenterol 2010;16:5845-51.
IV Mäkitalo L, Rintamäki H, Tervahartiala T, Sorsa T, Kolho KL. Serum MMPs
7-9 and their inhibitors during glucocorticoid and anti-TNF-α therapy in
pediatric inflammatory bowel disease. In press.
These articles were reprinted with the copyright holder’s permission. Some unpublished
data are also presented.
ACTH adrenocorticotropin hormone
ANA antinuclear antibodies
ANCA antineutrophil cytoplasmic antibodies
Anti-OmpC anti-outer membrane porin C IgA antibody
Anti-TNF-α anti-tumor necrosis factor-alpha
APC antigen presenting cell
ASCA anti-Saccharomyces cervisiae antibodies
BSA bovine serum albumin
CCP cyclic citrullinated peptide
CD Crohn´s disease
CD (4 etc.) cluster of differentiation
CDAI Crohn´s disease activity index
CDEIS Crohn´s Disease Endoscopic Index of Severity
CHAQ Child Health Assessment Questionnaire
CRP C-reactive protein
Ct threshold cycle
CTLA cytotoxic T lymphocyte associated antigen
DC dendritic cell
DMARD disease modifying antirheumatic drug
ECM extracellular matrix
ELISA enzyme-linked immunoabsorbent assay
ESR erythrocyte sedimentation rate
F-calprotectin fecal calprotectin
FOXP3 forkhead transcription factor 3 / forkhead box P3
GBA glucocorticoid bioactivity
GITR glucocorticoid-induced tumor necrosis factor receptor
GM-CSF granulocyte-macrophage-colony stimulating factor
GR glucocorticoid receptor
GRE glucocorticoid response element
HAT histone acetyltransferase
HDAC2 histone deacetylase
HLA human leukocyte antigen
HNE human neutrophil elastase
HsCRP high sensitive C-reactive protein
IACS intra-articular corticosteroids
IBD inflammatory bowel disease
IC indeterminate colitis
IFNγ interferon gamma
IKKβ inhibitor of nuclear factor kappa-beta kinase
IPEX immune dysregulation, polyendocrinopathy autoimmune enteropathy Xlinked
JIA juvenile idiopathic arthritis
MHC major histocompatibility complex
MMP matrix metalloproteinase
MR mineralocorticoid receptor
(m)RNA (messenger) ribonucleic acid
NFκB nuclear factor kappa-beta
NOD (2) nucleotide-binding-oligomerisation-domain (2)
NO(S) nitric oxide (synthase)
PBMC peripheral blood mononuclear cell
PBS phosphate buffered saline
PCR polymerase chain reaction
PGA Physician`s global assesment
RA rheumatoid arthritis
RF rheumatoid factor
RORC2 retinoic acid receptor-related orphan receptor C2
RT-PCR reverse transcriptase polymerase chain reaction
SF synovial fluid
SFMC synovial fluid mononuclear cells
STAT signal transducer and activator of transcription
SuPAR soluble urokinase plasminogen activator receptor
TA triamcinolone acetonide
TCR T-cell receptor
TGF-β transforming growth factor-beta
THA triamcinolone hexacetonide
TIMP tissue inhibitor of matrix metalloproteinases
TLR toll-like receptor
TNF tumor necrosis factor
Treg regulatory T-cell
UC ulcerative colitis
Crohn´s disease (CD) and ulcerative colitis (UC) are termed inflammatory bowel diseases
(IBD). The classical symptoms of IBD are diarrhea, stomach ache and rectal bleeding.
Intermittent fever can be related to Crohn´s disease. Extra-intestinal manifestations can
also exist, joint symptoms being the most common. In addition, IBD can affect growth,
fertility and mood. Juvenile idiopathic arthritis (JIA) is classified in seven subtypes, and
the clinical picture varies from inflammation of a single joint to the systemic disease.
The exact etiology of IBD and JIA are unknown, but they both seem to a have
multifactorial background. In IBD, it is suggested that genetic vulnerability together with
environmental triggers lead to altered immune response against normally tolerated
commensal flora. Disturbance of tolerance plays a role in immunopathogenesis of both
diseases, and inflammatory activity of the patient´s white cells contibutes to tissue
damage. Recently, the balance between T-helper (Th) cells, especially Th17 cells and
regulatory T-cells (Tregs), has received much attention in the pathogenesis of both IBD
Anti-inflammatory medicines, such as glucocorticoids (GCs) and anti-tumor necrosis
factor-alpha (anti-TNF-α) agents are used to induce and maintain remission in both IBD
and JIA. The mechanisms of action of GCs are not fully understood despite the long
history of GCs in the treatment of inflammatory diseases. The GC therapeutic effect seems
to arise from the inhibition of inflammatory gene reading. Restoring the balance between
Th-cells and Tregs has been shown as one therapeutic effect of anti-TNF-α agents. Up to
30% of adult IBD patients have been reported as treatment failures during systemic GC
therapy, and a similar number of adult patients have become steroid dependent. Among
pediatric IBD patients, the steroid denpendency can be even more common. During anti-
TNF-α therapy, approximately 60% of adult IBD patients have been reported to be shortterm
responders, and around 40% of the patients were able to maintain remission for one
year. In addition, both GC and anti-TNF-α therapies can have severe adverse effects. Sideeffects
have been described even after local delivery such as intra-articular GC injections
Several approaches have been tried to find early predictors for therapeutic response to
GC therapy. Of the laboratory markers, glucocorticoid receptor (GR) isotypes and GR
polymorphisms has been shown to be the most promising predictor in IBD. It has also
been shown that parameters of lymphocyte function can reflect individual steroid
sensitivity. There are very few studies exploring markers to predict anti-TNF-α therapy
effects. High pre-treatment CRP associated with good therapeutic response to anti-TNF-α
agents in adult IBD. In another study with adult CD patients, the high mucosal expression
of pro-inflammatory transcription factor nuclear factor kappa-beta (NFκB) preceeded
therapy relapse. There is no laboratory marker in clinical use to foresee those patients who
would benefit from GC or anti-TNF-α therapy. Due to a fear of side-effects, this would be
especially useful in pediatric patients.
In this thesis, a new biological assay was developed with the aim to find innovative
tools for predicting therapeutic response for GC and anti-TNF-α therapy. This assay is
based on detection of the treatment effect on the patient`s serum on donor derived
allogenic white cells that are refered as target cells. Patient serum immune-activation
potency is assessed by measuring a panel of Treg (forkhead transcription factor 3, FOXP3
and glucocorticoid-induced tumor necrosis factor receptor, GITR) and Th-cell (interferon
gamma, IFNγ, interleukin-5 and IL-17) markers in target cells or culture supernatants of
the target cells. Other experimental serum markers, including glucocorticoid bioactivity
(GBA), matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), alpha-2macroglobulin
(α2M), human neutrophil elastase (HNE) and myeloperoxidase (MPO)
were also studied with the aim to find new tools to optimize GC and anti-TNF-α therapy
in IBD and JIA.
Review of the literature
1 Inflammatory bowel diseases
1.1 Classification and clinical picture
Inflammatory bowel diseases (IBD) that are Crohn´s disease (CD) and ulcerative colitis
(UC), are lifelong, systemic inflammatory diseases of the gastrointestinal (GI) tract. CD
can affect the GI tract from the mouth to the anus. Histologically, the inflammation
patches in CD are submucosal or transmural (IBD Working Group of the European
Society for Paediatric Gastroenterology, Hepatology and Nutrition 2005, Baumgart DC
2007 b). The Vienna classification of CD divides the disease according to location into
disease of the ileum, colon, ileocolon or upper GI-tract. CD is further subclassified
according to phenotype into inflammatory, penetrating or stricturing disease (Gasche C
2000). In UC, the inflammation typically extends continuously from the rectum to the
proximal colon (Baumgart DC 2007 b). Histologically, the inflammation in UC involves
only mucosa (IBD Working Group of the European Society for Paediatric
Gastroenterology, Hepatology and Nutrition 2005). UC can be further divided into leftsided
colitis, where the inflammation is located distally to splenic flexure, and pancolitis
(Kugathasan S 2003). Those patients with colonic histological findings of inflammation
with architectural changes that cannot be defined as UC or CD and with no disease in the
small bowel, are diagnosed as unclassified or indeterminated colitis (IC) (IBD Working
Group of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition
2005). In two pediatric studies, IC was diagnosed in 3.3-14% of the IBD patients
(Hildebrand H 2003, Turunen P 2006), and in a study with an adult population in 7.8%
(Rubin GP 2000). The peak incidence age for CD is 15-25 years and for UC 25-35 years
(Vind I 2000, Jussila A 2012). Another peak incidence age is suggested to be in patients
over 75 years (Vind I 2000). In pediatric studies, the mean age at diagnosis varies between
11 to 13.5 years (Hildebrand H 2003, Kugathasan S 2003, Turunen P 2006). In adults, UC
predominates over CD in developing countries, but it seems that a pattern of IBD is
evolving (Rubin GP 2000, Bernstein CN 2008). In pediatric studies, the prevalence of CD
has risen above that of UC (Hildebrand H 2003, Kugathasan S 2003), although in Finland
UC predominates over CD (Lehtinen P 2011).
In IBD, the remission – relapse cycles follow each other (Baumgart DC 2007 b, Shikhare
G 2010). IBD can typically present with abdominal pain, diarrhea and rectal bleeding, but
the clinical picture can vary depending on the localization of the inflammation (Table 1)
(Baumgart DC 2007 b, Shikhare G 2010). Both adult and pediatric patients can have extraintestinal
manifestations such as arthritis, erythema nodosum, pyoderma gangrenosum,
episcleritis, uveitis or pancretitis (Table 1) (Shikhare G 2010). At the time of diagnosis,
13-88% of pediatric CD patients were reported to have impaired growth, meaning height
standard deviation scores ≤-1.96, or decrease in height velocity (Walters TD 2008).
Delayed and prolonged puberty are also reported among IBD, and especially CD patients
(Kirschner BS 2008).
Table 1 Clinical symptoms and extra-intestinal manifestations in pediatric Crohn´s disease (CD)
and ulcerative colitis (UC) (Kugathasan S 2003, Sawczenko A 2003, Gupta N 2008).
Symptom CD (%) UC (%)
Rectal bleeding 21-43 83-84
Diarrhea 30-56 74-98
Abdominal pain 42-72 43-62
Weight loss 21-58 31-38
Fatigue 8-13 2
Fever 13 -
Aphthous ulcers 3-5 13
Anorexia 21-25 6
Lethargy 27 12
Nausea/Vomiting 6 0.6
Constipation/Soiling 1 -
Growth failure/ Delayed puberty 4-8 -
Toxic megacolon - 0.6
Perianal fistulae 4-8 -
Anal fissure or anal skin tags 15 -
Cholangitis/liver disease/abnormal liver enzymes 1-3 3
Arthritis or arthropathy 2-7 2-6
Pancreatitis 1 -
Erythema nodusum/rash 1-2 0.6
Pyoderma gangrenosum 5 -
IBD diagnosis is confirmed by endoscopy with biopsy, or imaging techniques such as
wireless capsule endoscopy (Girardin M 2008, Bernstein CN 2010, Shikhare G 2010).
Serological and fecal laboratory tests (Table 2) can provide information on the disease
severity and activity, but the inflammatory parameters C-reactive protein (CRP) or
erythrocyte sedimentation rate (ESR) can also be at normal levels (Bernstein CN 2010,
Sidoroff M 2010, Turner D 2010 b). Fecal markers such as calprotectin (Røseth AG 1999)
or lactoferrin (Kayazawa M 2002) can be used to monitor the inflammation of the gut
(Fagerhol MK 2000, Bernstein CN 2010). In adult IBD patients, both fecal calprotectin
and lactoferrin correlated with endoscopic score and colon histology (Sipponen T 2008 a,
Langhorst J 2008). In pediatric IBD patients, fecal calprotectin correlated closely with
macroscopical and histological inflammation (Bunn SK 2001, Kolho KL 2006).
Table 2 Laboratory tests for suspected inflammatory bowel disease. Modified from Strople
Test Possible finding
Blood count Anemia, thrombocytosis, leukocytosis
Liver function test Elevated transaminases, alkaline phosphatase or gamma-glutamyl
Serum albumin Hypoalbuminea
Stool calprotectin Elevation
Stool lactoferrin Elevation
Serologic tests: ASCA, pANCA, anti-
OmpC May support the diagnosis
ESR= erythrocyte sedimentation rate, CRP= C-reactive protein, ASCA=anti-Saccharomyces cervisiae
antibodies, pANCA=perinuclear antineutrophil cytoplasmic antibodies, anti-OmpC=anti-outer membrane
porin C IgA antibody
The etiology of IBD is considered to be multifactorial in that genetic susceptibility
combined with environmental factors trigger a mucosal immune response against normally
tolerated antigens like commensal flora (Baumgart DC 2007 a).
A positive family history is considered to be the largest independent risk of IBD
(Baumgart DC 2007 a). In studies of twins, genetic concordance seems more abundant in
CD than in UC (Tysk C 1988). In familial aggregation studies, 5-22.5% of the IBD
patients are reported to have first degree relatives with IBD (Russell RK 2004). In
pediatric studies, 11-18.5% of the IBD patients had first- or second-degree affected
relatives (Kugathasan S 2003, Hildebrand H 2003, Turunen P 2009). Pediatric CD patients
with early onset disease more frequently had first-degree relatives with IBD than older CD
patients (42% versus 19%) (Weinstein TA 2003). The first identified genetic linkage was
the association between CD and a mutation of the NOD2/CARD15 gene in chromosome
16 that codes for intra-cellular microbe sensing pattern recognition receptor, nucleotidebinding-oligomerisation-domain
(NOD) 2 (Hugot JP 2001, Martinon F 2005). Today, the
developing genome-wide association techniques have revealed over 60 IBD susceptile
gene loci; potential candidate genes being for example IL23R, IL27 and toll-like receptor-
4 (TLR4) (Henderson P 2011, Thompson AI 2011). Approximately half of IBD susceptile
loci have been confirmed in both UC and CD (Thompson AI 2011), and 60% of adult loci
have been replicated in pediatric studies (Henderson P 2011).
The IBD incidence rates are highest in Western countries (Baumgart DC 2007 a), and in
most pediatric cohorts the incidence rates have continued to rise (Kugathasan S 2003,
Hildebrand H 2003, Lehtinen P 2011). Increased numbers of IBD in Asian countries have
been associated with socioeconomic changes in this area (Zheng JJ 2005). It is suggested
that the slow genetic changes cannot account for the fast increase in IBD incidence which
implies that environmental and lifestyle factors contribute to the IBD etiology (Bernstein
CN 2008, Nunes T 2011). In epidemiological studies, several environmental risk factors
such as host microbial environment, diet and smoking have been identified as modifying
the development of IBD, but it should be empasized that the studies are discrepant, and no
risk factor with high impact has yet been identified (Table 3) (Bernstein CN 2008, Nunes
Table 3 Role of environmental factors in development of ulcerative colitis (UC), Crohn`s
disease (CD) and juvenile idiopathic arthritis (JIA) (Baumgart DC 2007 a,
Bernstein CN 2008, Nunes T 2011, Ellis JA 2010).
Environmental risk factor UC CD JIA
Longer duration of breast feeding ↓ ↓ ↓/↑
Consumption of polyunsaturated fats ? ↑ -
Consumption of dietary fibers - ↓ -
Consumption of unpasteurized milk - ↑ -
Rural or low hygiene living ↓ ↓↓ ↓
Large or poor family ↓ ↓↓ ↓
Childhood pet contact - ↓ -
Stress ↑ ↑ -
Smoking ↓ ↑ ↑ 1
History of infections ↑ ↑ ↑
Oral contraceptives ↑ ↑ -
Non steroidal anti-inflammatory drugs ↑ ↑ -
Appendectomy ↓ ↑ -
Arrow up=incresed risk, arrow down=decreased risk, 1 maternal smoking during pregnanacy (>10 cigarettes
2 Juvenile idiopathic arthritis
2.1 Diagnosis and classification
JIA is defined as arthritis of unknown etiology which starts before the age of 16 and
persists for six weeks. For diagnosis, other causes of joint symptoms must be excluded.
