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Tinjauan Pustaka
Coeliac Disease:
Focus on Multisteps Immunopathogenesis
and Their Correspond Clinical
Investigations
I Gede Yasa Asmara
Faculty of Medicine, University of Mataram
Abstract: Coeliac disease is a chronic inflammatory intestinal disease as a result of gluten
hypersensitivity. It affects 1 in 200 people mainly in Western countries. There are four main
pathogenic steps of coeliac disease which correspond to each specific clinical investigation;
The first begin with the introduction of gluten into the intestinal mucosa which leads to the
development of antigliadin antibodies. Specific gluten peptides undergo deamidation by tissue
transglutaminase in the lamina propria of the small gut. Serologically, anti endomysial and
anti-tissue transglutaminase antibodies can be detected in this phase. Next, local macrophages
which act as professional antigen presenting cells (APCs) processed the deaminated peptides
and present it to the T cells via specific MHC type II. It is therefore molecular technique is able
to detect the present of particular HLA allele of the MHC type II. Finally, small bowel mucosal
and villi destruction occur and can be examined using endoscopy and biopsy. The last method of
investigations remains the gold standard of diagnosis of coeliac disease. An improved understanding of the immunopathogenesis of coeliac disease is likely to lead to the improvement of
diagnostic ability and the development of novel strategies for the treatment of coeliac disease.
Keywords: Coeliac disease, immunopathogenesis, clinical investigations, diagnosis
Maj Kedokt Indon, Volum: 58, Nomor: 12, Desember 2008
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Focus on Multisteps Immunopathogenesis and Their Correspond Clinical Investigations
Coeliac Disease:
Focus on Multisteps Immunopathogenesis and Their Correspond
Clinical Investigations
I Gede Yasa Asmara
Faculty of Medicine, University of Mataram
Abstrak: Penyakit soeliak adalah penyakit inflamasi kronik pada usus halus yang diakibatkan
oleh hipersensitivitas terhadap produk tepung. Penyakit ini mengenai 1 dari 200 orang terutama
di negara barat. Ada 4 tahapan penting dalam patogenesis penyakit seliak yang setiap tahapan
berkorelasi dengan pemeriksaan penunjang tertentu. Pertama, pemeriksaan antibodi anti-gliadin dapat dilakukan pada fase awal ketika mukosa usus halus terpapar oleh produk tepung.
Kedua, antibodi anti-endomisial dan anti transglutaminase jaringan dapat dideteksi dalam darah
pada fase deamidasi protein gluten di lapisan lamina propria usus halus. Ketiga, protein
terdeamidasi akan diproses dan dipresentasikan oleh makrofag jaringan kepada sel T melalui
komplek major histokompatibilitas tipe II. Pada fase ini pemeriksaan molekuler dengan
menggunakan PCR dapat mendeteksi adanya HLA alel tertentu pada pasien. Pada fase terakhir
saat mukosa dan vili usus halus telah mengalami kerusakan akibat respon imun, pemeriksaan
endoskopi dan biopsi dapat dilakukan. Pemeriksaan terakhir masih merupakan baku emas
diagnosis penyakit seliak. Pemahaman yang baik dalam proses imunopatogenesis penyakit ini
sangat penting dalam menentukan pemeriksaan penunjang yang diperlukan dalam diagnosis
dan pengembangan terapi terbaru.
Kata kunci: penyakit seliak, imunopatogenesis, pemeriksaan penunjang, diagnosis
Introduction
In some pathogenesis of diseases, environmental factors, genetics, as well as host response form complex interplay giving rise to the clinical signs and symptoms. Some
immunological disorders such as rheumatoid arthritis, systemic lupus erythematosus, irritable bowel syndrome and
coeliac disease also fit with this concept. Coeliac Disease
(CD) which will be discussed here is chronic intestinal inflammatory disorder.1 It occurs as a result of environmental
exposure (wheat gluten) to genetically susceptible individual.2 It is thought to be organ specific autoimmune disease since autoantibody is produced in its pathogenic process. Since CD was firstly described by Samuel Gee in 1888,3
there has been a lot of research exploring its immuno-pathogenesis, relation to other diseases and therapeutic interventions. However, some molecular mechanisms of CD remain
unresolved.
