1. sCD163 AS POTENTIAL BIOMARKER FOR
COELIAC DISEASE
Jazzy Marie Garania
April 2015
School of Biological Sciences
Dublin Institute of Technology
Kevin Street,
Dublin 8
This project was submitted in part fulfilment of the BSc Biosciences,
Dublin Institute of Technology.

2. i
Acknowledgements
I wish to express my outmost gratitude to my supervisor, Dr. Greg Byrne, Lecturer in
Clinical Immunology, School of Biological Sciences in Dublin Institute of Technology,
for providing me with all the necessary information and tools for this research. I am
sincerely grateful to him for sharing his expertise and valuable guidance extended to me.
I would like to acknowledge the School of Biological Sciences and the Institute for
providing the necessary tools and equipment needed to complete this task.
I also take this opportunity to express my gratitude to all of the people who have
supported me, my family, friends, and colleagues, who have helped me and encouraged
me throughout this venture.

3. ii
List of Abbreviations
Abs Antibody
AU Arbitrary units
AGA Anti-gliadin antibodies
APC Antigen Presenting Cells
CD Coeliac disease
CO Carbon monoxide
CV Coefficient Variations
DC Dendritic cells
DH Dermatitis Herpetiformis
ELISA Enzyme linked Immunosorbent Assay
EMA Anti-endomysial Antibodies
FasL Fas ligand
GFD Gluten Free diet
GIT Gastrointestinal tract
GM-CSF Granulocyte macrophage colony stimulating factor
Hb Haemoglobin
Hb-Hp Haemoglobin-Haptoglobin complex
HbSR Haemoglobin scavenger receptor
HIV Human immunodeficiency virus
HLA Human Leukocyte Antigen
HO Heme-oxygenase
IEL Intraepithelial lymphocyte
IFN Interferon

4. iii
IgA Immunoglobulin A
IL Interleukin
LPMC Lamina propria mononuclear cells
LPS Lipopolysaccharide
mAbs Monoclonal antibodies
MCSF Macrophage colony stimulating factor
MHC Major histocompatibility complex
MMPs Matrix metalloproteinases
mtTg Membrane-bound tissue transglutaminase
PST Proline-serine-threonine
NK Natural Killer cells
NO Nitric oxide
sCD163 Soluble CD163
sIGA Secreted Immunoglobulin A
SRCR Scavenger receptor cysteine-rich
TGF Transforming growth factor
Th T-helper
TLR Toll-like receptor
TNF Tumour necrosis factor
TNFR Tumour necrosis factor receptor
tTG Tissue Transglutaminase

5. iv
Table of Contents
Acknowledgements ..........................................................................................................................................i
List of Abbreviations...................................................................................................................................... ii
Table of Contents ...........................................................................................................................................iv
List of Figures .................................................................................................................................................1
List of Tables...................................................................................................................................................2
Abstract ...........................................................................................................................................................3
1 Introduction ............................................................................................................................................4
1.1 Coeliac disease ..............................................................................................................................4
1.1.2 Prevalence of Coeliac disease...........................................................................................4
1.1.3 Genetics ............................................................................................................................6
1.1.4 Symptoms .........................................................................................................................7
1.1.5 Diagnosis and Treatment and Coeliac disease..................................................................9
1.1.6 Immunopathogenesis of Coeliac disease ........................................................................12
1.1.6.1 Transport of gluten .....................................................................................................13
1.1.6.2 Modification and presentation of gluten.....................................................................14
1.1.6.3 Disease progression involving Innate and adaptive immune system..........................14
1.1.7 Coeliac Disease and future developments ......................................................................16
1.2 CD163..........................................................................................................................................16
1.2.1 Macrophages .......................................................................................................................16
1.2.2 CD163.................................................................................................................................17
1.2.2.1 Structure..........................................................................................................................18
1.2.2.2 Expression and Regulation .............................................................................................20
1.2.2.3 Functions ........................................................................................................................22
1.2.2.3.1 Homeostatic function: Haemoglobin clearance by CD163 ......................................22
1.2.2.3.2 Regulation of erythropoiesis by CD163...................................................................24
1.2.2.3.3 Tweek scavenging function of CD163.....................................................................25
1.2.2.3.4 CD163 as bacterial receptor.....................................................................................26
1.2.2.3.5 CD163 as a molecule with an Immunoregulatory function......................................26
1.3 Soluble CD163 (sCD163)............................................................................................................27
2 Methodology.........................................................................................................................................29
2.1 ELISA – Sandwich ......................................................................................................................29
2.2 Statistics.......................................................................................................................................31
3 Results ..................................................................................................................................................32
4 Discussion.............................................................................................................................................36
5 Concluding remarks..............................................................................................................................39
Bibliography..................................................................................................................................................40