JIA is classified into seven subtypes which are presented in Table 4 (Petty RE 2004).
The etiological triggering factor of JIA is unknown, but similar to other autoimmune
diseases, it seems that both genetic as well as environmental factors play a part in disease
onset. Studies of the environmental factors are rare and the results are not uniform. The
most evidence is for the role of preceding infections such as streptococcus and virus
infections for onset or flare-up of JIA (Table 3) (Ellis JA 2010). In a Finnish study which
monitored familial aggregated JIA for 15 years, a concordance was 25% for monozygotic
twins (Savolainen A 2000). To date, several human leukocyte antigen (HLA)
polymorphisms have been linked to an increased or decreased risk of various JIA
subtypes, as are other candidate genes such as genes coding for IL-6, macrophage
migration inhibitory factor and natural resistance-associated macrophage protein 1
(Borchers AT 2006, Berntson L 2008).
Table 4 Juvenile idiopathic arthritis (JIA) subtypes. Exclusions are: a.) psoriasis or
history of psoriasis of the patient or the first degree relative, b.) HLA-B27
positive male having arthritis after age of six years, c) ankylosing spondylitis,
enthesitis related arthritis, IBD related sacroiliitis, Reiter´s syndrome or acute
anterior uveitis in first degree relative, d.) positive rheumatoid factor (RF) twice
with three-month interval, e.) systemic JIA (Petty RE 2004)
JIA subtype Definition Exclusions
Arthritis with minimum 2 weeks fever, of which minimum 3 days daily,
and at least 1 systemic manifestation: 1. nonfixed erythematous rash, 2.
lymph node enlargement, 3. hepatomegaly / splenomegaly or 4. serositis.
2.Oligoarthritis Arthritis in 1-4 joints during the first 6 months. Persistent form does not
affect more than four joints throughout disease course while extended
form affects more than four joints after six months.
Arthritis of 5 or more joints during the first 6 months. RF negative. a-e
Arthritis of 5 or more joints during the first 6 months. At this time two
positive tests for RF with 3-month interval.
Arthritis combined with psoriasis and 2 of the following: 1. dactylitis, 2.
nail pitting or onycholysis, 3. psoriasis of a first degree relative.
Arthritis and / or enthesitis with 2 of the following: 1. presence or history
of inflammatory lumbosacral or sacroiliac joint pain, 2. positive HLA-
B27, 3. male over 6 years of age, 4. symptomatic anterior uveitis, 5. first
degree relative having history of ankylosing spondylitis, enthesitis
related arthritis, IBD related sacroiliitis, Reiter`s syndrome or
symptomatic anterior uveitis.
Arthritis that does not fit any of the categories listed above or fills the
criteria of more than 1 category.
HLA= human leukocyte antigen, IBD=inflammatory bowel disease
a, d, e
3 Immunological aspects in IBD and JIA
The following chapters briefly summarize some basic concepts of the immune system,
concentrating on the function of T-cells and the immunological features linked to the
development of IBD and JIA.
3.1 Evoking the immune response
Innate immunity cells, dendritic cells (DCs), macrophages and neutrophils, express
pathogen recognition receptors, for example TLRs (Medzhitov R 2000) and intra-cellular
NOD-like receptors (Martinon F 2005). In addition to receptor mediated endocytosis,
immature DCs in tissue can also take up pathogenic material by receptor independent
macropinocytosis (Janeway CA 2005 a). The binding of microbe derived structures to
TLRs leads to the activation of DCs and secretion of cytokines and chemokines
(Medzhitov R 2000). Mature DCs present antigens bound to membrane glycoproteins
called major histocompatibility complexes (MHC) to T –cells of adaptive immunity,
which leads to T-cell activation. By controlling the antigen presentation, the innate
immunity system has a central role in conducting the adaptive immunity response type and
magnitude (Medzhitov R 2000).
The hallmark of a mature T-cell is expression of T-cell receptor (TCR) and surface
molecules, cluster of differentiation-3 (CD3), and either of CD4 (effector cells) or CD8
(cytotoxic cells). Effector T-cell differentiation depends on presented antigen and cytokine
environment during the antigen presenting process (Janeway CA 2005 a).
Type 1 Th cells (Th1) secrete dominantly pro-inflammatory cytokine IFNγ, and
activate CD8+ T-cells and macrophages in response to infection with intracellular microorganisms
(Lazarevic V 2011). Th2 response is characteristic of asthmatic and allergic
conditions or helminth infection. Th2 cells secrete cytokines IL-4, IL-5, IL-9 and IL-13,
and can activate B-cell response (Okoye IS 2011).
During the last decade, a new helper T-cell subtype, Th17 cells, has been described
(Harrington LE 2005). Th17 cells secrete IL-17 cytokines that enhance the inflammatory
process by increasing local production of cyto- and chemokines, leading to increased
leucocyte recruitment, expansion of myeloid cell lines, and enhanced T-cell activation
(Aggarwal S 2002). There can be plasticity between Th17 and Th1 cells, and the balance
of cytokines like transforming growth factor-beta (TGF-β), IL-23 and IL-12 has been
reported to affect the cytokine profile secreted by Th17 cells; varying from purely IFNγ or
IL-17 secreting Th-cells to Th17 cells capable of secreting combination of IL-17, IFNγ
and IL-22 (Lee YK 2009). Th17 cells are typically induced in the mucosal surface as a
defence against bacteria and fungi (Hue S 2006, Lee YK 2009). Th17 cells and induced
Tregs are reported to share TGF-β dependent differentiation pathways, and the
differentiation to either of these cells can depend on the cytokine environment as IL-6
promotes the Th17, and all-trans retinoic acid promotes the Treg pathway (Lee YK 2009).
Further, Treg specific transcription factor FOXP3 expression is reported to inhibit the
retinoic acid receptor-related orphan receptor C2 (RORC2) a key transcription factor of
Th17 cells (Lee YK 2009, Bene L 2011, Strober W 2011).
3.3 Regulatory T-cells
CD4+CD25+FOXP3+ regulatory T-cells (Tregs) are described as a unique effector cell
line with a supressive function (Itoh M 1999). Natural Tregs develop in the thymus as an
individual T-cell population which acquires an antiproliferative (anergic) stage and
suppressive function before entering into circulation (Itoh M 1999). Adaptive Tregs
(iTregs) are described as cells that develop in periphery from CD25 - CD4+ T-cells upon
stimulation in the presence of TGF-β and IL-2 (Allan SE 2007, Horwitz DA 2008).
Several molecules commonly expressed on Tregs are used for identification of these cells
such as CD25, CD127, GITR, FOXP3, cytotoxic T lymphocyte associated antigen-4
(CTLA-4) and lymphocyte activation gene-3 (Corthay A 2009).
Figure 1 Functions of Tregs. Modified from Corthay A 2009.
In periphery, Tregs play a central role in maintaining self-tolerance and preventing
autoimmunity (Sakaguchi S 2006) (Figure 1). Tregs are suggested to have several
mechanisms of suppression including secretion of anti-inflammatory cytokines IL-10,
TGFβ or IL-35, or driving effector-Th cells to apoptosis due to consumption of
maintaining cytokines such as IL-2 (Boden EK 2008). Tregs isolated from IL-10 deficient
mice were unable to prevent experimental colitis similar to wild-type mice Tregs
(Asseman C 1999). Treg suppression can also be mediated by CTLA-4, a surface receptor
that interacts with CD80 and CD86 expressed on antigen presenting cells (APCs) (Read S
2000, Sansom DM 2006). However, CTLA-4 seems not to be required for Treg function
(Sansom DM 2006).
FOXP3 gene encodes transcription factor, originally called scurfin protein when
discovered in mice (Hori S 2003). FOXP3 is expressed in CD4+CD25+ regulatory T-cells
of mice and humans (Fontenot JD 2003, Hori S 2003). CD25 negative (CD4+CD25-) Tcells
can also express FOXP3, although to a much lesser extent (Fontenot JD 2003, Hori
S2003). FOXP3 is required for Treg development (Fontenot JD 2003). In a mice model,
exogenous FOXP3 induced regulatory activity in CD4+CD25- T-cells during expression
when cotransferred with retroviral infection-vector (Fontenot JD 2003, Hori S 2003).
These ectopic FOXP3-transduced cells were able to inhibit experimental colitis of mice
similarly to naturally occurring Treg cells (Hori S 2003). The importance of FOXP3
function in humans is underlined by the finding that the mutations affecting to FOXP3
function lead to a severe autoimmune syndrome called immune dysregulation,
polyendocrinopathy autoimmune enteropathy X-linked syndrome (IPEX). The symptoms
of IPEX arise in early infancy and include diabetes, eczema, enteropathy, hematological
abnormalities, endocrinopathies or renal dysfunction (Ruemmele FM 2004).
GITR is gene coding for a TNF receptor family protein. GITR was originally found in
murine T-cell hybridoma cultures, where dexamethasone (DEX) increased its expression
approximately sixfold (Nocentini G 1997). Thereafter, it was found that GITR expression
does not necessitate GC, but GITR is expressed in low levels in almost every lymhoid
tissue, and its expression is upregulated on unselected T-cell activators such as anti-CD3
or Concavallin A (Nocentini G 1997).
GITR has been suggested as an activity marker of Tregs (Corthay A 2009), although
Treg activity does not necessitate functional GITR because mouse GITR+ and GITR-
Tregs suppressed Th cells equally (Ronchetti S 2004). In mice, GITR is expressed in naïve
Tregs, and its expression increases upon stimulation with anti-CD3 combined to IL-2 or
anti-CD28 in mice (McHugh RS 2002, Shimizu J 2002, Kanamaru F 2004). GITR is also
described at low level in resting mice Th cells, and is upregulated in these cells upon
activation (McHugh RS 2002, Shimizu J 2002, Kanamaru F 2004). Despite a higher GITR
expression, activated Th cells do not transform into a suppressive phenotype (Shimizu J
2002). GITR is a TCR co-receptor and enhances both Th and Treg cells (Kanamaru F
2004, Ronchetti S 2004). A combination of anti-GITR-antibody (anti-GITR-Ab) to anti-
CD3 led to Treg proliferation in a dose-dependent manner (Kanamaru F 2004). In the
same study, activation of CD4+T-cell cultures with either anti-GITR-Ab or anti-CD28
enhanced similarly and dose dependently the secretion of cytokines IL-2, IL-4, IFNγ and
IL-10 (Kanamaru F 2004). GITR can also modulate T-cell apoptosis so that T-cell
hybridoma cells that over-expressed GITR became resistant to anti-CD3 driven apoptosis
(Nocentini G 1997).
GITR has been detected in several different immune cells such as natural killer-cells,
B-cells, macrophages and DCs (Hanabuchi S 2006, Shimizu J 2002, Kanamaru F 2004).
In vivo, GITR elimination, but also activation with anti-GITR-Ab led to development of
autoimmune gastritis in mice (Shimizu J 2002).
3.4 Immunopathogenesis of IBD
3.4.1 Innate immunity mediated IBD pathogenesis
TLRs are conserved transmembrane receptors expressed on innate immunity cells such as
DCs, macrophages and neutrophils, and also on epithelial and endothelial cells. TLRs
recognize pathogen associated molecule patterns such as bacterial wall components
(Medzhitov R 2000). TLRs enhance inflammation through an intra-cellular activation
cascade resulting in activation of pro-inflammatory transcription factor, NFκB, and
production of pro-inflammatory cytokines (Medzhitov R 2000, Martinon F 2005).
Modified expression of TLRs in the gut has been suggested to be significant for IBD
pathogenesis by leading to an altered response against normally tolerated luminal antigens
such as commensal flora (Baumgart DC 2007 a, Bene L 2011). TLR4 was upregulated in
IBD patients’ intestinal epithelial cells in comparison with controls (Cario E 2000). In the
same study, TLR3 was expressed on normal mucosa, but downregulated in mucosa of
active CD patients (Cario E 2000). In another study, TLR4 was up-regulated in biopsies of
both UC and CD patients, while TLR2 was up-regulated in ileal biopsies of UC patients
(Frolova L 2008). CD14, which is an up-stream molecule in the TLR4 pathway, was also
over-expressed in both UC and CD in comparison with controls (Frolova L 2008). A
further pathogen recognition receptor, NOD2, was expressed in CD patients’ inflamed
ileum (Ogura Y 2003). The significance of pathogen recognition receptors in
immunopathogenesis of IBD was underlined by the finding of a genetic association
between mutations in gene of NOD2 in chromosome 16 in CD (Hugot JP 2001).
Altered function of DCs can also affect to IBD immunopathogenesis due to the role of
DCs in tolerance induction and antigen specific T-cell activation. Sorted DCs from CD
patients’ intestinal specimens expressed increased number of TLR2 and TLR4 (Hart AL
2005). In the same study, DCs of CD patients inflamed biopsies over-expressed coreceptor
CD40, considered as marking of mature DCs (Hart AL 2005). IBD patients were
also reported to have a lower number of circulating immature DCs than the controls
(Baumgart DC 2005).
3.4.2 T-cell response in IBD
Traditionally, CD has been considered as a Th1 mediated inflammatory disease, and CU
as a Th2 mediated (or resembling) disease (Strober W 2011). This is based on findings
that in vitro activated lamina propria CD4+T-cells of CD patients secreted Th1 cytokine
IFNγ and IL-2, but the secretion of Th2 cytokines IL-4 and IL-5 was decreased in
comparison with controls. In the same study, CU patients’ lamina propria cells secreted
IL-5, but not another Th2 key cytokine IL-4, or IFNγ (Fuss IJ 1996). Later, increased
mucosal secretion of multiple cytokines was reported in IBD such as IL-12 and IL-18 in
CD, IL-13 in UC and TNF-α, IL-1β and IL-6 in both diseases (Baumgart DC 2007 a,
Strober W 2011, Eastaff-Leung N 2010).
In recent years, IL-17 immunity has undergone intensive research in IBD. The mucosal
expression of IL-17 and the number of circulating Th17 cells has been shown to have
increased in adult patients with active IBD (Annunziato F 2007, Hölttä V 2008, Eastaff-
Leung N 2010). In adult CD patients’ ileal biopsies, IL-17A, IL-6 and FOXP3 messenger
ribonucleic acid (mRNA) expression was increased in comparison with controls regardless
of the disease activity (Hölttä V 2008). In IBD, especially those Th17 cells that are
maturating in an IL-23 environment and capable of secreting both IL-17A and IFNγ may
contribute to the immunopathogenesis of the disease (Annunziato F 2007). A group of
adult CD patients’ gut Th 17 cells were shown to produce IFNγ, and were considered as
Th17/Th1 cells. Both Th17 cells and Th17/Th1 cells were found to express either RORC2
or T-bet, supporting the idea of the placticity between Th17 and Th1 cell lines
(Annunziato F 2007, Lee YK 2009). Th17 and Th17/Th1 cells were suppressed less by
Tregs than Th1 or Th2 cells (Annunziato F 2007).
Furthermore, the balance between Th17 cells and Tregs has risen as a potential
determinant of IBD immunopathogenesis (Eastaff-Leung N 2010). The role of Tregs in
IBD immunopathogenesis is based on mice models of experimental colitis. In a scurfy
mice model, a mutation of FOXP3 gene was shown to lead to lethal colitis (Brunkow ME
2001). In humans, the IPEX syndrome arising from the mutations impairing the FOXP3
function is presented with severe diarrhea due to eneropathy (Ruemmele FM 2004). In
immunodeficient colitis mice, a transfer of Tregs was shown to lead to resolution of
diseased mice gut infiltrates (Mottet C 2003). In adult IBD, the number of peripheral
Tregs was decreased in comparison with controls (Eastaff-Leung N 2010, Wang Y 2011).