Epidemiology
Coeliac disease or gluten sensitive enteropathy is quite
common, affecting 1 in 250 people in the world.1 It mainly
occurs in Western countries such as Europe and some part
of North America. For this reason, it is called a disease of
Caucasians.4 The incidence of CD is even higher based on
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serologic screening, where it is found in 1 in 200 populations.2 Other studies revealed that approximately 1% of population suffers from CD.5-7 This condition can be diagnosed
either in early childhood or late during the adulthood, with
peak age of diagnosis at forth or fifth decade of life. Women
are more likely to get CD, accounted for 3:1,6 whereas the
equal proportion may be the case in childhood.7 It seems
that CD is under-diagnosed because of considerable number of silent disease with mild pathogenic mucosa changes.
This has been attributed to physician delay in diagnosis
instead of patient’s delay in seeking health care.6
CD is acquired within the family and a good example of
disease related to HLA alleles.4 CD is likely to occur in 10%
of first degree of relatives. Concordance in identical twins is
around 70%.1,4,6 Several studies reported that HLA-DQ2
(DQA1*0501/DQB1*0201) and in some extent to HLA-DQ8
(DQA1*0301/DQB1 *0302) associated with CD.1-9 Roughly
95% of patients carry both HLA alleles, indicating CD is unlikely to develop in person with absent of these HLA alleles.5
In addition, other genetic factors such as abnormality in chromosome 5, 6, 11 and 19 have been investigated.1,5,8 However,
direct relation is not clearly defined yet.
There are certain people who have high risk for developing CD and serologic testing is strongly recommended.
Maj Kedokt Indon, Volum: 58, Nomor: 12, Desember 2008
Focus on Multisteps Immunopathogenesis and Their Correspond Clinical Investigations
For instance, first degree or may be second degree relatives
of affected individual, patient with type 1 diabetes, Down or
Turner syndrome, selective IgA deficiency and Sjõgren syndrome.3,9,10
Regarding environmental factor, gluten (protein from
wheat, barley and rye) has been known as the major factor
eliciting sequential immune response in small intestine in
CD.3 Moreover, a large epidemiological study in Sweden reported that lack of breast feeding, a large amount of gluten
in the infant formula and >3 infections during childhood
play an important role increasing the risk of CD.6 It seems
that breastfeeding delays the onset and alters the clinical
presentation of CD in children.6,11
Clinical Presentation
CD is classified based on its clinical features into two
categories namely symptomatic/classical/typical and asymptomatic/atypical/silent.6 The presence of diarrhoea with or
without malabsorption is characterised the classical disease,
whereas the silent disease lacks those symptoms.1,9,10
There are slightly different signs and symptoms between children and adult. In childhood, patients could
present diarrhoea, short stature, failure to thrive, anaemia
and sometimes constipation.11 In contrast, weight loss, diarrhoea and anaemia are still the common features in adulthood.10 Some other symptoms that have been reported are
osteoporosis, infertility, a variety of neurological disorders,
dilated cardiomyopathy and myocarditis.3,10,12
CD is also associated with other autoimmune diseases,
immunodeficiency and malignancy. Serum IgA deficiency
has been reported to have at least ten fold increased risk of
CD.3 Similarly, 1.7-2.6% of coeliac patient is also IgA- deficient.1 It becomes important in the interpretation when IgA
based serologic tests are employed to establish the diagnosis. The prevalence of CD in Insulin-Dependent Diabetes
Mellitus (IDDM) has been reported to be 2-7%.3 In addition,
CD increased risk for malignancy or mortality of two fold or
greater, particularly intestinal T-cell lymphoma.3,12
Immunopathogenesis
As mentioned earlier, the pathogenesis of CD is involving environmental factor (gluten from wheat), genetic susceptibility (HLA-DQ2 or -DQ8) and immune response (T cells
activation in the lamina propria).2,13,14 CD is characterised by
hypersensitivity to gluten.3 There are four main steps in the
development of CD, i.e., introduction of gluten to intestinal
mucosa, deamidation of gluten by endogenous tissue
transglutaminase enzyme (tTG), presentation of toxic gluten
peptide by Antigen Presenting Cells (APCs) bearing HLADQ2 or -DQ8 and destruction of intestinal mucosa (villus
atrophy and crypt hyperplasia).