6. 1
List of Figures
Figure 1 Global prevalence of CD.................................................................................................................... 5
Figure 2 Coeliac iceberg concept ..................................................................................................................... 6
Figure 3 Normal and healthy mucosa and abnormal mucosa caused by coeliac disease ................................ 9
Figure 4 Indirect Immunofluorescence of EMA ............................................................................................ 10
Figure 5 Marsh-Oberhuber Classification system. ......................................................................................... 11
Figure 6 Factors involving in the development of coeliac disease. A ............................................................ 13
Figure 7 SIgA-gliadin peptide complex CD71-mediated retrotranscytosis into lamina propria .................... 14
Figure 8 Mechanism of mucosa damage in coeliac disease ........................................................................... 15
Figure 9. Schematic representation of the CD163 gene. I. ............................................................................. 18
Figure 10. Representation of CD163 SRCR................................................................................................... 19
Figure 11. Representation of free haemoglobin elimination by CD163 positive macrophage.. ..................... 24
Figure 12. Soluble CD163 ELISA assay. ....................................................................................................... 31
Figure 13 Serum levels of soluble CD163 in healthy controls, Coeliac disease patients, and Crohn's disease
patients ........................................................................................................................................................... 33
Figure 14. Percent coefficient variation of Normal control, Coeliac disease, and Crohn's disease samples. . 35
Figure 15. Serum levels of soluble CD163, according to Marsh score of mucosal lesion ............................. 34

7. 2
List of Tables
Table 1 Symptoms manifested at different age groups. ....................................................................................7
Table 2 Gluten-Free Diet Guidelines. .............................................................................................................12
Table 3. Distribution of CD163 positive macrophages in most common studied tissues in human................20
Table 4. CD163 expression regulating factors. ...............................................................................................22

8. 3
Abstract
Coeliac disease is an autoimmune disorder of the small intestine that occurs in genetically
predisposed individuals, it is a common genetic condition in Europe. In Ireland, it is said
that 1 in 122 people have coeliac disease. It is triggered by gluten found in wheat and
grains such as barley and rye. It affects the small intestine and can also manifest itself in a
range of clinical manifestations such as anaemia, diarrhoea, and dermatitis herpetiformis.
Diagnosis of coeliac disease involves blood tests for Immunoglobulin A anti-tissue
transglutaminase and anti-endomysial antibodies, and a biopsy of the small intestine.
Treatment for this condition is to start and adhere to a strict gluten-free diet for life. It is
of great interest to investigate and develop a new method of diagnosing coeliac disease
with the use of a specific protein biomarker expressed during the disease. The suggested
biomarker being investigated in this study is CD163. CD163 is a transmembrane protein
belonging to group B of the scavenger receptor cysteine-rich superfamily. It is expressed
on monocyte/macrophages which are cells that possess strong anti-inflammatory
potential. CD163 expressed on cells of monocytic lineage and also its soluble form has a
role in down regulation of inflammatory response, it is also involved in homeostasis. Due
to this function CD163 can be a target for therapeutic control of inflammatory response.
In coeliac disease, CD163 is highly expressed and due to this elevated CD163 expression
it is said that this protein has the potential to be an inflammatory marker for the disease.
If a test that measures this inflammatory marker is investigated and developed this would
be very valuable.