However, in mucosa of adult IBD patients, the number of Tregs and FOXP3 mRNA and
protein expression was increased (Wang Y 2011). The imbalance between peripheral
Treg/Th17 compared with normal subjects is reported in both CD and UC (Eastaff-Leung
N 2010). A model of a network of effector T-cells and secreted cytokines in IBD is
presented in Figure 2.
Figure 2 Model of regulatory cytokines for immunopathogenesis of IBD. Modified from Bene L
2011. Arrow=enhancement, Blunt end=inhibition, Th=T-helper cell,
Treg=regulatory T-cell, IL=interleukin, IFN=interferon, TNF=tumor necrosis factor,
TGF=transforming growth factor
3.5 Immunopathogenesis of JIA
3.5.1 T-cell response in JIA
In JIA, the pathologically expanded synovial tissue and pannus are formed of proliferating
synoviocytes and patches of lymphocytes and APCs, while the clonally expanded CD4+
T-cells are the most abundant cells of the synovium (Grom AA 1993, Grom AA 2000). In
vivo, synovial CD4+ T-cells expressed surface molecules that are typical in an activated
state (Wedderburn LR 1999, Grom AA 2000). The flow cytometry analysis of synovial
fluid (SF) lymphocytes showed an over-expression of adhesion molecule recognizing,
“primed” CD4+CD29 T-cells in SF in comparison with peripheral blood (Silverman ED
1993). In JIA patients, 5-15% of inflitrating cynovial T-cells have been reported to express
γδ-TCR suggesting recognition of mycobacterial antigens and stress-related heat shock
protein 65 (Kjeldsen-Kragh J 1993, Grom AA 2000). Despite an activated phenotype,
synovial T-cells are reported to have impaired potency for proliferation and activation in
vitro (Grom AA 2000). In JIA, SF B-cells and CD4+ T-cells were decreased and cytotoxic
CD8+ T-cells increased when compared with peripheral blood (Silverman ED 1993). This
accumulation of CD8+ cells into joints suggests a difference from findings in adult
heumatoid arthritis (RA), and characteristic for oligoarticular JIA (Wedderburn LR
In synovium of JIA patients, T-cell cytokine profile is polarized to Th1 type response.
This has been shown in different JIA subtypes by comparing the IFNγ/IL-4 ratio of
synovial T-cells with peripheral blood, and also by increased expression of Th1 linked
chemokine receptors in synovial T-cells (Wedderburn LR 1999). JIA patients expressed
TNF-α and TNF-β on synovial tissue (Grom AA 1996). It is likely that polarization
toward Th1 response is not restricted to joints, as serum IL-12 measured with
immunoassays was increased in all subtypes of active JIA in comparison with controls
(Gattorno M 1998). During inactive stages, serum IL-12 was higher than in controls only
in patients with systemic JIA (Gattorno M 1998). The patients with systemic JIA, but not
other JIA subtypes, had elevated serum levels of IL-18, in comparison with controls
(Maeno N 2002). The reports of other cytokines have not been uniform (Borchers AT
Two studies have explored the role of Th17 and Treg cells in JIA. In the first, SF IL-17
was higher than in controls, or in serum of the same patients (Agarwal S 2008). The levels
of IL-17 in SF showed correlation with the clinical disease activity of the joints, but not
with the measured inflammatory cytokines IL-6, IL-1β, TNF-α (Agarwal S 2008). Another
study monitored the number of Tregs, and expression of FOXP3 and RORC2 in SF
mononuclear cells (SFMC) and in PBMCs from patients with JIA (Olivito B 2009). JIA
patients expressed lower number of Tregs in blood, but a higher number of CD4+CD25
bright T-cells in SF than normal controls. In the same study, RORC2 mRNA in SFMCs
showed an inverse correlation with SFMCs FOXP3 mRNA. In addition, IL-17 secretion
from SFMC cultures correlated inversely with the number of SF Tregs (Olivito B 2009).
These studies demonstrate that reciprocal actions between Tregs and Th17 effector cells
can have role in the pathogenesis of JIA.
Autoantibodies are not good diagnostic markers for JIA although antibodies, such as RF,
antinuclear antibodies (ANA), anticardiolipin and antiphospholipid antibodies, are
occasionally found in serum of JIA patients (Borchers AT 2006). RF, an immunoglobulin
(Ig) that reacts with IgG Fc fragment, can be found in all five Ig isotypes. Reported
numbers of RF positivity with tests in routine use in clinical laboratories vary between 2-
21% depending on the JIA subtype (Borchers AT 2006). RF positivity is most common in
polyarticular disease (Borchers AT 2006, Syed RH 2008). RF can be a predictor of severe
disease course seen as radiological joint destruction and difficulty in achieving remission
(Van Rossum M 2003 a, Van Rossum M 2003 b, Flatø B 2003,). ANAs can be detected in
approximately 30-50% of JIA patients, most commonly in pauciarticular or oligoarticular
JIA (Serra CR 1999, Borchers AT 2006). The presence of ANAa increases the risk of
uveitis (Kotaniemi K 2001) and longer duration of active disease, but not disability (Oen
K 2003). Cyclic citrullinated peptide (CCP) antibodies, including antiperinuclear factor or
anti-keratin antibodies, are targeted antibodies of citrullinated fillagrin, a protein that is not
expressed in joint cartilage, but during skin and esophagus epithelial cell differentiation.
Anti-CCP antibodies have been shown to have high disease specificity in RA, but in JIA,
the presented numbers for IgG anti-CCP positive patients vary highly, ranging from 2 to
77%, with anti-CCP positivity being highest in patients with seropositive polyarthritis
(Van Rossum M 2003 a, Syed RH 2008). In JIA, CCP positivity has been linked to
radiological joint destruction (Van Rossum M 2003 a).
4 Matrix metalloproteinases, their inhibitors and neutrophil
4.1 Basic concepts of MMPs and TIMPs
In humans, 24 MMP genes code for 23 different neutral zinc metalloproteinases that
cleave and remove the extracellular matrix (ECM). The majority of MMPs are
extracellular proteins. MMP-1, MMP-2 and MMP-11 can also have intra-cellular
functions. MMPs are grouped according to their structure and preferred substrates into
collagenases (MMP-1 -8 -13 -18), stromelysins (MMP -3, -10 -11), gelatinases (MMP-2
and -9), matrilysins (MMP-7 and -26), membrane-type MMPS (MMP-14-17, -24, -25) and
others (MMP-12, -19, -20, -21, -23, -27, -28) (Nagase H 2006).
MMP activity is low in normal tissue, and their expression is controlled by cytokines,
growth factors, hormones and interaction between cells and cells to ECM (Nagase H
2006). Most MMPs are secreted as pro-MMPs that become activated by proteolytic
removal of amino terminus or by oxidants or denaturing agents (von Lampe B 2000,
Nagase H 2006). In addition, MMPs can activate each other, for example MMP-14 is an
activator of MMP-2 (von Lampe B 2000). MMPs can act as sheddases and activate, by
cleving (solubilising), molecules on the surface of immune cells, which can result in both
anti –and pro-inflammatory effects. For example, MMP-7 that can activate protelytically
anti-bacterial peptide α-defensin in the gut, but also cleave pro-inflammatory cytokine
TNF-α independent of TNF-α converting enzyme (Burke B 2004).
MMPs are regulated by controlled activation of their precursors and expression of their
inhibitors such as TIMPs and α2M. The relationship between active MMPs to TIMPs is
considered to determine ECM degrading activity (von Lampe B 2000). Impaired
regulation ECM formation or degradation can play a part in pathogenesis of multiple
diseases such as arthritis, chronic ulcers, cancers and cardiovascular diseases (Nagase H
TIMPs are MMPs’ major cellular inhibitors (Baker AH 2002). TIMPs are secreted
proteins, but can on the cell surfaces bind to MMPs and affect and focalize their activity
(Baker AH 2002). TIMP1-4 share a similar structure of N-and C-terminal domains
(Nagase H 2006). In addition to inhibition of MMPs, TIMPS are suggested to affect cell
growth, apoptosis, tumorigenesis and angiogenesis (Baker AH 2002).
4.2 MMPs, TIMPS, α2M, MPO and HNE in IBD
4.2.1 MMPs and TIMPs
Altered immunological response plays a part in the pathogenesis of IBD leading to
increased expression of pro-inflammatory cytokines in the gut (Baumgart DC 2007 a,
Strober W 2011). Cytokines TNF-α, IL-1β and IFNγ can induce MMP expression in fetal
gut experiments, and colonic cell cultures (Pedersen G 2008). MMPs are found in
intestinal ulcers (Saarialho-Kere UK 1996) and are suggested to serve as a link between Tcell
mediated gut inflammation and GI ulcer formation (Pender SL 1997). Indeed,
artificial MMP-inhibitor had potency in the treatment of mice dextran sulfate sodium
induced colitis (Naito Y 2004). UC has been associated with single nucleotide
polymorphism of genes coding for MMP-3, MMP-8, MMP-10, and MMP-14, but these
results have not been confirmed in other studies (Morgan AR 2011). The central findings
of MMPs and TIMPs in IBD are summarized in Table 5. Until now, there have been very
few studies of MMP expression in pediatric IBD.
The non-specific inhibitor of endoproteinases, α2M, serves as the main plasma inhibitor of
MMPs (Baker AH 2002). α2M is a tetrameric plasma glycoprotein that is synthesised
mainly by liver hepatocytes, but also in other cells, for example macrophages (Baker AH
2002). During MMP inhibition, α2M binds covalently to MMPs (Baker AH 2002), after
which the α2M-MMP complex becomes bound into receptor (low density lipoprotein
related-protein-1) and endocytosed (Strickland DK 1990, Baker AH 2002). Previously,
serum α2M level was found to be lower in adult IBD patients than in normal controls
when detected with electrophoresis (Weeke B 1971), while the fecal α2M level in
immunonephlometry was increased (Becker K 1999). Fecal α2M was higher in active
IBD, and in CD patients correlated with Crohn´s Disease Activity Index (CDAI) (Becker
Table 5 Central findings of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in IBD patients’ gut tissue samples
Research Patients (n) Method Central findings in IBD Correlations to clinical activity parameters
Bailey CJ Adult IBD (11) 2 In CD MMP-9 was increased in inflammatory infiltrates, MMP-3 associated -
with tissue damage sites in CD and UC
Saarialho- Adult IBD (12) 1,2 High MMP-1 signal in IBD samples -
Kere UK 1996
Vaalamo M Adult IBD (24) 1,2 MMP-10, MMP-12-13 and TIMP-3 were similarly over-expressed in UC -
Baugh MD Pediatric and 2,3,6 MMP-1-3 and MMP-9 were over-expressed in IBD patient samples, -
1999 adult IBD (29)
neutrophil MMP-9 most abundantly
Von Lampe B Adult IBD (55) 2,3,4 MMP-1-3, MMP-14 and TIMP-1 expression was increased in active mucosa MMP-1 and MMP-3 correlated with
Louis E 2000 Adult IBD (38) 5 MMP-3 and TIMP-1 secretion increased in samples of inflamed IBD MMP-3 and TIMP-1 correlated with TNF-α,
IL-1β, IL-6 and IL-10 secretion and to
degree of inflammation
Matsuno K Adult UC (19) 2 MMP-1-3, MMP-7, MMP-9 and/or TIMP-1-2 were expressed in IBD MMP-7 expression in epithelial cells
correlated with degree of inflammation
Rath T 2006 Adult IBD (40) 4,5 MMP-2, MMP-7 and MMP-13 RNA expression, and MMP-2 and MMP-9 MMP-7 and MMP-13 expression was
protein level was increased in IBD samples
increased in non-IBD adenomatous polyps
Meijer MJ Pediatric and 5 MMP-1-3 and MMP-9 in relation to TIMP-1-2, and MMP activity increased MMPs or TIMPs expression did not
2007 a adult IBD
in inflamed IBD mucosa
correlate with sample fibrosis or
developement of fistulae
Pedersen G Adult IBD (19) 4,6 MMP-1, MMP-3, MMP-7, MMP-9-10 expression was increased in -
epithelial cells of inflamed mucosa
Mäkitalo L Adult CD (17) 2 MMP-9, MMP-26, TIMP-1 and TIMP-3 expression decreased during MMP-9, MMP-26, TIMP-1 and TIMP-3
showed correlations to histological score,
calprotectin and/or CDEIS
Mäkitalo L Pediatric IBD 2 Epithelial MMP-10 and stromal TIMP-3 expression were increased in IBD -
samples, MMP-7 was greater in CD than in UC samples
Methodology: 1= In situ hybridization, 2= immunohistochemistry, 3= Western blotting, 4=PCR, 5=enzyme linked immunoassay, 6= functional assays of enzyme activity;
CDEIS= Crohn´s Disease Endoscopic Index of Severity
MPO is an enzyme abundantly present in primary granules of myeloid cells such as
neutrophils, monocytes and macrophages (Sangfelt P 2001). Normally, reactive oxygen
metabolites are converted in a two-step manner. First, superoxide dismutases convert
superoxide anion into hydrogen peroxide which is then neutralized to water by catalase or
glutathione peroxidase. However, in inflammatory conditions like IBD, there is excessive
hydrogen peroxide (Nathan CF 1987), which can be delt by other reactions such as Haber-
Weiss reaction in the presense of transition metals (Kehrer JP 2000), or alternative
pathways such as MPO activity leading to formation of hypochlorus acid, a potential
cause of oxidative tissue damage (Kruidenier L 2003).
High gut MPO levels are associated with IBD. MPO enzyme activity and expression
was increased in gut biopsies of adult IBD patients, and higher in inflamed samples than
in non-inflamed or control samples (Kruidenier L 2003, Meijer MJ 2007 a). MPO detected
with immunoassay was increased 2-18 fold in sigmoid perfusion fluid of adult UC patients
in comparison with healthy controls, and this increase associated with the increase of IL-8
and TNF-α in perfusion fluid (Raab Y 1993). In adult IBD patients` gut specimens, MPO
activity correlated with activity of MMP-1, MMP-3 and MMP-9 (Meijer MJ 2007 b). In
line with this, fecal MPO expression detected with immunoassay was higher in adult IBD
patients in comparison with healthy controls. In this study, the highest fecal MPO levels
associated with UC and to CD with high Crohn´s Disease Endoscopic Index of Severity
(CDEIS>200) (Peterson CG 2002). Fecal MPO is also reported to correlate with
endoscopic score in adult UC patients (Peterson CG 2007).
HNE is a proteinase released from activated neutrophil azurophilic granules (Young RE
2004). Neutrophil elastase can affect leukocyte extravasation, cytokine release,
phagosytosis and endothelial cell injury (Smedly LA 1986, Young RE 2004). High HNE
levels detected with immunoassays were found in adult IBD patients in comparison with
controls, and especially in CD patients or patients with highly active disease (Adeyemi EO
1985, Fischenbach W 1987, Gouni-Berthold I 1999). In adult UC patients with active
disease, HNE enzyme activity was increased in both plasma and parallel gut biopsy
samples (Morohoshi Y 2006). Also in adult IBD, fecal HNE complexed to proteinase
inhibitor was higher than in controls and correlated with disease activity index in active
UC and in active colonic CD (Adeyemi EO1992). IBD patients’ plasma HNE level
correlated with CRP and CDAI (Adeyemi EO 1985). In a mice model with induced colitis,
neutrophil elastase inhibitor,ONO-5046, presented with reduced colonic inflammation in
autopsy (Morohoshi Y 2006).