Gluten is the term for the storage protein of wheat, which
made up of gliadin and glutenin. In general, wheat gluten
Maj Kedokt Indon, Volum: 58, Nomor: 12, Desember 2008
protein consists mainly of proline and glutamine residues.2,4,5
Gliadin, an alcohol soluble fraction of gluten is toxic, inducing activation CD4 T cell in intestinal lamina propria.2,15 Why
only gluten elicits this harmful type of immune response is
still unclear. However, some evidence indicated that gluten
might have an additional activity inducing T cell responses
such as increasing HLA-DR expression and Intercellular Cell
Adhesion Molecules (ICAM).2,15 Another reason could be
gluten peptide is absorbed in higher concentration than peptides of other proteins.2 Thus their level in lamina propria is
enough to be recognised by CD4+T cells. In addition, gliadin which has been described as 33mer sequence of α gliadin is resistant to intestinal peptidase such as pepsin and
chymotrypsin. It is because gastric and pancreatic enzymes
lack post-proline cleaving activity.5,16 Hence, it is able to reach
lamina propria and stimulate T cells. At this first pathogenic
step, an evidence of antigliadin antibody could be obtained
and useful for diagnosis CD.
During the first response of innate immunity against
toxic wheat gluten, this peptide should be recognised by
intestinal APCs and presented to CD4 T cells. At this point,
APCs bearing HLA-DQ2 or -DQ8 play an important role in
efficiently present the peptide to CD4 T cells. In order to be
presented efficiently, gluten needs to be deamidated by so
called tTG.1,17,18 Deamidation is modification of glutamine to
glutamic acid leading to a mass increase of 1 Da and negatively charge molecules.2 Deamidation of gluten occurs just
before gluten is endocytosed by APCs. Tissue transglutaminase (tTG) is a Ca2+ dependent enzyme which is ubiquitous just beneath the epithelium in the gut wall as well as
other tissues and organs.2,18 At this second pathogenic step,
antibody to endomysium (tTG as an auto antigen) could be
detected as a very good marker for diagnosing CD.
As for following pathogenic sequence, HLA-DQ2 or DQ8 has prefential to bind peptides with negatively charge
amino acids present in key positions. APCs with HLA-DQ2
or -DQ8 then efficiently present this toxic peptide to CD4 T
cells.8 As a result of the CD4 T cells activation, cytokines
such as interferon gamma (IFNγ) and Tumour Necrosis Alfa
(TNFα) are produced.4,16,19 TNFα activates stromal cells to
produce Keratinocyte Growth Factor (KGF), and KGF causes
epithelial proliferation and crypt cell hyperplasia. Furthermore, TNFα and IFNγ can jointly have a direct cytotoxic
effect on intestinal epithelial cells. In conclusion, those
cytokines are responsible for not only epithelial cell destruction but also crypt cells proliferation,4 which classically
characterise the last pathogenic step of CD (villous atrophy
and crypt cell hyperplasia).9,12
Interestingly, gliadin itself also has ability to induce the
production of IL-15 by APCs. IL-15 is then induce the over
expression of a stress molecule, MICA, on the surface of
enterocytes and up regulate NKG2D receptor on
intraepithelial lymphocytes (IEL).7,20 The interactions between
these two molecules lead to direct enterocytes killing
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Focus on Multisteps Immunopathogenesis and Their Correspond Clinical Investigations
(apoptosis). Another direct effect of gliadin is the increase
of intestinal permeability via release of zonulin and effects
on intracellular tight junction.7,15 The net result is more uptake of gliadin to the lamina propria.
