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Studies of experimental biomarkers during ... - Helda - Helsinki.fi

Pediatric Graduate School

Children’s hospital

Helsinki University Central Hospital

University of Helsinki

Helsinki, Finland

Studies of experimental biomarkers during

glucocorticoid and

anti-tumor necrosis factor-alpha therapy in

inflammatory bowel disease and

juvenile idiopathic arthritis

Hanne Rintamäki

ACADEMIC DISSERTATION

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

and Adolescents

on 20 th April 2012, at 12 noon.

Helsinki 2012


Supervised by Docent Kaija-Leena Kolho

Children`s Hospital

Helsinki University Central Hospital

Helsinki, Finland

and

Professor Outi Vaarala

Immune Response Unit

National Institute for Health and Welfare

Helsinki, Finland

Reviewed by Professor Yrjö T. Konttinen

University of Helsinki

Helsinki, Finland

and

Docent Katri Kaukinen

University of Tampere

Tampere, Finland

Official opponent Professor Eeva Moilanen

University of Tampere

Tampere, Finland

ISBN 978-952-10-7917-7 (pbk.)

ISBN 978-952-10-7918-4 (PDF)

http://ethesis.helsinki.fi

Unigrafia Oy University Printing House

Helsinki 2012


To Matti, Pyry and Otso

To Kaija-Leena


Abstract

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

agents.

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.


Contents

Abstract 4

List of original publications 10

Abbreviations 11

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

potency 47

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

Conclusions 68

Acknowledgements 69

References 71


List of original publications

This thesis is based on the following publications that are referred to in the text by their

roman numerals:

I Rintamäki H, Salo HM, Vaarala O, Kolho KL. New means to monitor the

effect of glucocorticoid therapy in children. World J Gastroenterol

2010;16:1104-9.

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.

10


Abbreviations

α2M α2-macroglobulin

Ab antibody

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

DEX dexamethasone

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

GC glucocorticoid

GI gastrointestinal

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

HPA hypothalamic-pituitary-adrenal

HsCRP high sensitive C-reactive protein

IACS intra-articular corticosteroids

IBD inflammatory bowel disease

11


IC indeterminate colitis

IFNγ interferon gamma

IFX infliximab

Ig immunoglobulin

IKKβ inhibitor of nuclear factor kappa-beta kinase

IL interleukin

IPEX immune dysregulation, polyendocrinopathy autoimmune enteropathy Xlinked

syndrome

JIA juvenile idiopathic arthritis

MHC major histocompatibility complex

MMP matrix metalloproteinase

MP methyprednisolone

MPO myeloperoxidase

MR mineralocorticoid receptor

(m)RNA (messenger) ribonucleic acid

MT metallothionein

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

PHA phytohemagglutin

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

Th T-helper

THA triamcinolone hexacetonide

TIMP tissue inhibitor of matrix metalloproteinases

TLR toll-like receptor

TNF tumor necrosis factor

Treg regulatory T-cell

UC ulcerative colitis

12


Introduction

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

and JIA.

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

in JIA.

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

13


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.

14


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

15


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 -

1.2 Diagnosis

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).

16


Table 2 Laboratory tests for suspected inflammatory bowel disease. Modified from Strople

J 2008.

Test Possible finding

Blood count Anemia, thrombocytosis, leukocytosis

Liver function test Elevated transaminases, alkaline phosphatase or gamma-glutamyl

transferase

Serum albumin Hypoalbuminea

ESR Elevation

CRP Elevation

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

1.3 Etiology

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).

17


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

T 2011).

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

daily)

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).

18


2.2 Etiology

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

1.Systemic

arthritis

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

3.Seronegative

polyarthritis

4.Seropositive

polyarthritis

5.Psoriatic

arthritis

6.Enthesitis

related

arthritis

7.Undifferentiated

arthritis

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

19

a-d

a-e

a-c, e

b-e

a, d, e

no


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).

3.2 T-cells

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

20


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

21


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).

3.3.1 FOXP3

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).

3.3.2 GITR

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

22


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).