5 Therapeutic options of IBD and JIA
IBD and JIA share similar treatment goals, which are induction and maintaining of
remission, preventing destructive effects like joint damage in JIA, improvement of the
quality of life, and maintaining normal growth and development in pediatric patients with
minimal adverse effects (Hashkes PJ 2005, Wilson D 2010). Medical therapy of IBD and
JIA include anti-inflammatory and immunomodulatory medication, often used in both
diseases (Table 7) (Hashkes PJ 2005, Baumgart DC 2007 b, Wilson D 2010, Beukelman T
2011). Other therapeutic options for IBD are operations and nutritional therapy (Wilson D
2010, Bernstein CN 2010), and in JIA inta-articular GC injections, physiotherapy,
occupational therapy, and operations (Hashkes PJ 2005, Beukelman T 2011). For the
management of systemic and polyarthritic JIA not responding to other medication,
autologous stem cell transplantation has been tested (Hashkes PJ 2005), as is also in
severe CD (Duijvestein M 2010).
6 Glucocrticoids in IBD and JIA
The main human endogenous GC is cortisol that is synthesized in the adrenal cortex in a
circadian and stress-related fashion (Shirtcliff EA 2011, Sapolsky RM 2000). GCs widely
affect the human genome, and glucocorticoid receptor (GR) is expressed in nearly all
tissues of the body (Janeway CA 2005 b). Synthetic GCs differ mainly with their receptor
affinity and balance between anti-inflammatory and mineralocorticoid effect (Table 6).
The medical use of GCs began in 1948 when the first RA patient received cortisone
acetate intra-muscularly (Hench PS 1949). For the treatment of IBD, GCs have been used
since 1950 (Truelove SC 1954). In IBD, GC rarely leads to mucosal healing, which was
seen in studies where clinical healing due to GCs was not associated with endoscopical or
histological remission (Beattie RM 1996), or with normalization (
Table 7 Medical therapies in adult and pediatric inflammatory bowel disease (IBD) and juvenile idiopathic arthritis (JIA) (Hashkes PJ 2005, Baumgart DC
2007 b, Wilson D 2010, Beukelman T 2011, Goebel JC 2011).
Medicine type Product IBD JIA
5-aminosalicylates and Sulphasalazine Induction and maintainance of remission of mild to moderate Optional 1 .line DMARD for enthesitis related JIA, 2. line DMARD for oligo –and
inflammatory drugs and coxibs
IBD. Prevention of colorectal carcinoma.
Mesalamine Not indicated
Not indicated 1. line medicine for oligoarthritis and seronegative polyarthritis. If not disease
modifying during 4-6 weeks used later for symptom relief and to control fever.
Glucocorticoids Systemic Induction of remission in moderate to severe IBD As bridging regimen at the start of DMARDs, induction of remission in systemic JIA
Local Rectal Intra-articular THA for treatment of active joints
Azathioprine and 6-
1.line immunomodulators as maintenance therapy in moderate
to severe IBD
Azathioprine as optional DMARD in uveitis
Methotrexate 2.line immunomodulator of moderate to severe CD 1.line immunomodulator in polyarthritis and extended oligoarthritis
Leflunomide Not indicated 2. line immunomodulator of polyarthritis
Anti-TNF-α agents Infliximab Induction and maintaining of remission for moderate to severe
Other biologic agents Abatacept,
adult and pediatric CD and adult UC
Adalimumab Induction and maintaining of remission for moderate to severe
Etanercept Not indicated
Active arthritis of ≤ 4 joints not responding to DMARDs in six months or in three
months if features of poor prognosis.
Arthritis of ≥ 5 joints not responding to DMARD within three months is moderate or
high disease activity and in six months if low disease activity
Not indicated History of arthritis ≤ 5 joints with poor prognosis not responding to TNFα inhibitors.
Rituximab secondary to abatacept. Anakinra in systemic JIA not responding to
Acute or penetrating CD Not indicated
Probiotics - Unclear Not indicated
DMARD=disease modifying antirheumatic drugs, THA=triamcinolone hexacetonide, TNF=tumor necrosis factor
The use of GC therapy is complicated due to the fact that some patients do not respond to
therapy, and can be considered as steroid resistant. In addition, some patients become
steroid-dependent, with a flare-up of the symptoms shortly after GC tapering leading to
prolonged GC therapy. Approximately 40-60% of the adult IBD patients on oral GCs have
been reported as achieving remission, but 25-30% became steroid-dependent (Creed TJ
2007), and the number of steroid-dependent patients can be even higher in pediatric IBD
patients (Hyams J 2006, Jakobsen C 2011) (Table 8).
Table 8 Recent studies of glucocorticoid (GC) response in pediatric Crohn`s disease (CD)
and ulcerative colitic (UC). Short-term response is defined after one month and
long-term response after one year systemic GC therapy unless otherwise stated.
J 2006 1
A 2011 2
C 2011 3
Short-term response % Long-term response %
62/50 27/29 12/21 42/57 31/14 27/29
60 27 2 50 45 4
60 24 17 61 31 8
55 37 8 nd 41 5
63/64 29/28 8/8 58/49 37/49 nd
1 Short-term response defined at 90 days; 2 Long-term response at 180 days; 3 Prolonged response defined as
remission for 30 days after the the therapy
It is thought that GC pharmacokinetics, for example impaired absoption from the inflamed
gut, did not explain the differences in GC responsiveness in pediatric IBD (Faure C 1998).
Several molecular mechanisms during GC-GR mediated gene reading process can
modulate GC responsiveness. For example, GR expression and ligand binding or affinity,
GR isoforms (GRα, GRβ), single nucleotide polymorphism (BcII) of GR gene and altered
function of GC transmembrane glycoprotein pump (P-glycoprotein 170) have been studied
regarding GC therapy response in IBD (Barnes PJ 2009, Sidoroff M 2011). Several other
approaches such as clinical, tissue specific, bioassays and gene studies have tried to find a
method to predict GC therapy response in IBD (Sidoroff M 2011; Table 9) and other GC
treated diseases such as asthma (Barnes PJ 2009, Leivo-Korpela S 2011).
GCs can present with adverse effects (Figure 3). Budesonide is a GC molecule with
higher than 90% first-pass metabolism leading to low systemic bioavailability, and there
are two oral preparations with controlled ileal release (Seow CH 2008, Vihinen MK
2008). A Cochrane review has shown budesonide to have fewer side-effects, but also to be
less effective in induction of remission in CD than conventional GCs (Seow CH 2008). In
pediatric patients, GC effect on bone mineral content and growth has been a particular
concern (Boot AM 1998, Hämäläinen H 2010). GC side-effects are mediated by GR
transactivation, as in diabetes mellitus and glaucoma, or transrepression during
suppression of the hypothalamic-pituitary-adrenal axis, or both as in osteoporosis
(Schäcke H 2002, Buttgereit F 2005). Until now, there has been no laboratory test in
clinical use to predict therpeutic response to GCs.
Figure 3 Adverse reactions to glucocorticoids (Buttgereit F 2005). HPA= hypothalamicpituitary-adrenal.
Drawing by Elina Vanninen reproduced with permission from
Table 9 Examples of the studies monitoring glucocorticoid (GC) response in pediatric and adult inflammatory bowel disease (IBD)
Flood L 2001 Adult UC
Studied markers Methods Association to glucocorticoid therapeutic response
Inhibition of thymidine uptake T-lymphocyte stimulation test;
GR mRNA and MTIIa mRNA,
suppression of thymidine uptake due to
DEX was measured
Low-dose ACTH test, mRNA quantitation
with hybridization assay
T-cells from steroid responders were suppressed more by
DEX than T-cells from partial responders or treatment
Non-responding patients expressed more MTIIa on
leukocytes before and their serum cortisol level decreased
more during GC therapy compared with steroid responders
STAT-5 Immunohistochemistry of colon biopsies Biopsies of GC-resistant patients expressed STAT-5
while the biopsies of GC responders did not
GBA Bioassay No association between serum GBA and GC therapy
Panel of single nucleotide
polymorphisms related to
Genotyping and haplotype analysis In CD patients polymorphism of SLC22A5 gene allele -
associated to steroid resistance
Adiponectin, leptin Immunoassays Patients with early appering GC related adverse effects
had high serum adiponectin
GBA Bioassay No association between serum GBA and GC therapy
UC=ulcerative colitis, STAT= signal transducer and activator of transcription, GR=glucocorticoid receptor, MT= metallothionein GBA= glucocorticoid bioactivity,
DEX=dexametasone, ACTH= adrenocorticotropin hormone
6.1 Intra-articular corticosteroid therapy in JIA
Intra-articular corticosteroid therapy (IACS) is effective in the relief of pain and limited
range of motion. IACS are indicated either for oligoarticular JIA or for treatment of flareups
of individual joints in other JIA subtypes in combination with disease modifying
antirheumatic drugs (Bloom BJ 2011). All patients seem to respond clinically to IACS
within 48 hours (Padeh S 1998, Bloom BJ 2011). In JIA, IACS are disease modifying
which was seen as relieved joint effusion and pannus in magnetic resonance imaging
seven weeks after the injection (Huppertz HI 1995). Approximately 70-90% of
triamcinolone hexacetonide (THA) treated, and 40-50% of methylprednisolone (MP)
treated patients maintained remission over six months (Honkanen VE 1993, Padeh S 1998,
Bloom BJ 2011). THA can provide longer remission than triamcinolone acetonide (TA)
(Zulian F 2003, Zulian F 2004). Most commonly reported adverse effects of IACS are
local flare or subcutaneous atrophy in approximately 3% of injected patients (Padeh S
1998, Bloom BJ 2011). Cushinoid appearance was reported in a chart review in two of 61
patients (Bloom BJ 2011), as was also in a case report of two other JIA patients after TA
injections (Kumar S 2004). To date, there has only been one study designed to explore the
systemic effects during IACS in JIA. It showed that after IACS, endogenous cortisol
production measured from saliva was suppressed for 10-30 days, and that circadian
fashion of GC secretion was also disturbed (Huppertz HI 1997). In another study in RA,
serum MP levels increased and cortisol level decreased in a dose dependent manner after
intra-articular MP injection, and was restored to normal within one week (Armstrong RD
1981). In two pediatric arthritis patients that developed a Cushinoid appearance after intraarticular
TA injection, TA was detectable in plasma and urine for five months after the
injection, using mass spectrometry (Kumar S 2004).
6.2 Immunological effects of GCs
After absorption, GC diffuses to cytoplasm where it binds to GR. Ligand binding leads to
activation of GR, quick release from chaperone complex and enterance of the GR-GC
complex into the nucleus after which it either participates in activation of antiinflammatory
genes (Figure 4), or suppression of activated pro-inflammatory genes
(Figure 5) (Barnes PJ 2009). GC leads to an increase of circulating leukocytes, mainly
neutrophils, and a decrease of eosinophils (Zhang X 2000, Franchimont D 2004, Sbiera S
2011). GCs’ therapeutic effect is a result of a broad anti-inflammatory effect causing the
attenuation of inflammation (Table 10) (Franchimont D 2004).
Figure 4 Activation of anti-inflammatory target genes by GCs. Modified from Barnes PJ 2009.
GR=GC receptor, GRE= GC response element, HAT=histone acetyltransferase
Figure 5 GCs can switch off activated pro- inflammatory genes by interacting with corepressor
molecules that attenuate coactivator activity of proinflammatory
transcription factors (NFκΒ or activator protein 1) and lead to decreased histone
acetylation and down-regulation of gene reading. Modified from Barnes PJ 2009.
NFκβ= nuclear factor kappa β, IKKβ=inhibitor of NFκβ kinase, HAT=histone
acetyltransferase, HDAC2=histone deacetylase, *down-regulation
Table 10 Anti-inflammatory effects of glucocorticoid therapy (Ahluwalia A 1998, Janeway
CA 2005 b, Franchimont D 2004).
Effect Physiological effect
Down-regulation of IL-1, TNF-α, GM-
CSF, IL-4, IL-5, IL-12 etc.
Down-regulation of inflammation
Down-regulation of NOS Down-regulation of NO
Up-regulation of lipocortin 1 leading to Down-regulation of prostaglandins,
down-regulation of phospholipase A2 and leukotrienes, platlet activating factor and
Down-regulation of adhesion molecules Reduced leukocyte extravasation
Up-regulation of endonucleases Induction of leukocyte and eosinophil
IL=interleukin, TNF=tumor necrosis factor, GM-CSF=granulocyte-macrophage-colony stimulating factor,
NO(S)= nitric oxide (synthase)
6.2.1 GC effects on cytokine profile
GCs have a suppressive effect on inflammatory cytokines (including both Th1 and Th2
class cytokines) (Table 10) (Franchimont D 2004, Janeway CA 2005 b). Non-specific
cytokine suppression of GCs is likely to be a result of disturbed antigen presentation
leading to impaired activation of T-cells. GC treated DCs were expressed more in
immature phenotype with higher endocytotic activity but lower antigen presenting and
cytokine secreting capacity than untreated DCs (Piemonti L 1999). A gene profiling study
monitoring the effect of DEX on human PBMCs using microarray analysis, PCR and flow
cytometry found that GC down-regulated MCHII and co-stimulatory molecules like CD40
on mature DCs, which could lead to impaired DC antigen presentation and T-cell
activation (Galon J 2002). Further, the shift toward Th2 reponse can occur due to a
modulated innate immunity response by GCs. Mainly secretion of Th1 response regulating
IL-12 (Lazarevic V 2011) in monocytes and DCs is downregulated (Blotta MH 1997,
Vieira PL 1998), and anti-inflammatory (IL-10, TGF-β) cytokines are up-regulated (Galon
During the adaptive immunity reponse, the shift toward Th2 response is mediated by lack
of IL-12. IL-12 receptor expression in human PBMC cultures was down-regulated by
DEX. This down-regulation was independent from the cytokine environment (IL-4, IL-10,
TGF-β), and led to impaired IL-12 binding (Wu CY 1998). However, down-regulation of
IL-12 receptors was not confirmed in another study (Franchimont D 2000). Instead, when
human T-lymphocytes and two commercial cell lines were cultured with DEX, DEX
inhibited IL-12 induced phosphorylation of signal transducer and activator of transcription
4 (Stat4) mediating Th1 cytokine response, with no effect on phosporylation of Stat6 that
is linked to Th2 signaling (Franchimont D 2000). GCs can also have pro-inflammatory
effects such as an up-regulation of several pro-inflammatory receptor genes (IL-1RI, IL-
8R and TNF-receptor) and down-regulation of the anti-inflammatory decoy receptor IL-
1Ra gene (Galon J 2002).
6.2.2 GC effect on Tregs
GCs are effective medicines in various autoimmune diseases. This could be mediated by
an enhanced action of Tregs. However, until now the findings of the effect of GCs on
Tregs have been confusing. Two studies have explored the effect of GC therapy on normal
humans. The first in vitro study found a decrease of Treg surface marker CTLA-4 when
DEX was added in the culture of tetanus toxoid activated PBMCs (Xiang L 2011).
Another study monitored the effect of a 14-day high dose GC therapy on patients with
sudden hearing loss, and found only a modest increase in the number of circulating
FOXP3+T-cells accompanied with a decrease in frequency of these cells in relation to the
whole CD4+ T-cell population (Sbiera S 2011). An increase in the number of Tregs or
expression of their activity markers during GC therapy has been shown in several clinical
studies in different inflammatory or autoimmune disorders (Karagiannidis C 2004, Suárez
A 2006, Braitch M 2009, Sbiera S 2011).