9. 4
1 Introduction
1.1 Coeliac disease
Coeliac disease (CD) is a gluten-sensitive enteropathy; it is an autoimmune condition due
to gluten sensitivity characterized by inflammatory damage to the small intestine caused
by exposure to gluten in a susceptible individual. It is an immune system mediated
hypersensitivity response to gluten and other foods based on barley and rye. Oats,
according to (Cooper et al., 2012) , does not activate the disease however other studies
suggest that CD activation by ingestion of oats occurred if consumed in ample amounts
and over a long period of time.
CD develops in genetically susceptible individuals where they can present
gastrointestinal symptoms, extra intestinal symptoms or be asymptomatic (Gujral et al.,
2012); untreated patients that are asymptomatic have an increased risk of disease
intensification, gastrointestinal or haematological cancers, and can present secondary
autoimmune disorders at a later stage. CD appears in early childhood with severe
symptoms such as diarrhoea, bloating and failure to thrive however, not all patients
develop symptoms until they reach adulthood. CD is also associated with many other
autoimmune conditions as it is a common feature of autoimmunity to have many
conditions that overlap. Due to CD being complex hardship is not rare in dealing with
this condition as it affects quality of life as strict gluten free diet (GFD) is the only
current effective treatment (Gujral et al., 2012).
1.1.2 Prevalence of Coeliac disease
It was previously thought that CD affects Westerners and Europeans however, now CD is
widely distributed globally (Figure 1). According to Gujral et al., CD distribution
followed wheat consumption and migratory flows where tribes originating from the
Middle East where wheat and barley grains grew naturally have migrated towards the
west and spread through the Mediterranean and Central Europe which means that
populations from these areas share the same genetic backgrounds and may have created
populations that are inclined to develop CD.

10. 5
In Europe and USA, CD is one of the most common disorders where the condition affects
all age groups with an estimated prevalence of 1 in 200 (Schuppan et al., 2003). 0.6 to
1% is affected by CD worldwide where in European areas there are wide differences
between regions that ranges from 1:88 to 1:262. CD clearly affects genetically
susceptible individuals, up to 20% of the first degree relatives of CD patients are affected
by the disease (Leon et al., 2005).
Figure 1 Global prevalence of CD. Adapted from Gujral et al. Map shows prevalence however, in Asia data is not
available hence N/A
Globally, incidence and prevalence of CD have increased steadily which are due to
awareness, a shift to more Western diet where gluten is one of the main components of
food stuffs, and also better and improving diagnostic and screening tests.
The Coeliac Iceberg is a concept where many people have CD and remain undetected
while only a few present severe symptoms which are diagnosed.

11. 6
Figure 2 Coeliac iceberg concept. Adapted from Hall & Yates, 2010. Bottom of iceberg represent the high population
of CD patients that has the latent or asymptomatic form of CD and as CD becomes more symptomatic populations that
have this form of CD decreases hence forming an iceberg. Figure also shows what can be seen and detected in different
stages of CD.
1.1.3 Genetics
Genetic factors and background do not predict development of the disease but provide
susceptibility of an individual to CD. Genetic association relies on the variants in the
Human Leukocyte Antigen (HLA) region and non-HLA variants (Heap & van Heel,
2009). HLA is the human version of the major histocompatibility complex (MHC)
responsible for identifying foreign epitopes or antigens and produce an immune response
to protect the body.
HLA-DQ genes are strongly associated with CD; the haplotypes HLA-DQ2 and HLA-
DQ8 are necessary predisposing variables that contribute to a risk of 40% in development
of the disease (Guandalini & Assiri, 2014). Among CD patients 5% express HLA-DQ8
and 95% express HLA-DQ2 where different isoforms exist of which are DQ2.2 and
DQ2.5 this makes the association of HLA-DQ2 to CD complex. In Gujral et al., DQ2.5
binds and presents deamidated gliadin while DQ2.2 combined with DQ7.5 show a protein
identical of which DQ2.5 expresses. Many genes predispose an individual to CD,
COELIAC1 locus on chromosome 6, COELIAC2 on chromosome 5, COELIAC3 on
chromosome 2 and COELIAC4 on chromosome 19. CD patients carry a HLA variant
where most have DQ2 and others carry DQ8 located in COELIAC1 locus. HLA genes
and CD have a strong association compared to other diseases where the genetic attribute