The presence of serum autoantibodies directed against
gliadin, tTG and endomysium is very helpful in establishing
the diagnosis of CD. Moreover, tTG antibody is a specific
marker for active disease.3 Nevertheless, the mechanism of
antibody formation is still a mystery. One possible hypothesis could be complex between gluten peptide and tTG act in
a hapten carrier like fashion.2 The complex is then taken up
by tTG specific B cell and after intracellular degradation,
gluten-derived cells is presented to T cell through HLADQ2 or -DQ8. The activated T cells, in turn, provide help to
the B cell in producing specific antibodies.4,5
Clinical Investigation
In correlation to the four steps of immunopathogenesis
in CD, there are several laboratory examinations which can
be performed corresponding to each step. In the first step in
which wheat gliadin peptide is introduced to intestinal mucosa, antigliadin antibody (AGA) can be detected. Both IgA
and IgG AGA were detected using indirect immunofluorescence.3 Subsequently, the introduction of Enzyme Linked
Immunosorbent Assay (ELISA) made diagnosis of CD more
rapidly and conveniently performed. However, IgA and IgG
AGA have a lower sensitivity and specificity than IgA
Endomysial Antibody (EMA) assay and tTG IgA assay, especially in adult and children over the age of two years.3,7 On
the contrary, in children under the age of two years, IgG
AGA may still useful since serum IgA slowly raise in children toward adulthood. Similarly, in patient with selective
IgA deficiency, IgG AGA may also beneficial.3,9,11
The second step of pathogenesis of CD is deamidation
of gliadin by tTG. In this stage, both IgA EMA and IgA tTG
antibodies play a pivotal role. Since EMA antibody assay
was introduced by Chorzelski et al in 1980s,3 it has been
widely used in part of diagnosing CD. It has very high specificity (99%) and sensitivity (90%).1,3,7 It was performed initially by indirect immunofluorescence with the lower third of
monkey oesophagus as a tissue substrate. However, the
test is labour intensive and qualitative, this requires money,
time and expertise to perform.3,7 Moreover, due to ethics and
cost, human umbilical cord has replaced monkey oesophagus as a substrate. In addition, it has been revealed that the
human umbilical assay demonstrated a better interlaboratory
reproducibility.3 IgA EMA antibody assay can be used in
the monitoring of CD, whereas IgG EMA could not. It because IgG EMA still recover in the serum despite gluten-free
diet.21
The development of ELISA also brings about measurement of tTG which is the autoantigen of EMA. Although
tTG is not necessary a single antigen for EMA, this assay
may still have a comparable sensitivity and specificity to the
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EMA antibody test.3,16 tTG antibody assay is easier, quicker
and quantitative so that it is superior than EMA antibody
test.7 Some previous studies used guinea pig liver tTG (gpltTG) as substrate, whereas now it has been replaced by purified erythrocyte and recombinant human tTG. Furthermore,
human tTG has a higher specificity than gpl-tTG even where
purified gpl-tTG was used. 3
Serotype based association study revealed that the association of HLA-DQ2 and -DQ8 in CD. A European study
reported than 90% coeliac patient possessed HLA-DQ2 and
the rest possessed HLA-DQ8.7 This has lead to the development of HLA-DQ typing, which is performed using PCR with
sequence specific primer. Some studies mentioned the involvement of non HLA-DQ alleles such as HLA-DR3, -DR5
and -DR7 in the pathogenesis of coeliac disease.4,8 However,
the evidence is not conclusive. Since 40% of general population has HLA-DQ2 or -DQ8, HLA-DQ typing appears to be
a necessary but not sufficient, factor in CD diagnosis.6,9
Therefore, in the case where the diagnosis of coeliac disease
is unclear, screening for patient’s family member and excluding coeliac disease when the patient are already on a gluten
free diet, HLA-DQ typing may be essential.9
The last step of CD pathogenesis is the destruction of
intestinal mucosa as a result of chronic inflammatory response. In this stage, biopsy remains the gold standard in
the diagnosis of CD. Since the nature of villous atrophy is
patchy, multiple biopsies are necessary which focus on the
descending duodenum rather than the more distal intestine.9,12
The spectrum of histologic changes in CD is quite wide
from mild to severe villous atrophy. Marsh9 classified histopathology change in coeliac disease into three stages
namely:9 Marsh I is characterised by normal villous architecture with IEL (>30 lymphocytes per 100 enterocytes); Marsh
II is recognised as both IEL and crypt hypertrophy; Marsh
III includes the evidence of moderate to severe reduction in
villous height.