7 Anti-TNF-α agents in IBD
TNF-α antagonist agents, infliximab (IFX) and adalimumab were introduced for treatment
of adult CD (Hanauer SB 2002, Hanauer SB 2006) and IFX for UC (Rutgeerts P 2005) at
the end of the 20 th century. IFX is also recomended in the guidelines for the management
of steroid refractory pediatric UC (Turner D 2011), and small studies have been published
of use of adalimumab in pediatric IBD (Wilson D 2010). IFX is a chimeric TNF-α
antibody, and adalimumab a humanized IgG TNF-α antibody (Hanauer SB 2002, Hanauer
SB 2006). Both IFX and adalimumab bind to TNF-α with high affinity. IFX binds to both
soluble and transmembrane TNF-α, while adalimumab binds to soluble TNF-α (Hanauer
SB 2006, Owczarek D 2009). IFX induces mucosal healing of endoscopic lesions in adult
and pediatric CD patients (D'haens G 1999, Borelli O 2004). In pediatric CD patients, the
weight and height Z scores increased 6 months after one to three IFX infusions (Borelli O
2004). Of adult CD patients with moderate CD, 58% responded to first IFX infusion
(Hanauer SB 2002), and the responder rates in pediatric CD patients were similar or better
(Borelli O 2004, Wilson D 2010). Of adult CD patients, 45% maintained remission for a
year after IFX induction (Hanauer SB 2002), while a three month remission rate in
pediatric CD patients was reported to be lower at 30% (Wilson D 2010). Rate of induction
and maintenance of remission for one year with adalimumab in adult CD was 36% and 41
% respectively (Hanauer SB 2006, Colombel JF 2007). Of adult CU patients, 69%
responded to IFX therapy within eight weeks, and 45% mainained remission for one year
(Rutgeerts P 2005). Serious side effects such as infections, malignancies, autoimmunity
and liver toxicity have been associated with the use of anti-TNF-α agents in IBD patients
(Stallmach A 2010, Wilson D 2010). Compared with the other immunosuppressants, the
risk of side-effects is not increased when anti-TNF-α agents are used alone, but the risk
increases in a combination treatment with GCs or other immunosupressants (Stallmach A
7.1 Immunological effect of anti-TNF-α agents
TNF-α is a pro-inflammatory cytokine secreted by monocytes, macrophages and T-cells
(Van Deventer 1997). In three studies with pediatric patients with active IBD, increased
level of TNF-α protein or immunostaining was found in feces, mucosal amina propria and
serum (Braegger CP 1992, Murch SH 1993, Murch SH 1991). TNF-α has been reported to
serve as a “downstream” activator of other proinflammatory cytokines, but also to enhance
“upstream” production of IL-12 (Strober W 2011), thus the immunosupressive effect of
anti-TNF-α agent is extended beyond the down-regulation of TNF-α.
In adult CD, IFX down-regulated IL-6 and up-regulated inactive TNF-α in serum, but
had no effect on PBMC capacity to secrete IFNγ or TNF-α upon in vitro activation
(Cornillie F 2001). The decrease in the number of lamina propria mononuclear cells
secreting IFNγ or TNF-α after IFX treatment ex vivo was demonstrated in two studies with
adult CD patients (Plevy SE 1997, Cornillie F 2001). One study monitored the IFX effect
in vivo to sorted DCs, and found that T-cell co-activator CD-40 on DCs was downregulated
with no correlation to inflammatory activity in the gut biopsies (Hart AL 2005).
Recently, three studies reported a restored function of Tregs to mediate anti-TNF-α agent
effect in adult IBD (Li Z 2010, Boschetti G 2011, Veltkamp C 2011). The number of
blood CD4+FOXP3+ T-cells (both CD25+ and CD25-) and FOXP3 expression detected
with flow cytometry increased two weeks after the start of anti-TNF-α therapy (Li Z 2010,
Boschetti G 2011), and this increase in Tregs associated with a higher suppressive effect
on activated Th-cells (Boschetti G 2011). In the third study, the number of circulating
Tregs was decreased before IFX therapy in CD patients in comparison with the controls,
and enhanced apoptosis was suggested to be the reason based on annexin V staining.
Increased apoptosis was also detected in lamina propria of CD patients (Veltkamp C
2011). In the same study, IFX was shown to restore peripheral Treg cells by normalizing
apoptosis with simultaneous decrease of serum caspase-3/-7 activity (Veltkamp C 2011).
These findings of an anti-TNF-α therapy effect as restoring Tregs supressive capacity is in
line with a previous report with RA (Ehrenstein 2004).
7.2 Predicting therapeutic response to anti-TNF-α agents in IBD
CRP and fecal calprotectin have been studied as markers for predicting therapeutic
response to anti-TNF-α agents. CRP is an acute phase protein secreted by liver
hepatocytes upon inflammatory activation with IL-6, TNF-α and IL-1β (Vermeire S
2006). Two studies with adult CD patients at IFX induction resulted in different outcomes
of the CRP predictive value for the anti-TNF-α response. The first study with 226 patients
found the therapy responding patients to have higher baseline CRP than non-responding
patients (Louis E 2002), while the other study with 279 patients did not find a significant
difference in baseline CRP level regarding the therapy response (Ester N 2002). The use
of CRP as a prognostic marker to anti-TNF-α therapy in IBD is also restricted because
CRP does not rise similarly in UC and CD patients (Vermeire S 2006).
Calprotectin and lactoferrin are neutrophil relased proteins that are secreted in feces
and measurable in IBD (Roseth AG 1999, Kayazawa M 2002). In adult CD patients,
during three months of anti-TNF-α therapy, the decrease of fecal calprotectin and
lactoferrin correlated with the decrease of CDEIS (Sipponen T 2008 b). In another study
with adult CD patients, the decrease of fecal calprotectin correlated with the decrease of
high sensitive CRP one week after the first IFX infusion, but not with the decrease of the
Harvey Bradshaw index of disease activity (Lönnkvist MH 2011).
There were no studies to be found exploring markers to evaluate anti-TNF-α
therapeutic response in pediatric IBD. Several serological markers have been studied to
monitor the effect of anti-TNF-α therapy in adults, but these have not been shown to be
superior to CRP or fecal calprotectin (Table 11). Studies exploring immunological
pathways during anti-TNF-α therapy are also presented in Table 11.
Table 11 Studies monitoring anti-TNF-α therapy response in adult inflammatory bowel disease
CD (24) Whole blood
Tissue Studied markers Methods Predictive value to anti-TNF-α therapy
TNF-α, NFκB Immunoassay, Western blot Increase of NFκB in biopsies or TNF-α secretion in supernatants
Esters N 2002 CD (279) Serum ASCA and pANCA Immunoassay, immunofluorescence ASCA or pANCA did not predict infliximab response
Gao Q 2007 CD (83) Blood, serum, gut MMP-2, MMP-9 Immunohistochemistry, immunoassays,
CD (12 with
Li Z 2010 CD (23), UC
Gut MMP-1, MMP-7, MMP-9-10,
MMP-26, TIMP-1, TIMP-3
Blood, gut Treg cells, CD25 mRNA, CD4
mRNA, FOXP3 (mRNA,
MMP-2 or MMP-9 expression in the gut or blood did not associate
to infliximab response
Immunohistochemistry Of therapy responding markers, stromal MMP-9, MMP-26 and
Flow cytometry, PCR,
TIMP-1 correlated with histological score and MMP-26, TIMP-1
and TIMP-3 with fecal calprotectin
During maintenance, number of blood FOXP3 positive cells
increased more and FOXP3 and CD25 expression in the gut
decreased more in treatment reponders
CD (16), UC (9) Blood Tregs Flow cytometry and suppression assay No correlation was found between the change of Treg number and
CD (32), UC
Blood, serum, gut Apoptotic Tregs, caspase-3 and
CD (22) Serum, plasma Calprotectin, total nitrite,
suPAR, ghrelin, endothelin
cytometry, luminescent substrate assay
the clinical scores during anti-TNF-α therapy
Blood Treg count increased and caspase -3/-7 activity decreased
more in anti-TNF-α responding CD patients
Immunoassays, Griess reaction The change of calprotectin and suPAR after infliximab induction
correlated with the decrease of hs-CRP but not with clinical activity
CD=Crohn`s disease, UC=ulcerative colitis, TNF=tumor necrosis factor, NF=nuclear factor, MMP=matrix metalloproteinase, TIMP=tissue inhibitor of MMPs, Treg=regulatory T-cell, CD25/4=cluster of
differnetation 25/4, ASCA=anti-Saccharomyces cervisiae antibodies, pANCA=perinuclear antineutrophil cytoplasmic antibodies, suPAR=soluble urokinase Plasminogen Activator Receptor, hs=high sensitive
Aims of the study
The ultimate aim of this thesis was to find new means to monitor the decline of the
inflammation during GC and anti-TNF-α therapy, and to find new tools for recognizing
patients who would benefit from these therapies.
The specific aims were:
I To investigate pediatric IBD patient`s serum induced immune responses on donor
derived allogenic peripheral blood mononuclear cells during GC therapy.
II To study whether intra-articular GC injections have systemic effects that would reflect
glucocorticoid bioactivity or patient serum immune activation potency in JIA patients.
III To study whether pre-treatment serum immune activation potency in adult CD patients
is associated with treatment response during anti-TNF-α-therapy.
IV To monitor the effect of GC and anti-TNF-α-therapy on serum levels of MMPs and
their inhibitors in pediatric IBD patients.
Study subjects and methods
1 Study subjects and samples
The summary of patient characteristics is presented in Table 12, and the order of the
sample collection is shown in Figure 6. The samples were collected in sodium-heparine
tubes. Serum for GBA measurements were collected between 8-11 am. After collection,
all serum samples were stored at -20 or -70ºC. All patients or their parents had given their
written consent. Study protocols were approved by the ethics committee of the Helsinki
University central hospital.
Table 12 The study subjects
Patient group Number of
on GC therapy
IBD children on
patients with anti-
shared in I and
13 (4-18) 6/13 in
Diagnosis (n) Study
UC (16+11), CD (2+6), IC (1+2) I+IV
16 14 (8-18) 9/7 UC (5), CD (11) IV
13 13 (9-18) 6/7 UC (6), CD (6), IC (1) IV
15 25 (19-44) 6/9 CD III
21 11 (7-17) 16/5 Oligoarthritis (13), seronegative
polyarthritis (5), seropositive
polyarthritis (1), psoriatic arthritis
(1), undifferentiated arthritis (1)
Pediatric controls 19 12 (5-15) 8/11 Familial hypercholesterolemia (2),
arthritis (1), healthy (16)
IBD=inflammatory bowel disease, GC=glucocorticoid, TNF-α=tumor necrosis factor-alpha, JIA=juvenile
idiopathic arthritis, IACS= intra-articular corticosteroid therapy, UC=ulcerative colitis, CD=Crohn`s disease,
Figure 6 Timing of the samples and clinical activity parameters in studies I-IV. Pediatric
patients with oral GC therapy provided control samples between week two to four.
Serum was used for immunoassay (I-III) and for detection of MMPs (IV). Fcalprotectin=fecal
calprotectin, CDEIS=Crohn´s disease endoscopic index of
severity, CDAI=Crohn´s disease activity index, GBA=glucocorticoid bioactivity
(Raivio T 2002), PGA=Physician’s global assessment.
1.1 Pediatric IBD patients (I, IV)
Forty-four pediatric patients with active IBD were recruited for studies I and IV at the
Childrens Hospital, University of Helsinki, Finland, between 2004 and 2010. Basic
characteristics of the patients are presented in Table 12. The exclusion criterion for the GC
treatment group was preceding systemic GC medication within one month. Patients in
publication I have been described earlier (Vihinen MK 2008) (Table 12). GC treated
patients had oral prednisolone with a median dose of 0.8 (IV) - 1 (I) mg/kg. Study IV
included one patient with oral budesonide with 0.4mg/kg. Sixteen anti-TNF-α naïve
patients were introduced to IFX 5mg/kg (IV). Concomitant medicines were used on a
clinical basis and included 5-aminosalisylate products, azathioprine/methotrexate,
antibiotics or oral GC therapy at the start of anti-TNF-α therapy, and maintained
unchanged between the study samples. Patients were examined by pediatric
gastroenterologists before, and two to four weeks after the start of the therapy. When
available, ESR, CRP and fecal calprotectin were registred before and by the time of the
control visit. In study IV, a chart review was made to evaluate clinical disease activity
with a three-step scale of physician’s global assessment (PGA) defined as 1=no or minor
symptoms, 2=occasional diarrhea, pain or rectal bleeding and 3=diarrhea several times a
day/night or daily rectal bleeding as described (Haapamäki J 2011).
1.2 Adult CD patients (III)
Characteristics of 15 adult CD patients from the Clinic of Gastroenterology, Helsinki
University Central Hospital, Finland, have been described before (Sipponen T 2008 b)
(Table 12). IFX was given with a dose of 5mg/kg at week 0 and 8. One patient had
subcutaneous adalimumab 80mg at week 0, and 40mg every other week until week eight.
When clinically indicated, concomitant medication including 5-aminosalisylate products,
azathioprine/methotrexate, antibiotics or GCs were given, but maintained unchanged
between the study samples. Clinical disease activity graded with CDAI (Best WR 1976,
Sipponen T 2008 b) and endoscopic activity graded with CDEIS (Mary JY 1989) were
evaluated before the therapies and at a three months control visit. CRP, ESR and fecal
calprotectin were measured at the same time.
1.3 Patients with JIA (II)
Twenty-one outpatients with JIA from the Heinola Rheumatism Foundation Hospital
(Heinola, Finland) entered the study with inclusion criteria of active joint inflammation
with need of IACS, but not preceding GC injection within a month (Table 12). The
number of injected joints (2-7) and the injected dose (sum GC dose median 2.5mg/kg)
were judged on clinical grounds. IACS were performed either under local (n=4) or general
anesthesia (n=15). All patients received systemic immunosuppressive and/or
immunomodulating therapy on clinical grounds. The background medication was
unchanged between the study samples. A pediatric rheumatologist examined the patients
before injection, and at two months. During these visits, the number of joints with
swelling, pain or limited range of motion was recorded, and patient and physician global
assessments of the disease activity were evaluated, and Childhood Health Assessment
Questionnaire (CHAQ) values were calculated (Pelkonen P 2001). Serum for GBA,
cortisol and immunological assay was collected before the therapies, 7 (at 3pm) and 24
hours (at 7-8am) thereafter, and within a two months control visit (Figure 8). ESR and
CRP were measured before IACS, and at a two months control visit.
1.4 Controls (IV)
The control group consisted of two different groups: pediatric IBD patients with inactive
disease (n=13), and pediatric patients visiting hospital outpatient clinics for other reasons
(n=19) (Table 12). PGA was determined from controls with IBD. There were no
statistically significant differences in any of the measured markers between the two
control groups, thus the two control groups were united for analyses.
2 Laboratory methods
A summary of the used laboratory methodology is presented in Table 13.
2.1 T-cell stimulation assay for measurement of serum immune-activation
A new biological assay for detection of serum immune activation potency was developed
in this thesis. The effect of the patient`s serum on activation markers of peripheral blood
mononuclear cells (PBMCs) from an allogenic donor (referred to as target cells) was
studied. The PBMCs were isolated from whole blood withdrawn from healthy donors.
PBMCs were separated by Ficoll-Paque (Amersham Biosciences) gradient centrifugation
(800G, 25min). The PBMC layer was washed three times with phosphate buffered saline
(PBS) and centrifuged 10min with 400G between the washes.