12. 7
of HLA to CD is 53% (Leon et al., 2005) which suggest that HLA is only part of the
cause and need other factors to develop CD.
HLA genes that code for HLA-DQ2 and HLA-DQ8 MHC Class II molecules are
expressed on the surface antigen presenting cells (APC) such as dendritic cells and
macrophages in the lamina propria of the gut. These APCs then present bound antigens
which in CD is gliadin peptides to CD4+ T cells which then induces an immune
response. This immune response gives rise to various clinical manifestations of CD
which can make diagnosis of the disease difficult.
1.1.4 Symptoms
Coeliac Disease can present itself in many ways and forms and is associated with many
autoimmune conditions. It is well known that manifestations of coeliac disease vary
which makes recognition and diagnosis difficult. Different age groups also present
different symptoms which contribute to diagnostic difficulties (Table 1).
Table 1 Symptoms manifested at different age groups. Adapted from Food Safety Authority of Ireland
Age Group
Infants and young children
under 2 years
Childhood 2 – 16 years Adults
Symptoms
Diarrhoea/steatorrhoea Poor growth (small for age) Short stature
Vomiting Delayed puberty Anaemia (esp. in pregnancy)
Anaemia Anaemia Osteoporosis
Cranky Osteoa/rickets Dyspepsia
Bloated belly Diarrhoea/Steatorrhoea Weight loss
Wasted buttocks Lethargy Mouth ulcers
Mouth ulcers Diarrhoea/steatorrhoea
Infertility
Dermatitis herpetiformis
Tetany

13. 8
The range of clinical presentation of CD is wide (Di Sabatino and Corazza, 2009),
categorised into silent, minor, and major CD. Individuals who do not complain of any
symptoms are said to have silent CD, these are relatives of CD patients or of the general
population that are positive for anti-endomysial antibodies (EMA). Minor CD patients
have transient or trivial symptoms few of which are dyspepsia, bloating, fatigue, anaemia
and more, these patients are positive for EMA. Major CD patients have severe symptoms
and complain about malabsorption, steatorrhoea, and features of malnutrition, tetany, and
more. Biopsy is performed based on symptoms.
Many other conditions are linked with CD. Autoimmune and immune-related diseases
such as type 1diabetes, thyroiditis, myocarditis, IgA deficiency, inflammatory bowel
disease, juvenile idiopathic arthritis are reported in conjunction with CD. Neurological
and psychiatric disorders are also reported with CD such as ataxia, dementia, depression,
and peripheral neuropathy. In addition, male patients encounter problems in fertility and
the loss of libido and in females recurrent and unplanned miscarriages, a delay in
menarche, early menopause and amenorrhoea (Di Sabatino and Corazza (2009).
Dermatitis herpetiformis (DH) is present in CD patients where itchy and blisters occur on
the body. In DH, sub-epidermal transglutaminase react with IgA antibodies that result in
inflammation (Thom et al., 2009).
CD is characterised by chronic inflammation in the small intestine causing atrophy of the
villi and nutrient malabsorption (Thom et al., 2009). Inflammatory immune response
cause loss of nutrients and degradation and digestive enzymes, and lose of surface area
needed for effective absorption (Figure 3).

14. 9
Figure 3 Normal and healthy mucosa (left) and abnormal mucosa caused by coeliac disease (right). Villi
flattening which cause malabsorption. Adapted from Thom et al., 2009 .
1.1.5 Diagnosis and Treatment and Coeliac disease
Presentation of CD is complex which can lead to an incorrect diagnosis, differential
diagnosis is used to rule out the possibility of manifested symptoms coming from a
different cause. Differential diagnoses for CD include: diarrhoea caused by infection and
drugs, irritable bowel syndrome, and Crohn’s disease. It is important to detect CD as it
starts in childhood and can commence at any age.
In 1970s the criteria for diagnosis involved three biopsies of flat mucosa, restored mucosa
on GFD, and mucosa after gluten challenge in children. This criteria is now revised to
which symptoms are analyzed with serological tests, and a small bowel biopsy followed
by a follow-up testing to where favourable clinical and serological response to GFD is
acceptable to confirm CD diagnosis (Gujral et al., 2012).
Serological tests are used to determine the presence of two major autoantibodies which
are anti-tissue transglutaminase (tTG) Immunoglobulin A (IgA) and EMA (Guandalini
and Assiri, 2014). Other screening antibodies that can be measured are anti-reticulin and
anti-gliadin (AGA) (Leon et al., 2005).