23


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.

24


Th2

Treg

IL-4

IL-10

Th17

IL-22

TGFβ

IL-17

IBD

IL-23

IFNγ

25

IL-13?

TNF-α

Th1

IL-27?

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

1999).

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

2006).

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.

3.5.2 Autoantibodies

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

26


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

released enzymes

4.1 Basic concepts of MMPs and TIMPs

4.1.1 MMPs

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

2006).

27


4.1.2 TIMPs

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.

4.2.2 α2M

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

K 1999).

28


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 -

1994

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 -

1998

and CD

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

2000

samples

histological score

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,

mucosa.

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

2003

samples

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

(142)

developement of fistulae

Pedersen G Adult IBD (19) 4,6 MMP-1, MMP-3, MMP-7, MMP-9-10 expression was increased in -

2008

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

2009

immunosuppressive therapies

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 -

2010 (33)

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

29


4.2.3 MPO

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).

4.2.4 HNE

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).

30


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

sulphasalazine

Non-steroidal anti-

inflammatory drugs and coxibs

IBD. Prevention of colorectal carcinoma.

32

polyarthritis

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

Other immunosupressants/

DMARDs

Local Rectal Intra-articular THA for treatment of active joints

Azathioprine and 6-

mercaptopurine

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

adult CD

Etanercept Not indicated

Rituximab

Anakinra

Antibiotics Metronidazole,

Ciproflixacin

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

systemic glucocorticoids.

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.

Study Patients

Tung J

2006

Hyams J

2006 1

Markowitz

J 2006 1

Krupoves

A 2011 2

Jakobsen

C 2011 3

(n)

Pediatric

CD

(26)/UC

(14)

Pediatric

UC (62)

Pediatric

CD

(109)

Pediatric

CD

(364)

Pediatric

CD

(87)/UC

(77)

Short-term response % Long-term response %

Remission Partial

response

Treatment

failure or

operated

Remission Steroid

dependency

Treatment

failure or

operated

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

33


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

Papunet.

34


Table 9 Examples of the studies monitoring glucocorticoid (GC) response in pediatric and adult inflammatory bowel disease (IBD)

Research Patients

Hearing SD

1999

(n)

Adult UC

(18)

Flood L 2001 Adult UC

Löwenberg

M 2006

Vihinen MK

2008

Nakahara S

2008

Vihinen MK

2009

Turner D

2010 a

(21)

Adult UC

(9)

Pediatric

IBD (22)

Adult IBD

(188)

Pediatric

IBD (19)

Pediatric

UC (50)

Studied markers Methods Association to glucocorticoid therapeutic response

Inhibition of thymidine uptake T-lymphocyte stimulation test;

GR mRNA and MTIIa mRNA,

cortisol

suppression of thymidine uptake due to

DEX was measured

Low-dose ACTH test, mRNA quantitation

with hybridization assay

35

T-cells from steroid responders were suppressed more by

DEX than T-cells from partial responders or treatment

failures

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

steroid responsiveness

response

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

response

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).

36


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

37


Table 10 Anti-inflammatory effects of glucocorticoid therapy (Ahluwalia A 1998, Janeway

CA 2005 b, Franchimont D 2004).

Glucocorticoid therapy

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

arachidonic acic

cyclooxygenase activity

Down-regulation of adhesion molecules Reduced leukocyte extravasation

Up-regulation of endonucleases Induction of leukocyte and eosinophil

apoptosis

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

J 2002).

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

38


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

39


(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

2010).

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

40


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.