For cell culturing, the target PBMCs were diluted in cell culture media to 2x10 6 cell/ml
containing RPMI media with hepes buffer (Gibco), 2μM glutamine (Gibco) and 25�l/ml
gentamycin (Gibco). Target cells, unstimulated or stimulated with phytohemagglutin
(PHA) 5�g/ml, were cultured as duplicates in the presence of the IBD patient’s inactivated
serum (56�C for 35 minutes) at a concentration of 8%, for 72 hours at 37�C in a humified
atmosphere with 5% carbon dioxide. After the culture, the supernatants and the target cells
were collected and centrifuged with 400G for 7 minutes. Lyses buffer (Sigma) was added
on the target cells and the lysed cells, and the supernatants were stored at -70�C. During
methodological testing, the target cell assay was repeated with two different healthy
2.2 Enzyme-linked immunoabsorbent assays (ELISAs)
2.2.1 Cytokine ELISAs
The concentration of IL-5 and IFNγ was studied with an in-house ELISA (Halminen M
1997) from the supernatants collected from the target cell cultures incubated with patient
serum (see above). Detection was performed in duplicate wells. For the ELISA, flatbottomed
96-well plates (Nunc) were coated 24 hours earlier with a cytokine-specific
antibody (ENDOGEN monoclonal anti-human IFNγ or PharMingen anti-human IL-5)
diluted in 0.1M Na2HPO4. The following day, the wells were washed with PBS/0.05%
TWEEN, which was also used in the later washes. Then the filtered 1% bovine serum
albumin (BSA)-PBS was added, and after 30 minutes the wells were washed 3 times. The
samples and the standards were diluted in 1% BSA-PBS/0.05% TWEEN (later the diluting
buffer) and incubated on the plates for two hours. After washing the wells four times, the
second antibody (ENDOGEN monoclonal biotinylated anti-human IFNγ or PharMingen
purified anti-human IL-5) was added and incubated for 1.5 hours. After washing four
times, Streptadivin-phosphatase (Zymed) with the diluting buffer (1:1000) was added and
the wells were incubated for 30 minutes. The substrate Nitrophenylphosphate tabs (Sigma)
in sterilized water (2mg/ml) was added, and the detection was performed with a Thermo
Labsystems Multiscan Ascent. IL-17 was measured with an ELISA-kit according to the
protocol set by the manufacturer (R&D Systems). In cytokine ELISAs, Δ-value describing
the effect of the PHA stimulation was calculated by subtracting the non-stimulated value
from the stimulated value of every detected serum sample.
2.2.2 ELISAs for MMPs, TIMPs, α2M, HNE and MPO
Commercially available ELISAs according to the instructions of the manufacturer were
used for analysis of total MMP-7 (R&D Systems), total MMP-8 (Medix Biochemica),
total pro-MMP-9 (Amersham Biosciences), HNE (Bender MedSystems mbH), and MPO
(Immunodiagnostic AG) as described earlier (Tuomainen AM 2007, Rautelin H 2009,
Rautelin H 2010). Commercially available ELISAs according to the manufacturer’s
instructions were also used for detection of TIMP-1 (GE Healthcare), TIMP-2 (GE
Healthcare) and total α2M (Immunodiagnostic AG). The calculation for MMP-7-9/TIMP-
1 and MMP-7-9/TIMP-2 ratios was performed as mol/L.
2.3 Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
FOXP3, GITR and IFNγ spesific mRNA were measured in donor derived PBMCs
stimulated with patient serum as described above. In addition, IFN�, IL-5, IL-10, FOXP3,
GITR, GATA3, STAT-5b and t-bet specific mRNA were detected in PBMCs from
pediatric IBD patients. Total RNA was isolated with GenElute Mammalian total RNA
miniprep kit (Sigma) and RNA concentration was measured by spectrophotometer. For
reverse transcription, Tagman Reverse Transcription reagents (Applied Biosystems) were
used, combined with treatment of total RNA at 10 ng/μl with DNAse I (0.01U/μl) (Roche
Diagnostics) for eliminating genomic deoxyribonucleic acid. Quantitative RT-PCR was
performed by predesigned FAM –labelled TaqMan Gene Expression Assay reagents
(Applied Biosystems) and the ABI Prism 7700 Sequence Detection System (Applied
Biosystems) in triplicate wells. Threshold cycle (Ct) indicates the number of PCR cycles
at which the target molecule exceeds a predefined fluorescence threshold value. The
difference value (ΔCt) is achieved by subtracting the housekeeping gene’s (18s) Ct value
from that of the target gene’s. As an interassay standard was used, exogenous cdeoxyribonucleic
acid collected from PHA stimulated PBMC. ΔΔCt is the difference
between the ΔCt of the analyzed sample and ΔCt of the calibrator. 2 -ΔΔCt then gives a
elative amount of the target gene in the analyzed sample compared with the calibrator.
For presentations, the relative amount of target genes was multiplied by 1000.
2.4 Glucocorticoid bioactivity (GBA)
Bio-assay for GBA measurement was performed as duplicates as published (Raivio T
2002). GBA measurement is a recombinant cell assay in which 10μl of patient serum is
incubated overnight with COS-1 cells that are transfected with human GR coding
expression vector, ARIP3, nuclear receptor co-activator, and reporter gene (lusiferace and
galactosidase). The detection limit is
Table 13 Applied laboratory methodology
Aim or measured marker Methodology Performer Study
Separation of PBMCs from
T-cell stimulation assay T-cell stimulation assay
Detection of IFNγ and IL-5 from
(described detailed in
Detection of IL-17 from supernatants Commercially available
mRNA of IFNγ, IL-5, IL-10,
FOXP3, GITR, GATA3, STAT-5b
In-house ELISA Author I-III
RT-PCR PhD Harri Salo at professor
Outi Vaarala`s laboratory
GBA Bioassay Professor Olli A Jänne`s
MMP-7-9, TIMP-1-2, HNE, MPO
Cortisol Commercially available
Fecal calprotectin Commercially available
PhD, DDS Taina
Tervahartiala at professor
Timo Sorsa`s laboratory
Clinical laboratory II
Clinical laboratory I, III-IV
ESR, CRP Routine protocol Clinical laboratory I-IV
PBMC=peripheral blood mononuclear cell, IFN=interferon, IL=interleukin, FOXP3=forkhead transcription
factor 3, GITR= glucocorticoid-induced tumor necrosis factor receptor, STAT=signal transducer and
activator of transcription, GBA=glucocorticoid bioactivity, MMP=matrix metalloproteinase, TIMP=tissue
inhibitor of MMPs, HNE=human neutrophil elastase, MPO=myeloperoxidase, α2M=α2-macroglobulin,
ESR=erythrocyte sedimentation rate, CRP=C-reactive protein, ELISA=enzyme-linked immunoabsorbent
assay, RT-PCR= reverse transcriptase-polymerase chain reaction
Results and discussion
1 Serum immune activation potency on target cells (I-III)
For this study, a new biological assay was developed with the aim to describe the
individual sum effect of GC and anti-TNF-α therapies in patient serum samples. In this
assay, naïve or activated PBMCs from healthy donors were stimulated in the presence of
the patient’s serum, and up-regulation of cytokines and transcription factors were
measured at protein level with ELISA, and at mRNA level with quantitative PCR. It is to
be noted that the T-cell activation markers were not measured directly from patient serum
or the patient`s immune cells, thus the results are not directly comparable with the
immunological responses described earlier during GC or anti-TNFα therapy.
1.1 The assay development
The first pilot study was made by stimulating two different donors’ derived allogenic
PBMCs (referred to as target cells) with pediatric IBD patients’ plasma withdrawn before
and two to four weeks after the start of GC therapy. IFNγ secretion was median 75 000
ρg/ml (range 53 000-93 000 ρg/ml) before therapy, and median 28 000 ρg/ml (range
53 000-93 000 ρg/ml) after therapy (p=0.012; n=8; unpublished) when PHA activated
PBMCs derived from a 31-year-old male were cultured with pediatric IBD patients’
plasma. IFNγ secretion was median 131 ρg/ml (range 0-9500 ρg/ml) before therapy, and
median 0 ρg/ml (range 0-9400 ρg/ml; p=0.018; n=10; unpublished) when the same
patient’s serum samples were cultured with PHA activated PBMCs derived from a 26year-old
female. This study was repeated with similar results (data not shown). The
immunosuppressive effect mediated by GC treated patient serum was found regardless of
the target cell responsiveness. It is to be noted that PBMCs derived from the female donor
were not used in later experiments due to limited proliferation. The decision to continue to
test the effect of patient serum instead of plasma was based on the easy availability of the
serum samples. It was found that patient post-treatment serum suppressed PBMCs
cytokine response similar to the plasma (below).
During the study protocol, there were several steps designed for avoiding technical
bias. First, those serum samples that were compared with each other (before and after
therapy) were always cultured on the same plates. The serum samples were pipeted on the
plate’s minimum as duplicate wells, and the supernatants derived from the same serum
sample were collected as a pool for analysis. For the third work (III), the assay protocol
was modified to contain a control sample derived from one healthy individual on every
cell culture plate. The in-house cytokine ELISA also contained a standard sample on each
plate. The ELISA was performed as duplicates, and if the intra-assay variation became
higher than 15% between the duplicated wells, the sample was re-analyzed. The final
ELISA result represents the mean of the two analyzed wells.
The idea for this bioassay arose from three sources: first, from the knowledge that altered
immunological response plays a major role in pathogenesis of IBD (Baumgart DC 2007
a); second, the finding that patient serum could be used in a bioassay to elicit a GR
mediated response in COS-1 cell culture that was measurable with transactivation assay
(GBA; Raivio T 2002); and third, the observation that in IBD, GC resistance reflected Tcell
suppressive capacity, seen ex vivo as PBMCs derived from GC resistant patients
showed lower suppression of proliferation induced by increasing DEX concentration
(Hearing SD 1999). Altough not with the same protocol, the activation of peripheral blood
lymphocytes with patient plasma has been previously described as an ex vivo model of
acute respiratory distress syndrome (Meduri GU 2002). As GCs widely affect the human
genome, and multiple mechanisms related to GR and tissue responsiveness can mediate
GC biological effects (Sidoroff M 2011), a bioassay, as presented here, could offer a
promising tool for describing the individual sum effect of GC therapy.
1.2 The effect of GC treated patient`s serum on target cell cytokine profile
IFNγ and IL-5 secretion from the target cells and changes in cytokine secretion during oral
GC therapy and IACS are presented in Table 14. IL-5 secretion from unstimulated target
cells was low or below the detection limit.
1.2.1 Pediatric IBD patients with oral GC therapy (I)
The median secretion of IFNγ induced by pediatric IBD patient serum from both
unstimulated and PHA stimulated target cells decreased during a 2-4 week oral GC
therapy (p=0.017 and p=0.006 respectively), and the trend was similar in IL-5 secretion in
stimulated target cells (p=0.055) (Table 14). No correlations were found between weight
related dose of GC and serum induced target cells IFNγ or IL-5 secretion, or with the
change of these markers during the therapy (ΔIFNγ, ΔIL-5; all p=ns).
Table 14 Cytokine secretion from healthy donor derived peripheral blood mononuclear
cells in the presence of serum withdrawn from pediatric inflammatory bowel
disease (IBD) and juvenile idiopathic arthritis (JIA) patients at baseline and 2-4
weeks after the start of oral glucocorticoid (GC) therapy or 24 hours after
receiving intra-articular corticosteroid therapy (IACS).
Patient group Cytokine Baseline
on oral GC
Children with JIA
IFNγ 0 (0-308) 0 (0-29) 1/7/11 p=0.017
62000 (6600-122000) 4/13/2 p=0.006
IL-5 PHA 69 (0-148) 17 (0-164) 4/13/2 p=0.055
IFNγ 35 (0-95) 27 (0-75) 7/8/2 ns
IFN=interferon, IL=interleukin, ns= p>0.05
31 (2-144) 22 (0-135) 6/11/1 p=0.085
The finding that patient serum induced changes in target T-cell responses during GC
therapy is in line with a previous report that plasma from MP treated acute respiratory
distress syndrome patients decreased TNF-α and IL-1β, and increased IL-10 expression in
peripheral lymphocyte cultures detected with Western blot and PCR (Meduri GU 2002).
Here, the post-treatment serum mediated decrease in Th1 type cytokine IFNγ secretion
from target cells, which is in line with the reported effect of GC as suppressing Th1
immunity (Franchimont D 2004). Interestingly, when the effect of GC therapy was studied
as patients` serum induced changes in IL-5 secretion from the target cells, a trend of
decrease in IL-5 secretion was seen (Table 14). In the literature, the reported effect of GC
therapy on Th2 cytokines such as IL-4, IL-5 and IL-10 is mainly enhancing (Franchimont
D 2004). Enhanced Th2 response can result from concomitant Th1 suppression, especially
lack of IL-12, demonstrated previously in vitro (Franchimont D 2004). Activated APCs
treated with DEX showed a decreased capability to secrete IL-12, and in co-cultures these
APCs increased IL-4 and IL-10 secretion from CD4+ T-cells compared with APCs not
treated with DEX (Blotta MH 1997). GCs can have direct effects on Th2 transcription
factors as DEX was shown to up-regulate Th2 transcription factor c-maf (Galon J 2002).
The noted Th2 suppression could reflect GCs direct suppressive effect on lymphocytes.
Previously, DEX was shown to inhibit T-cell proliferation in vitro in a dose-dependent
manner (Hearing SD 1999). However, it is to be noted that here no GC was added to
PBMC cultures, but instead PBMCs were treated with patient serum containing
therapeutic doses of metabolized GC. Opposing reports of GC mediated Th2 supression
also exist. In vitro IL-4 secretion and IL-4 mRNA expression decreased in activated
human PBMC and purified T-cell cultures after adding physiological doses of
hydrocortisone (Wu CY 1991). In line with this report are two reports on asthma: the first
showing a significant decrease in IL-5 secretion after a single oral GC dose detected with
immunoassay from PBMC culture supernatants (Majak P 2009); and the second reporting
the decrease of IL-4 and IL-5 specific mRNA in cells of bronchoalveolar lavage fluid in
steroid sensitive asthma patients (Leung DY 1995). The finding here, that GC treated
patient serum showed a trend to decrease normal PBMCs IL-5 response, underlines GCs
multidirectional effect and supports the concept of the benefit of GCs in the treatment of
Th2 immune mediated diseases such as asthma or allergies.
In pediatric IBD patients, weight-related dose of achieved oral GC had no correlations
with serum induced target cells IFNγ, or IL-5 secretion from the target cells or Treg
markers expression (reported below); thus the observed suppression of target cell
responses was not solely due to exogenous GC concentration in serum, but reflects the net
effect of individual inflammatory activity in the patient’s serum. It is suggested that in
IBD, therapeutic response did not directly associate to dose of GC. This was based on the
finding that no difference in therapeutic response could be associated with doses of
intravenous GCs reaching equivalent plasma cortisol levels (Kaplan HP 1975). Further,
the availability of biologically active GC did not associate with therapeutic response in
pediatric IBD (Vihinen MK 2008).
1.2.2 JIA patients with IACS (II)
The total number of injected joints was 69 in 21 studied JIA patients. The doses in a single
large joint (shoulder, knee or hip) ranged between 0.6-1.4mg/kg for MP, and 0.4-0.9mg/kg
for THA. The median total sum dose of achieved GC (both MP and THA) of a single
patient was 2.5mg/kg (range 1.3-7.4 mg/kg). 24 hours after IACS, the serum induced
response on target cell IFNγ secretion was not seen (Table 14), and IL-5 secretion was
marginally decreased (p=0.085). After IACS, the weight-related total GC dose showed an
inverse correlation with post-treatment IFNγ and IL-5 secretion from activated target cells
(r=-0.691, p=0.003 and r=-0.482, p=0.043 respectively), but not with the change of target
cells’ cytokine secretion (ΔIFNγ or ΔIL-5; both p=ns).
The systemic GC treatment in IBD patients was seen as a serum mediated decrease in
IFNγ and IL-5 responses in the target cells. However, in JIA patients, this kind of effect
was only observed as a trend for IL-5 response after IACS, thus the systemic effect of
IACS was not comparable with that seen during oral GC therapy. In RA, it is reported that
after IACS the degree of GC systemic absorption was determined by the GC dose and by
the area of inflamed synovial membrane (Armstrong RD 1981). The inflammation of the
joint increases the joint permeability which can affect drug efflux (Middleton J 2004).
1.3 The effect of GC treated patient serum on target cell FOXP3 and GITR
Serum withdrawn from pediatric IBD patients two to four weeks after the start of GC
therapy induced lower levels of GITR specific mRNA expression in target cells when
compared with the serum withdrawn at the start of treatment. A decrease in patient serum
induced FOXP3 expression after GC therapy was seen in naїve, but not in activated target
cells (Figure 7). Weight-related GC dose had no correlations with FOXP3 or GITR
specific mRNA expression after two weeks GC therapy, or with the fold change of
FOXP3 and GITR during therapy (all p=ns).