15. 10
For initial testing serum IgA anti-tTG antibodies is measured; this test is 94% sensitive
and 97% in specificity. Anti-tTG is detected by monospecific test such as Enzyme linked
Immunoabsorbent Assay (ELISA). If patient is IgA deficient then anti-tTG IgG and anti-
gliadin IgG antibodies is measured as substitute (Briani et al., 2008).
EMA testing is more specific as specificity is close to 100% and has over 90%
sensitivity; recommended use is on patients with uncertain diagnosis. Before testing
ingestion of gluten proteins must be done to ensure response caused by antibodies is
present (Thom et al., 2009). EMA are determined using indirect immunofluorescence
(Figure 4) however, this requires well-versed staff as evaluation can be difficult, when
positive for serological tests the patient is subjected to a biopsy.
Figure 4 Indirect Immunofluorescence of EMA. EMA shown as green fluorescence on image. Adapted from Gosink,
2012
Histology of the small intestinal mucosa is always performed as a confirmatory test.
Biopsy is performed when serological testing is positive to confirm diagnosis. CD is
characterised by abnormal architecture of mucosa: high number of intraepithelial
lymphocyte (IEL), crypt elongation, and villous atrophy (Figure 3) (Fasano and Catassi,
2012).

16. 11
In Gujral et al., biopsy samples are characterised by Marsh-Oberhuber classification:
Type 1 as infiltrative lesions by normal mucosal architecture with high numbers of IEL,
Type 2 as hyperplastic lesions with increase depth of crypts and no villous atrophy, Type
3a, 3b, and 3c with destructive lesions with mild, marked and complete villous atrophy,
and Type 4 with hypoplastic lesions with normal crypt height and IEL count (Figure 4). It
should be mentioned that a biopsy may result in false results due to inter-observer
variability, low-grade histopathological abnormalities and limitations in technology.
Figure 5 Marsh-Oberhuber Classification system. Adapted from Franco (2010)
Treatment for coeliac disease is a strict lifetime gluten free diet (GFD). Patients are
educated in regards to diet and lifestyle and are given guidelines of gluten-free foods
(Table 2) (Thom et al., 2009). Follow-up serology using AGA is performed after one
year and a follow-up biopsy after two to five years on strict adherence to GFD. Strict
adherence to GFD results in tissue healing and diminished symptoms.

17. 12
Table 2 Gluten-Free Diet Guidelines. Adapted from Thom et al., 2009
Avoid (in any form)
All wheat forms (germ, bran, spelt, semolina, durum, faro, graham, einkorn, bulgar, couscous), rye,
barley, triticale, oat
Lactose products during acute irritation
Allowed
Vegetables, fruits, fish, and meat, rice, corn (maize), potato, tapioca, quinoa, amaranth, flax, nut, flours,
arrowroot, beans, garfava, lentil, sorghum, buckwheat, millet, teff, xanthum gum, guar gum
Caution with grain-based products
Food starch, malt, icing sugar, soy sauce, filler, gum base, hydrolysed vegetable or plant protein, white
vinegar, fat substitutes, some medications
Avoiding gluten in food products is difficult, expensive, and affects style of living. New
therapeutic alternatives are needed. In Lerner and Gujral et al., features new therapeutic
strategies which is safe, effective and affordable; these comprise of: dietary
manipulations to detoxify wheat using genetic modification, enzymatic degradation of
gluten removing immunogenic activities, tTG and HLA-DQ blockage using false
peptides, gliadin peptide hydrolysis using enzymes and microorganisms, prevent gliadin
peptide absorption using drugs and anti-gliadin egg yolk antibodies, vaccine to restore
immune tolerance towards gluten, and immune response modulation using IL-blocker,
and NKG2D antagonists. These treatment approaches are still in development as testing
is needed to ensure no side-effects in vivo come with these, studying CD pathogenesis is
vital to understand processes and steps occurring during the disease.
1.1.6 Immunopathogenesis of Coeliac disease
Development of CD is determined when genetic factors are combined with
environmental factors such as gluten ingestion as well as early infections, gut flora in
infants and amount and timing of initial gluten introduction (Guandalini & Assiri, 2014).
Studies of pathogenesis are focused on the mechanisms where gluten peptides are
deamidated by the tTG and presented to CD4+ T cells by APCs that express HLA-DQ2/8
induce an immune response that results in coeliac lesions, crypt hyperplasia, and villous
atrophy. Mechanisms are categorized into three events: transport of gluten, modification
of gluten, and presentation to APCs.