Diagnosis
Diagnosis of CD has inproved as the development of
serologic testing as well as endoscopic and biopsy procedures. In patients who display classical disease, high index
of clinical suspicion is immediately established. In contrast,
screening with serologic testing is required to find out silent
cases. The use of IgA and IgG EMA and tTG are recommended in screening CD. However, duodenal biopsy should
be subsequently performed in serologic positive patients.21
In few cases, coeliac patients may be derived from incidental
endoscopic procedures which are performed because of other
diseases. The diagnosis of coeliac disease depend on demonstration of the typical biopsy changes of untreated coeliac disease in the small intestinal mucosa and improvement
with dietary gluten restriction.9,10
In a rare case where a strong suspicion of the condition
exists but in which all test are negative, a rectal gluten chalMaj Kedokt Indon, Volum: 58, Nomor: 12, Desember 2008
Focus on Multisteps Immunopathogenesis and Their Correspond Clinical Investigations
lenge may yield a positive result.9 It is based on the fact that
the mucosal immune systems involve the rectal as well as
the small bowel mucosa. Additionally, a rectal gluten challenge is an easy office procedure and a direct provocation
test of gluten sensitivity while other tests are indirect.9
Treatment
The current treatment for CD is a gluten free diet for a
long life.12,19,22 In this diet, any food product containing
wheat, barley and rye must be avoided. It is unclear whether
oat is toxic to the patients. In severe cases, patient do require admission, replacement of fluids and electrolytes and
occasionally, steroid.12,19
Tabel 1. Main Pathogenic Steps of Coeliac Disease and Their
Correspond Test
Main pathogenic steps
Clinical investigations
Ingestions of gluten-containing food
Deamidation of gluten peptides by
tissue transglutaminase
Anti gliadin antibodies
Anti-endomysial and antitissue transglutaminase anti
bodies
HLA-DQ typing
Presentation of modified peptides by
HLA class II
Induction of autoimmune mucosal
injury
Small bowel biopsy
Education of the patient and his/her supportive household is inevitably important. Patient should know the fluors
and grain that are naturally gluten free such as rice, corn,
potato, chestnut, fluor, millet as well as buckwheat.10 Since
fluor from wheat is usually fortified with iron, thiamine, riboflavin and niacin. It is recommended that gluten free diet is
supplemented by those vitamins and trace elements.6,10
There are several novel treatments of CD being developed based on its pathophysiologic mechanism. Since gliadin which is the 33mer is toxic, enzyme supplementation of
bacterial prophyl endopeptidase is developed to degrade
the T cells stimulatory peptide.7 Alternatively, producing
wheat with absent or reduced immunogenicity by selective
breeding or genetic modification is also on research.7,22 Another strategy is also targeting tTG, zonulin and HLA peptide
interaction.7,19 However, none of them is in clinical use yet.
Summary
Coeliac disease is a good example of autoimmune disorder where environmental and genetic factors play an equal
role in its pathogenesis. There has been a lot of evidence
revealing the multisteps of host immune response against
wheat gluten resulting in the disease. We all now know that
each step can be demonstated by spesific clinical investigations leading to the diagnosis of coeliac disease. Moreover,
a good understanding of the immunopathogenesis of coeliac disease might help the clinician in deciding the correct
intervention. However, there is still a major challenge ahead
in transfering this knowledge into practical benefit for the
patients.
Reference
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Future 1. Sequence Evens in the Immunopathogenesis of Coeliac disease17 1. Gluten is digested to yield peptides, which
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5. Lamina pro pria lymphocytes proliferate and recruit
cellular infiltrate; 6. CD8 T cells with cytotoxic markers
increase in mucosa; 7. Fibroblasts are activated and produce
metalloproteinases to degrade matrix; 8. Plasma cells pro
duce disease-specific coeliac antibodies; 9. The role of primitive intra-epithelial lymphocytes remains unclear.
Maj Kedokt Indon, Volum: 58, Nomor: 12, Desember 2008
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