Relative units of mRNA
Relative units of mRNA (PHA)
Figure 7 Glucocorticoid-induced tumor necrosis factor receptor (GITR) and forkhead
transcription factor 3 (FOXP3) mRNA expression on donor derived peripheral blood
mononuclear cells cultured with pediatric IBD patient serum received before and
within a month after the start of oral glucocorticoid therapy. a.) Unstimulated target
cells, b.) Phytohemagglutin (PHA) stimulated target cells. Line shows the median
value. Modified from Study I.
No previous reports were to be found describing GC effect on Treg markers FOXP3 or
GITR in IBD. In a recent study in mice, DEX treatment decreased the number of
circulating Tregs in vivo without effect on Tregs suppressive capacity in vitro (Sbiera S
2011). Similarly, GC therapy on allergic rhinitis down-regulated FOXP3 expression on
nasal mucosal biopsies (Malmhäll C 2007). Two studies in asthmatic patients did not find
changes in Treg expression after GC exposure. The study with 54 asthmatic children
examined the effect of a single oral GC dose combined with regularly inhaled steroid
therapy and showed no effect on Treg number or FOXP3 expression measured at three
months (Majak P 2009). In another small study with adult asthmatic patients, no change
was observed in Treg cell counts or GITR expression during four weeks inhaled GC
therapy (Zhang Q 2008).
Here, the patient serum induced FOXP3 expression decreased at a level of statistical
significance on naїve donor PBMCs, but the decrease of FOXP3 did not achieve the level
of significance on activated donor PBMCs. It has been previously suggested that the
PBMC activation stage would affect GC response (Galon J 2002). In addition, healthy
PBMCs have been suggested to respond differently to GC therapy than cell derived from
diseased individuals (Galon 2002, Sbiera S 2011). Indeed, contrary to the findings here,
the majority of in vivo studies have reported increased Treg numbers or FOXP3
expression after GC treatment in patients with asthma (Karagiannidis C 2004) or
autoimmune disorders such as systemic lupus (Suarez A 2006) and multiple sclerosis
(Braitch M 2009).
1.4 Clinical implications of serum immune activation potency (II, III)
1.4.1 In JIA patients (II)
Post-treatment clinical disease activity had several inverse correlations with target cell
IFN� and IL-5 secretion induced by patient serum withdrawn either before, or 24 hours
after IACS (Table 15). No correlations were found between pre-treatment clinical disease
activity and pre-treatment target cell cytokine secretion (all p=ns).
Table 15 An inverse correlation between IFN� and IL-5 secretion from phytohemagglutin
activated donor derived peripheral blood mononuclear cells cultured with patient
serum withdrawn before and 24 hours after intra-articular corticosteroid therapy
(IACS) and clinical picture assessed at two months.
Clinical picture at two months Before IACS After GC IACS
Number of active joints ns p=0.044
IFNγ IL-5 IFNγ IL-5
Number of joints with limited range of motion ns p=0.049
Number of swollen joints ns ns p=0.005
Patient’s global assessment p=0.014
ns ns ns
ns ns ns
Physician`s global assessment ns ns ns ns
IFN=interferon, IL=interleukin, CHAQ= Child Health Assessment Questionnaire, ns= p>0.05
Because an inverse correlation between clinical disease activity parameters and target cell
cytokine secretion was seen both before and after IACS, this finding cannot solely reflect
the differences caused by the exogenous GC which has leaked out from the joint. Contrary
to the inverse correlations seen here between clinical disease activity and target cell
cytokine responses induced by patient serum, serum level of IL-12 protein was shown to
be elevated in active JIA (Gattorno M 1998, Yilmaz M 2001), and correlated positively
with ESR and CRP (Gattorno M 1998). In addition, in systemic and oligoarticlular JIA, a
positive correlation was found between serum IL-12 level and the number of swollen
joints or the joints with limited range of motion (Yilmaz M 2001). The inverse correlation
seen here as serum mediated impaired cytokine response in target cells could reflect the
chronic inflammation induced immune suppressive state, or also the existence of negative
feedback mediators in patient serum due to inflammatory activity of the patient.
1.4.2 Serum immune activation potency related to CDEIS and ESR in adult CD
Before anti-TNF-α therapy, adult CD patient serum induced FOXP3 specific mRNA
expression in target cells correlated inversely to CDEIS and ESR (r=-0.621, p=0.013 and
r=-0.548, p=0.034 respectively). GITR specific mRNA expression correlated inversely
with CDEIS (r=-0.625, p=0.013). The low target cells FOXP3 and GITR specific mRNA
expression at baseline correlated with the beneficial change of CDEIS during three months
anti-TNF-α therapy (r=0.600, p=0.018 and r=0.589, p=0.021 accordingly).
Tregs participate in controlling Th responses against pathogens of the gut, and their
impaired function plays a part in IBD immunopathogenesis (Boden EK 2008). In adult
IBD, the frequency of peripheral blood Tregs was decreased (Wang Y 2011, Boschetti G
2011) and there was an imbalance between expressions of circulating Treg/Th17 in
comparison with controls (Eastaff-Leung N 2010). As Tregs suppress Th cells (Thornton
AM 1998), it seemed logical that those patients whose serum mediated enhanced
induction of Treg markers FOXP3 and GITR in vitro had clinically milder disease in vivo
seen as low CDEIS and ESR. We did not measure Treg markers on the patient PBMCs,
but it is possible that allogeneic PBMCs derived from healthy donors were capable of
responding differently to patient serum than patient derived PBMCs in vivo.
Interestingly, the low FOXP3 and GITR specific mRNA expression in activated target
cells at baseline associated with better treatment response during three months, seen as a
decrease of CDEIS. Recently, anti-TNF-α therapy has been shown to restore the number
and function of circulating Tregs in IBD (Li Z 2010, Boschetti G 2011, Veltkamp C
2011), previously shown also in RA (Ehrenstein MR 2004). These results suggest that
there can be group of CD patients with low systemic Treg activity that may benefit from
anti-TNF-α therapy induced Treg activation, but those with high Treg activity already
before therapy would not benefit from further Treg activation achieved with biological
It is also possible that FOXP3 and GITR expression here reflected the activation of
target cells rather than direct up-regulation of Treg activity (Allan SE 2007, Corthay A
2009). In this case, the high expression of FOXP3 and GITR is associated with impaired
therapeutic response as similarly reported in adult RA patients in whom the high
inflammatory activity, seen as a high number of blood chemokine receptor -3 and -5
expressing CD4+T-cells, or high serum IL-2 receptor level at the start of the anti-TNF-α
therapy, associated with a worse therapeutic effect to anti-TNF-α agents (Nissinen R 2003,
Kuuliala A 2006).
2 GBA (II)
2.1 The effect of IACS to serum GBA and cortisol
Serum GBA rose and cortisol decreased after the IACS; these changes were maintained 24
hours but were normalized within two months (Figure 8).
Serum GBA (nM)
0 7-hours 24-hours2 months
Serum cortisol (nM)
0 7-hours 24-hours
Figure 8 The effect of intra-articular corticosteroid therapy to the level of juvenile idiopathic
arthritis patients’ serum glucocorticoid bioactivity GBA (a.) and cortisol (b). Line
shows the median value. Modified from Study II.
Endogenous GBA correlated with serum cortisol (r=0.821, p
JIA. After IACS, endogenous cortisol production measured from saliva was suppressed
for 10-30 days and the circadian fashion of GC secretion was disturbed (Huppertz HI
1997). Taken together, the finding that GBA rose and cortisol decreased after IACS
demonstrates that intra-articular GC delivery is evoking a systemic GC effect in the
patient. This systemic GC effect further seemed to modulate the immune activation
potency of the JIA patient’s serum (above).
2.2 Comparison between GBA and serum immune activation potency
At baseline, GBA correlated positively with potency of JIA patients’ serum to induce
IFNγ secretion from activated target cells (r=0.503, p=0.047). Similar pre-treatment
correlation was not seen in pediatric IBD patients (p=ns; unpublished). In JIA patients, the
increase of GBA (ΔGBA) due to IACS associated with the decrease of serum induced IL-
5 secretion (ΔIL-5; r=-0.772, p
Above, in chapter 2.1, serum GBA was shown to correlate highly to serum cortisol.
Cortisol is an anti-inflammatory hormone secreted upon stress, and its secretion is
stimulated by cytokines such as IL-2, IL-6, IFNγ and TNF-α (Sapolsky RM 2000). Proinflammatory
cytokine IL-1 directly increased cortisone levels in a rat model (Besedovsky
H 1986). GC effect has been suggested to vary from activating (permissive) to suppressive
actions depending on the level of the released hormone (Figure 10; Sapolsky RM 2000).
Figure 10 Model of glucocorticoids permissive (activating) and suppressive actions.
GR=glucocorticoid receptor, MR=mineralocorticoid receptor. Modified from
Sapolsky RM 2000.
Here, a similar model of GC action was found, as at baseline endogenous GBA correlated
positively with JIA patient’s serum immune activation potency, but after administration of
exogenous GC, a classical GC effect of immune suppression (Franchimont D 2004) was
seen as a rise in serum GBA associated with a pronounced decrease in serum induced
cytokine secretion in pediatric IBD and JIA patients (Figure 9).
2.3 Clinical implications
In JIA patients receiving IACS, no clinical correlations were found between GBA at any
time point and measurements for clinical disease activity or the inflammatory parameters
ESR and CRP (all p=ns; data not shown). This is in line with previous reports during oral
GC therapy in pediatric IBD where GBA did not associate with the treatment effect or to
side effects (Vihinen MK 2008, Turner D 2010 a). The upper limit of endogenous
circulating GBA in children is defined as 118 nM cortisol equivalents (Vihinen MK
2008), thus one clinical application of GBA measurement could be a monitoring of the
3 MMPs, their inhibitors and neutrophil released enzymes during
GC and anti-TNF-α therapy (IV)
Matrix metalloproteinases are enhanced by pro-inflammatory cytokines and overexpressed
in active IBD (Pedersen G 2008). Here the serum profile of total MMP-7, total
MMP-8, total pro-MMP-9, TIMP-1 and -2, total α2M, HNE and MPO was studied in
pediatric IBD patients’ serum at baseline and within a month after the start of oral GC
therapy or at week two after the first IFX infusion.
3.1 Comparison between patients with active IBD and controls
Before therapies, all measured MMPs, MMP-7-9/TIMP-1 ratios, MMP-8-9/TIMP-2 ratios,
α2M, HNE and MPO were higher in active IBD patients than in controls (all p≤0.022;
Table 16). At baseline, TIMP-1 was higher than in controls only in those IBD patients
with GC therapy (p=0.007) and MMP-7/TIMP-2 was higher in patients with anti-TNF-α
In line with the results here, unbound-plasma MMP-9 measured with gelatin zymography
was increased in adult CD patients in comparison with controls (Kossakowska AE 1999).
In mucosal biopsies, MMP-9 has been shown to be over-expressed in adult IBD in
comparison with controls (Baugh MD 1999, Mäkitalo L 2009), but this finding was not
consistent in pediatric IBD (Baugh MD 1999, Mäkitalo L 2010). In line with the finding
of a higher TIMP-1 level in pediatric IBD patients (GC group) than in controls, plasma
and serum level of TIMP-1 was shown to be higher in adult IBD patients compared with
controls (Wiercinska-Drapalo A 2003, Kapsoritakis AN 2008, Wang YD 2009).
Circulating TIMP-1 levels also correlated with clinical and endoscopic activity indices
(Wiercinska-Drapalo A 2003) and to CRP (Wiercinska-Drapalo A 2003, Kapsoritakis AN
2008). In one study where unbound-plasma TIMP-1 was measured with zymography, no
difference was seen in TIMP-1 between CD patients and controls, nor an association with
the disease activity index (Kossakowska AE 1999). This discrepancy between the results
might be related to the different form of the measured protein, for example total, free or
complexed; this information is rarely given in the text. Here, the GC treatment group had
higher pre-treatment PGA than the anti-TNF-α group (PGA median 3 and 2 respectively).
The lower disease activity is probably the reason why TIMP-1 was not statistically
different between the anti-TNF-α treatment group and the controls.
Contrary to the finding of high serum total α2M level in pediatric IBD patients, in
adult IBD patients’ serum, α2M level was lower than in the controls when detected with
electrophoresis (Weeke B 1971). It is possible that the differences in findings with serum
α2M level related to different disease activity between the studies, as 43% of the IBD
patients in Weeke´s study had inactive disease, whereas in this study α2M level was
compared between IBD patients with active disease to controls. Fecal α2M level detected
with immunonephlometry was increased in IBD patients in comparison with controls and
correlated with CDEIS (Becker K 1999).
Previously, in line with this study, HNE plasma level has been shown to be increased
in adult IBD patients in comparison with controls and to be higher in active disease
(Adeyemi EO 1985, Fischenbach W 1987, Gouni-Berthold I 1999, Morohoshi Y 2006).
Similarly, the finding of an increased level of another enzyme found in neutrophils, MPO,
in IBD patients’ serum in comparison with controls, was in line with a previous report of
higher MPO level in adult UC patients’ serum than the normal level as reported by the
manufacturer (Peterson CG 2007).
3.2 Changes in measured serum markers during oral GC and IFX therapy
In pediatric patients with active IBD two to four weeks after the start of oral GC therapy,
the serum level of MMP-7, TIMP-1 and MMP-7/TIMP-2 ratio decreased (all p≤0.001) to
the level of the controls, while MMP-9/TIMP-1 increased (p=0.0018). Although the
decrease of serum α2M was clearly not significant (p=0.658), after one month GC therapy
serum α2M level was comparable with the controls (p=0.185; Table 16). During GC
therapy, there were no statistically significant changes in other measured MMPs, TIMP-2,
the MMP/TIMP ratios, HNE or MPO (all p=ns). Two weeks after the first IFX infusion,
serum α2M and HNE had increased (p=0.023 and p=0.026 respectively) and MMP-7
presented a trend of decrease (p=0.063). There were no statistically significant changes in
other measured markers or MMP/TIMP ratios (all p=ns). Although the decrease of MMP-
7/TIMP-2 during anti-TNF-α therapy was not statistically significant (p=0.438), after two
weeks of the first anti-TNF-α infusion, MMP-7/TIMP-2 was no longer statistically
different from the controls (p=120; Table 16).
No previous reports were found designed to monitor the effect of GC therapy on serum or
tissue expression of MMPs or TIMPs in IBD. Here it was found that serum TIMP-1 level
decreased within a month of the start of GC therapy. In line with this current study, the
decrease in TIMP-1 level due to GCs has been previously demonstrated in vivo in
astmathic patients treated with inhaled and/or oral GCs that had lower TIMP-1 level in
bronchoalveolar lavage fluid than the non-treated subjects (Mautino G 1999), and in vitro
in bovine chondrocyte culture with lower TIMP mRNA expression and activity level after
treatment with DEX (Sadowski T 2001). In addition, Cushing’s syndrome patients from
cultured skin fibroblasts expressed TIMP-1 at a lower level than normal cells (Zervolea I
Table 16 MMPs, TIMPs, α2-macroglobulin (α2M), human neutrophil elastase (HNE) and myeloperoxidase (MPO) in pediatric inflammatory bowel disease
patients’ serum introduced to glucocorticoid or anti-TNF-α therapy. * p≤ 0.001 and **p
The observed decrease in serum TIMP-1 could reflect the suppression of inflammation
due to GCs with a concomitant decrease in the need of TIMP-1 mediated inhibition rather
than a direct GC effect. In support of this assumption, DEX activated the TIMP-1
promoter area through GR in COS-1 cell cultures (Förster C 2007).