18. 13
Figure 6 Factors involving in the development of coeliac disease. Adapted from Guandalini and Assiri (2014)
1.1.6.1 Transport of gluten
Gluten present in wheat, barley and rye, is the environmental stimulus that activate CD in
a genetically predisposed individual. It is composed of different components which are
glutenins and gliadins (Leon et al., 2005). Gliadin, a prolamin, is rich in proline and
glutamine which is difficult to break down with proteolytic enzymes. Prolamins are toxic
in vitro in mucosal explant cultures and in vivo in proximal and distal intestine (Howdle
et al., 1984; Marsh, 1992; and Ciclitira et al., 1984). The direct effect of gluten on
enterocyte and IEL epithelium play a role in the beginning of the mucosal immune
response.
Incomplete digestion of gluten results in gluten taking the appearance of a gluten-derived
gliadin peptide such as 33mer (Gujral et al., 2012) where characteristics overlap with T-
cell epitopes. These peptides pass into the lamina propria where immune reaction occurs.
Gut permeability is increased in CD and gluten peptides pass through using the spaces in
between enterocytes; tight junctions are opened due to increased levels of zonulin
(University of Maryland, 2010), a factor contributing to CD development released by
enterocytes when in contact with α-gliadin and increase permeability. This paracellular
route allows non-digested gluten to pass through in addition to α-gliadin (Gujral et al.,
2012).
Other gluten transportation is by transcytosis and retrotranscytosis. The α2-gliadin-33mer
translocates via an interferon-γ-dependent transcytosis (Corazza and Di Sabatino, 2009).
Secreted IgA (sIgA) is retrotransported into intestinal mucosa through CD71 transferrin

19. 14
receptor (Matysiak-Budnik et al., 2008). Retrotranscytosis allow gliadin-sIgA complex to
be transported across epithelium, resulting in further immune responses such as elevated
levels of AGA.
Figure 7 SIgA-gliadin peptide complex CD71-mediated retrotranscytosis into lamina propria
1.1.6.2 Modification and presentation of gluten
After translocation of gluten peptides and gliadin 33mers these gain access to Peyer’s
patches and is processed. tTG is an enzyme that catalyses post-translational modification
of proteins is released during inflammation, it deamidates glutamine rich peptides by
cross-linking glutamine residues to lysine taking out NH2 groups. After deamidation the
peptides stimulate APCs such as laminal macrophages and dendritic cells (DC) that
generate residues bound to HLA-DQ2 or HLA DQ8, APCs then present deamidated
peptides to naïve CD4+ T-cells. Presentation is not limited to macrophages and DC,
process can also be performed by B cells and enterocytes that express HLA class II (Leon
et al., 2005).
1.1.6.3 Disease progression involving Innate and adaptive immune system
Innate and adaptive immune system is stimulated by gliadin peptides. In innate reaction,
a Th-1 pathway takes place where T-cell and cytolytic activity is active, IL-15 cytokines

20. 15
and non-classic MHC class I molecules are expressed by epithelial cells which activates
CD8+ cytotoxic T-cells expressing up-regulated natural killer receptors NKG2D that
interact with epithelial cells releasing IFN-γ and cytotoxic molecules that contribute to
cell death. Matrix metalloproteinases (MMPs) is released and contributes to tissue
remodelling. In adaptive reaction, Th-2 pathway takes place where active T-cells
stimulate B –cell production of various antibodies that contribute to mucosal damage and
cell apoptosis (Figure 8).
Figure 8 Mechanism of mucosa damage in coeliac disease (adapted from Di Sabatino and Corazza, 2009).
Gluten peptides transported across epithelium by paracellular route (blue), transcytosis (green), and
retrotranscytosis(red). Gluten peptides deamidated by tTG reinforce presentation of peptides by APCs to CD4+ T-cells
associated with HLA-DQ2/8 molecules. CD4+ T-cells are activated which produce pro-inflammatory cytokines that
results in an IFN-γ dominated T-helper-cell-type-(Th)1. Th-1 cytokines boost inflammatory effects including lamina
propria mononuclear cell (LPMC) matrix metalloproteinases (MMPs) secretion that degrade extracellular matrix and
basal membrane, and a cytotoxicity increase of intraepithelial lymphocytes (IEL) or natural killer (NK) T-cells. These
facilitate apoptosis of enterocytes via Fas/Fasligand(FasL) system or IL-15-induced perforin-granzyme and NKG2D-
MIC signalling pathways. Released IFN-α maintain reaction by encouraging production of IFN-γ. Th-2 cytokines
activate B cells that differentiate to plasma cells and produce antibodies (Ab) which react to gliadin, membrane-bound
tTG (mtTG), and tTG. Enterocyte cytoskeleton changes and actin redistribution caused by tTG Ab deposits lead to
epithelial damage.