41


Table 11 Studies monitoring anti-TNF-α therapy response in adult inflammatory bowel disease

Research Diagnosis

Nikolaus S

2000

(n)

CD (24) Whole blood

Tissue Studied markers Methods Predictive value to anti-TNF-α therapy

cultivation

supernatants, gut

TNF-α, NFκB Immunoassay, Western blot Increase of NFκB in biopsies or TNF-α secretion in supernatants

42

preceeded relapse

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,

Mäkitalo L

2009

CD (12 with

infliximab)

Li Z 2010 CD (23), UC

Boschetti G

2011

Veltkamp C

2011

Lönnkvist MH

2011

(17)

Gut MMP-1, MMP-7, MMP-9-10,

MMP-26, TIMP-1, TIMP-3

Blood, gut Treg cells, CD25 mRNA, CD4

mRNA, FOXP3 (mRNA,

protein)

PCR

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,

immunohistochemistry

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

(24)

Blood, serum, gut Apoptotic Tregs, caspase-3 and

CD (22) Serum, plasma Calprotectin, total nitrite,

-7

suPAR, ghrelin, endothelin

Immunohistochemistry, flow

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

index


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.

43


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

IBD children

on GC therapy

IBD children on

anti-TNF-α

therapy

IBD children

in remission

Adult CD

patients with anti-

TNF-α therapy

JIA patients

with IACS

subjects

19+19

(10 patients

shared in I and

IV)

Age

(median,

range)

Sex

(f/m)

13 (4-18) 6/13 in

both

studies

44

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,

IC=indeterminated colitis

II

IV


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

45


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

46


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

potency

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

donors PBMCs.

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

47


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

48


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

peripheral blood

Ficoll gradient

centrifucation

T-cell stimulation assay T-cell stimulation assay

Detection of IFNγ and IL-5 from

supernatants

(described detailed in

text)

Detection of IL-17 from supernatants Commercially available

mRNA of IFNγ, IL-5, IL-10,

FOXP3, GITR, GATA3, STAT-5b

and t-bet

50

Author I-III

Author I-III

In-house ELISA Author I-III

ELISA

Author 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

and α2M

Commercially available

ELISA

Cortisol Commercially available

ELISA

Fecal calprotectin Commercially available

ELISA

laboratory

PhD, DDS Taina

Tervahartiala at professor

Timo Sorsa`s laboratory

Clinical laboratory II

I, III

II

IV

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.

51


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

(I,II)

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).

52


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

Children with

IBD

on oral GC

(n=19)

Children with JIA

receiving IACS

(n=21)

(ρg/ml; median,

range)

53

After therapy

(ρg/ml; median,

range)

Increase/

Decrease/

no

change

p-value

IFNγ 0 (0-308) 0 (0-29) 1/7/11 p=0.017

IFNγ

PHA

91000

(7200-

122000)

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

IFNγ

PHA

IL-5 PHA

49000 (2900-

16900)

IFN=interferon, IL=interleukin, ns= p>0.05

52000

(122-152000)

6/10/0 ns

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).

54


1.3 The effect of GC treated patient serum on target cell FOXP3 and GITR

expression (I)

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

a.)

200

150

100

50

0

p=0.033 p=0.050

GITR FOXP3

Relative units of mRNA (PHA)

4000

3000

2000

1000

55

b.)

0

p=0.005

p=ns

GITR FOXP3

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

56

r=-0.479

Number of joints with limited range of motion ns p=0.049

r=-0.471

p=0.025

r=-0.557

p=0.001

r=-0.743

Number of swollen joints ns ns p=0.005

CHAQ p=0.030

r=-0.541

Patient’s global assessment p=0.014

r=-0.582

r=-0.660

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

p=0.018

r=-0.552

p=0.001

r=-0.718

p=0.022

r=-0.536

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

patients (III)

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

therapy.

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

57


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)

a.)

1500

1000

500

p=0.001

p=ns

p�0.001

0

0 7-hours 24-hours2 months

Sampling

Serum cortisol (nM)

58

b.)

600

400

200

0

p=0.002

p=ns

p=0.003

0 7-hours 24-hours

Sampling

2 months

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

60


2008), thus one clinical application of GBA measurement could be a monitoring of the

medication adherence.

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

therapy (p=0.036).

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

61


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

2005).

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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).

64


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

65


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.

66


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

area.

67


Conclusions

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

therapies.

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

after IACS.

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.

68


Acknowledgements

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

laboratory.

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

School.

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

Tervahartiala.

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.

69


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.

70


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