Also MMP-7 and MMP-7/TIMP-2 ratio decreased during GC therapy, but the decrease
of MMP-7/TIMP-2 reflects the decrease of MMP-7, since TIMP-2 level remained
constant during the therapy. In line with this observation, in human macrophage cultures,
GC suppressed MMP-7 mRNA expression studied with Northern blot (Busiek DF 1995).
A possible mechanism linking the decrease of MMP-7 to GCs therapeutic effect in IBD
could be down-regulation of MMP-7 mediated proteolytic cleavage of innate immunity
related molecules (Burke B 2004). MMP-7 is reported to proteolytically activate
antimicrobial peptides such as α-defensin-5 expressed in Paneth cells of the intestine
(Wilson CL 1999, Lopez-Boado YS 2000), but also to cleave TNF-α family members and
pro-apoptotic Fas-ligand (Burke B 2004).
Within two weeks of the first IFX infusion, the serum level of α2M and HNE had
increased. No human studies were found designed to monitor the effect of anti-TNF-α
agents to these markers. Increase in serum α2M level during anti-TNF-α therapy seems
reasonable since MMPs are over-expressed in IBD (Table 5), which has been suggested to
contribute to IBD pathogenesis. As α2M is considered to serve as the MMPs major plasma
inhibitor (Baker AH 2002), it is possible that increased α2M mediated suppression
participates in the anti-TNF-α agents’ therapeutic effects in IBD.
The finding of increase of serum HNE level during anti-TNF-α therapy is rather
confusing, as plasma and gut HNE activity was shown to be elevated in active IBD.
Furthermore, in mice, intra-peritoneal delivery of NE-inhibitor had a therapeutic effect on
treatment of induced colitis (Morohoshi Y 2006). The increase of serum HNE level during
anti-TNF-α therapy could relate to increased apoptose of inflammatory cells reported to
lead to HNE release (Vandivier RW 2002).
During anti-TNF-α therapy, serum MMP-7 level presented a trend of decrese. The
anti-TNF-α treated patients were allowed to have GCs as concomitant medicines and had
lower pre-treatment PGA than the patients at the start of GC therapy. This lower disease
activity is probably the reason for not finding any significant decrease in serum MMP-7
level in this patient group similar to the GC treatment group. After the first IFX infusion,
the MMP-7/TIMP-2 ratio resembled that of the controls. This may reflect the resolution of
inflammation, as in the adult UC patients gut samples the high MMP-7/TIMP-2 ratio was
associated with high histological activity in the gut specimens (Matsuno K 2003).
It is to be noted that here, IFX therapy showed no effect on serum pro-MMP-9 level.
The only previous study monitoring the effect of anti-TNF-α therapy with ELISA on the
serum MMP-9 level in adult CD patients with active disease found a decrease in serum
MMP-9 just at the border of statistical significance in an in-house study, but no decrease
was seen in a larger patient group (Gao Q 2007). Previously in adult CD, mucosal
expression of MMP-9 decreased during anti-TNF-α therapy (Gao Q 2007, Mäkitalo L
2009). Also in an in vitro study, IFX down-regulated MMP-9 secretion into supernatants
in intestinal tissue cultures (Meijer MJ 2007 b).
3.3 Clinical implications
At baseline, serum MMP-7 correlated with ESR in pediatric patients with active IBD
(r=0.420, p=0.026, n=28). Also among all IBD patients, high serum MMP-7 associated
with clinically active disease assessed with PGA (p=0.047, n=48). There were no other
statistically significant correlations between measured pre-treatment serum markers and
assessed clinical parameters in this study (above; all p=ns). During the GC therapy, the
change of MMP-9/TIMP-1 (ΔMMP-9/TIMP-1) correlated positively with the decrease of
ESR (ΔESR; r=0.543, p=0.045, n=14). No other correlations relative to changes in MMPs
and clinical activity markers during GC or anti-TNF-α therapy were found (all p=ns).
Previously, several associations between MMP and TIMP expression in the intestinal
mucosa samples of IBD patients and disease activity have been reported (Table 5). The
studies of serum MMP levels are limited. In adult UC patients, the plasma level of TIMP-
1 was associated with three-step disease activity (Wang YD 2009). No studies were found
associating serum MMP-7 level to clinical disease activity in IBD, but the finding that
MMP-7 level reflected the clinical disease activity is in line with reports at tissue level. In
pediatric IBD patients, MMP-7 expression in colon samples was higher in CD than in UC
samples. Five UC samples that were later re-diagnosed as CD presented higher MMP-7
and MMP-1 expression than the other UC samples, and MMP-7 expression was suggested
to be useful in differentiating between UC and CD (Mäkitalo L 2010). MMP-7 expression
in epithelial cells at wound edges in adult UC patients correlated with the inflammatory
activity grade in these samples (Matsuno K 2003). Here, no direct correlations were seen
between serum MMP-9 level and disease activity measurments. In adult CD patients’ gut
biopsy samples, MMP-9 expression in neutrophils correlated with the histological score,
CDEIS and CRP (Mäkitalo L 2009).
4 General discussion
4.1 Strengths and weaknesses of the study
The methodology for the assesment of the immunomodulatory effect of the patients`
serum was designed by the study group, and the clinical use of the method was analyzed
in these studies, which could thus be called pilot studies.
4.1.1 Subjects and samples
This thesis is based on four individual studies, all of which included only 15 to 21
patients. The limited number of patients is a common problem in pediatric studies. During
the sample collection, all consecutive pediatric IBD patients from the Helsinki University
Children’s Hospital (I, IV) and the Heinola Rheumatism Foundation Hospital (II)
fulfilling inclusion criteria were assessed. The endoscopically evaluated adult CD patients’
samples were received as an extension of another study (Sipponen T 2008 b). Due to the
small number of study subjects, no power calculations were made, thus the risk of a false-
negative result is relatively high.
Studies I, II and III were designed to evaluate the effect of GC and anti-TNF-α
therapy, and paired samples at different time points were considered to serve as reciprocal
controls, thus no control group of healthy subjects were included in these studies. In
retrospect, this is a clear weakness of these studies. In work IV, two control patient groups
were included: patients with inactive IBD, and pediatric patients visiting Helsinki
University Children’s Hospital for other reasons. In addition, the measurement of drug
concentration in the study samples would have been useful to objectively evaluate the
individual biological effect induced by patient serum on target PBMCs. With a lack of
healthy controls and knowledge of exogenous GC concentration in the samples, the gained
data must be considered as observational instead of explanatory.
In the last work (IV), it would have been informative to examine MMP expression in
gut biopsies and MMP serum level as parallel samples, as the majority of previously
published data on the role of MMPs in IBD concerns findings in the intestinal biopsies.
4.1.2 Pitfalls in biological assay for monitoring the treatment effects
A new cell culture assay was applied in studies I-III. The methodology is based on
activation of PBMCs from healthy donors with PHA in the presence of patient serum. A
panel of T-cell activation markers was measured from the supernatants and the target cells.
The origin of target cells from different donors causes variation in the assay. Despite this,
the serum mediated modulation of cytokine response of different target cells was repeated
relatively well. For a character of a biological assay, the intra-assay variation must also be
taken into consideration. To diminish the bias during the cell culture process, the same
serum sample was cultured in minimum of the duplicate wells and supernatants and cells
derived from one serum sample were collected into pools for analyses. During the ELISA,
samples were analyzed in duplicates and the intra-assay variation limit between duplicated
samples was set at 15%. The comparison of paired serum samples (pre-treatment and posttreatment)
was always performed in the same assay, and thus the results should not be
affected by the interassay variation.
4.1.3 Drug of choice in JIA
In work II, MP is the primary GC injected into the joints of the JIA patients. Of the IACS,
THA is shown to be superior to other GCs and it is recommended as a first line GC to be
used in intra-articular injections (Bloom BJ 2010, Beukelman T 2011). However, at the
time of the sample collection 2004-2005, there were limitations in THA availability in
Europe, affecting the drug of choice in the JIA patients.
4.2 Future prospective
Here a new biological assay based on activation of normal PBMCs in the presence of
patient serum has been described. This assay might be useful in the evaluation of the
systemic effect of GC therapy in patients. Measured cytokine secretion or transcription
factor expression in donor derived PBMCs did not correlate to the dose of systemically
administrated GC. Further, the activation level of donor PBMCs showed some correlations
to clinical disease activity. These findings warrant further studies with larger patient
groups to establish the methodology. This kind of assay could also be tested in other
patient groups with allergic or autoimmune type diseases necessitating the use of GCs.
Regarding the future development of the assay, testing with other types of cells, possibly
from established and commercially available cell lines could be suggested. It is to be noted
that the use and continuation of both GCs and anti-TNF-α agents is based on clinical need
and patient response. Until now, the clinical evaluation and patient-centered parameters
such as PUCAI in pediatric IBD have been the best ways to monitor the treatment effect
(Turner D 2010 b).
As a future investigation line regarding the MMPs, a paired testing with serum and gut
biopsies is recommended. In the gut biopsies’ MMP expression, some disecrepancy
between pediatric and adult IBD has been reported, thus studying of the effect of GC or
anti-TNF-α on adult serum samples would be recommended.
As previously suggested, perhaps no single serum marker will be able to reflect the
wide GC therapy effect (Sidoroff M 2011), thus development of a multivariable analysis
of several of the most promising markers could be a reasonable future prospect in this
Glucocorticoids (GCs) and anti-tumor-necrosis-factor-α (anti-TNF-α) agents are antiinflammatory
medicines used for the treatment of inflammatory bowel diseases (IBD) and
juvenile idiopathic arthritis (JIA). Only part of the IBD patients responds to GC or anti-
TNF-α therapies. These medications can have adverse effects, and these have also been
reported after intra-articular corticosteroid therapy (IACS). Until now, there has been no
clinical test predicting therapeutic response to these medicines. With this aim, a new cell
culture assay was developed. This assay is based on monitoring the patient’s serum
induced responses in normal allogenic target white cells. Further, other experimental
biomarkers such as glucocorticoid bioactivity (GBA), matrix metalloproteinases (MMPs)
and tissue inhibitors of MMPs (TIMPs) were monitored during the GC and anti-TNF-α
In the first study (I), a new biological assay for measuring immune-activation potency in
patient serum was presented. During GC therapy, the treatment effect was seen as a serum
mediated decrease in target cell interferon-gamma secretion and forkhead transcription
factor 3 (FOXP3) and glucocrticoid induced tumor necrosis factor receptor (GITR)
expression. This effect was independent of the GC dose. It was concluded that the
individual net effect of GC therapy could be studied by monitoring the patient serum
induced target cell responses.
In the second study (II), it was found that in JIA patients, IACS led to a rise of serum
GBA and a decrease of cortisol and a reduction in serum induced target cells interleukin-5
secretion. It was concluded that there was a systemic leakage of biologically active GC
In the third study (III), serum immune-activation potency was studied in adult Crohn´s
disease (CD) patients at the start of anti-TNF-α therapy. At baseline, the high FOXP3 and
GITR expression in target cells associated with a low Crohn´s disease endoscopic index of
severity (CDEIS) and erythrocyte sedimentation rate. Interestingly, low pre-treatment
expression of FOXP3 and GITR associated with good treatment response seen as a
decrease of CDEIS during three months. It was concluded that the target cells FOXP3 and
GITR expression showed potency to reflect clinical disease activity in adult CD patients
during anti-TNF-α therapy.
In the final study (IV), the serum levels of MMP-7-9, TIMP-1, α2-macroglobulin (α2M),
human neutrophil elastase (HNE) and myeloperoxidase were found to be higher in
pediatric IBD patients with active disease than in controls. During GC therapy, MMP-7
and TIMP-1 decreased. During anti-TNF-α therapy, α2M and HNE increased. It was
concluded that active IBD reflected serum MMP levels. GC and anti-TNF-α therapy had
different kind of effect to serum MMP profiles that possibly reflect the therapeutic
mechanisms of these medicines.
This study was carried out at the Children`s Hospital, Helsinki University Central Hospital
and at the Immune Response Unit of the National Institute for Health and Welfare during
the years 2006-2012.
My sincere gratitude is due to the management of the Children`s Hospital, Helsinki
University Central Hospital and the National Institute for Health and Welfare for
endorsing and providing research facilities. I thank Docent Jari Petäjä, Head of the
Children`s Hospital, Professor Mikael Knip, Chair of the Children`s Hospital and Docent
Eero Jokinen, the Head of Department of Pediatrics. I thank Professor Markku
Heikinheimo, Head of the Institute of Clinical Medicine and former Head of Pediatric
Graduate School and Docent Jussi Merenmies the Head of Pediatric Graduate School for
their valuable work in gathering, and guiding young investigators.
For this thesis I owe my deepest gratitude to my supervisors Docent Kaija-Leena Kolho,
Children`s Hospital, Helsinki University Central Hospital and Professor Outi Vaarala
National Institute for Health and Welfare, Immune Response Unit. I thank you both for the
excellent facilities to do the research, and the teaching and guidance during the work. Dear
Kaija-Leena, this thesis would not been carried out without your positive and encouraging
attitude. Dear Outi, it was honor to have a glimpse to the work of your professional
Professor Yrjö T Konttinen from University of Helsinki and Docent Katri Kaukinen from
University of Tampere are warmly thanked from thorough review of the thesis and from
inspiring discussions. Docent Pekka Lahdenne and Professor emeritus Erkki Savilahti are
thanked for valuable advises as members of the follow-up group in Pediatric Graduate
I thank all the co-authors: PhD Harri Salo, Docent Taneli Raivio, Professor Olli A Jänne,
Docent Visa Honkanen, MD Katariina Tamm, MD, PhD Marianne Sidoroff, MD, PhD
Taina Sipponen, MD, PhD Laura Mäkitalo, Professor Timo Sorsa and DDS, PhD Taina
I thank Professor Erkki Savilahti, Docent Matti Verkasalo and MD, PhD Paula Klemetti
for providing patients for this study. I also warmly thank Anne Nikkonen and Sari
Honkanen for their excellent work in collecting and managing the patient data, but as well
for the warm atmosphere created in Kaija-Leena’s group. I thank Anneli Suomela and
Harry Lybeck for their work in the PCR-laboratory of the Immune Response Unit. Maria
Kiikeri and Sinikka Tsupari from the Immune Response Unit and Berta Davydova are
thanked for providing valuable advice with the laboratory work. I also want to thank Tarja
Alander from from the Immune Response Unit. I thank Professor Seppo Sarna for his
advices concerning the statistics. Mr Colin Jackson is thanked for language edition.
I want to thank all my collegues and friends. Besides all the practical help and advises you
have provided to me, you are the ones that give the good taste for everyday work. From
Biomedicum 2C 6 th floor I thank Anne, Helena O, Maija, Teija, Anssi, Hanne, Pirjo,
Maarit, Sonja, David, Hanna, Silja, Helena V, Suvi and all the others not mentioned here.
From Outi´s laboratory “Vimo” I thank Tomi, Veera, Janne, Jarno, Terhi, Laura P, Tuija,
Laura O and Kristiina.
I want to thank my sons, Pyry and Otso, you bring joy and laugh into every day. My
darling Matti, you have provided me all the facilities, home, and also many good scientific
advices. I thank my mother Heli and my sister Milja of your unwavering support and
friendship. My father Ilkka is thanked for scientific advises and interesting discussions.
Harry, Christina, Pirjo, Jorma, Hanna, Robin, Leena, Mika and Kari are all thanked for
practical help, taking care of the children, support and joyfull moments during these years.
I´m lucky to have such a family! Of my friends I want to thank Mari, Oona, Tytti, Elina,
Saara and Anna-Sofia. I hope coming years will bring us many shared adventures.
This study was financially supported by the Finnish Pediatric Research Foundation,
Helsinki University Central Hospital Research Foundation (EVO), Helsinki University
Clinical Graduate School in Pediatrics and Obstetrics, the Päivikki and Sakari Sohlberg
Foundation, the Finnish Medical Society Duodecim, and Orion Research Foundation.
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