21. 16
1.1.7 Coeliac Disease and future developments
As stated previously, diagnosis of CD can be complex, it would be ideal to have a
diagnostic tool to make this process easier and more convenient to patients as biopsies
would not be required or not be needed in mild CD. A biomarker that can be detected and
quantified easily and accurately can be ideal for both patient and medical professionals. A
serum or blood sample is easier to obtain, and analyze than a biopsy.
1.2 CD163
1.2.1 Macrophages
Macrophages originate from their progenitor cells the monocytes; these cells play an
important role to the immune system. They are also key players in normal homeostasis
and also in pathological conditions including inflammation, infection, and cancer. Both
macrophages and monoctyes originate from a myeloid progenitor cell within the bone
marrow; mature macrophages are a part of a very heterogenous population of cells.
Macrophages present in the tissue form the first line of defence; these cells can recognize
and eliminate pathogens and foreign material that gain access to the body. They show
homeostatic function and are also capable of phagocytic action, degradation of self and
foreign materials, establishing cell to cell interactions and producing inflammatory
mediators (Gordon et al., 1995)
Specialized functions of macrophages depend on the molecular tools they express, these
tools include Fc receptors, complement receptors, scavenger receptors, adhesion
molecules, and soluble mediator receptors such as cytokines, chemokines, and growth
factors. Tissue localization and macrophage activation status play a role in the expression
of mentioned tools in the cells.
Haemoglobin (Hb) is an abundant protein present in higher organisms, it is the oxygen
carrier protein found within red blood cells that allows for transport and exchange of
gases. Isolation of haemoglobin within the red blood cells minimizes accumulation of
free haemoglobin in the plasma which limits its toxicity. In a pathological condition,

22. 17
when lysis of red blood cells occur Hb is released from these lysed cells, free Hb in
circulation binds with haptoglobin (Hp) which is a glycoprotein in serum that forms a
haemoglobin-haptoglobin (Hb-Hp) stable complex (Kristiansen et al, 2001).
Haemoglobin is efficiently bound and removed by Hp via gating Hb to a high affinity
receptor for Hb-Hp complexes namely CD163 which is found on macrophage monocyte
lineage cell surfaces.
In intravascular haemolysis or Hb solution transfusion, Hb precipitates in renal tissue,
this can lead to acute renal failure and depletion of Hp. This shows that Hp slows down
passage of Hb through the glomeruli into the renal tubular cells which protects the kidney
from peroxidative injury. When Hp levels are low CD163 becomes operative, in this
pathway, isolated parenchymal liver cells take up Hb in circulation at a faster rate
compared to the rate of which Hb-Hp complexes do, which results in a faster rate of
clearing free Hb from circulation (Weinstein MB & Segal HL, 1984).
1.2.2 CD163
CD163 was first identified in 1987 (Graverson et al, 2002). This antigen is a member of
the scavenger receptor cysteine rich (SRCR) super family class B (Fabrick et al., 2005,
Sarrias et al., 2004). It was previously called M130 and P155 before it received its CD
designation, according to Bruce & Zarev, 2005 and Sarrieas et al., 2004, several names
can be used to refer to CD163 these names include haemoglobin scavenger receptor
(HbSR), haemoglobin/haptoglobin complex receptor, RM3/1 antigen, M130 antigen
precursor, MM130, Ki0M8, Ber-MAC3, SM4, and GHI/61. CD163 is expressed on most
subsets of myeloid cells and mature tissue macrophages.
The SRCR super family is a family of structurally related transmembrane glycoproteins
(Fabriek et al., 2005). This super family is divided into two groups, Group A and Group
B. Both groups A and B contain three disulfide bridges, group B contains a fourth
disulfide bridge which enable us to distinguish one between the other where group A
SRCR molecules have 6 cysteine residues and group B have 8 cysteine residues (Aruffo
et al., 1997; Resnick et al.,1994). Group A SRCR molecules are encoded by two exons
and Group B is encoded by a single exon (Van Gorp et al., 2010). Group A SRCR

23. 18
molecules include SR-AI, Mac-2 binding protein MARCO, lysyl oxidase related protein,
complement factor I and enterokinase. Some molecules of group B SRCR molecules
include CD5, CD6, SPα, gp-340, M160, and CD163 (Fabriek et al., 2005). Group B is
subdivided into two subgroups depending on the presence or absence of extracellular
domains other than the SRCR domains (Sarrias et al., 2004). According to van Heuvel et
al., 1999, within group B SRCR CD163 and M160 are the only members which are
selectively expressed on monocytes and macrophages.CD163 belongs to the first
subgroup within group B as it is a protein that exclusively comprises of none SRCR
domains in the extracellular region. Little is known about the functions of members of the
group B SRCR family.
It is clear that the SRCR domains recognize a wide variety of structurally different
ligands, with some having a narrow specificity and some having broad specificity.
Exploring the SRCR family in context of its ligand recognition and physiological
relevance of their interactions is a challenge.
1.2.2.1 Structure
The CD163 gene chromosomal location was mapped to region p13 on chromosome 12,
which consists of 17 exons and 16 introns and spans at least 35kD. The start codon and
the N-terminal of the signal peptide are encoded by exon 1. The C-terminal is encoded by
exon 2 and two nucleotides of exon 3 (Figure 9). The nine SRCR domains of CD163 are
encoded by a separate exon for each domain. These exons vary in length from 309 to 324
nucleotides and are separated by introns (Law et al., 1993).
Figure 9. Schematic representation of the CD163 gene. Individual structures and parts of CD163 are shown. Adapted
from Kowal et al., 2011.

24. 19
CD163 is a 130kDa human-macrophage associated antigen which is defined by five
different antibodies, it is extensively glycosylated with predominant N-linked glycans
where a reduction in it’s molecular weight after endoglycosidase F treatment is observed
as it becomes 110kDa (Fabriek et al., 2007; Hogger et al., 1998). It is a glycoprotein that
contains a single transmembrane element, a short cytoplasmic tail and a large
extracellular region of nine SRCR domains (Law et al., 1993). According to Sarrias et al.,
2004 and Onofre et al., 2009, CD163 is a membrane bound protein that contains a leader
peptide of 40 residues. The extracellular part contains 1003 amino acids, the
transmembrane single segment is made up of 24 amino acids and the short cytoplasmic
domain comprises of 49 amino acids. As previously mentioned there are nine type B
SRCR domains in the extracellular region of CD163, a group B SRCR domain has eight
cysteine residues with disulphide bridges linked in a 1-4, 2-7,3-8 and 5-6 pattern, in
CD163 SRCR8 lacks the 2-7 bridge. The SRCR6 and 7 domains are separated by a
proline-serine-threonine rich (PST) polypeptide which is 35 amino acids. A short PST
linker connects SRCR9 with a transmembrane domain and an intracellular cytoplasmic
tail (Akila et al., 2002).
Figure 10. Representation of CD163 SRCR. CD163 membrane protein composed of nine SRCR domains, and PST
domains. A characteristic of CD163 is the long-range repeat of five consecutive SRCR domains with a small PST
linker which separates the second and third SRCR domain referred to as [b-c-d-e-d] cassette. Adapted from van Gorp et
al., 2010.
There are five different isoforms of CD163 has been described so far according to Onofre
et al., 2009. These isoforms differ in the structure and length of the cytoplasmic tail
domains and their putative phosphorylation sites. Three out of the five display alternative
splicing forms of the cytoplasmic domain that vary in the number of amino acids 49 to
84 or 89; Common in all three isoforms if the first 42 amino acids after the membrane
spanning segment (Gravensen et al., 2002). The other two isoforms display alternative
splice sites in the extracellular part where one generates a stop codon which produces a

25. 20
truncated form of the protein, and the other where an additional 33 amino acids are added
between SRCR5 and 6 domains (Sarrias et al., 2004; Vila et al., 2000).
1.2.2.2 Expression and Regulation
In 1999 van den Huevel et al. extensively studied the expression of CD163 in humans.
CD163 has also been described in other animals such as mouse (Schaer et al., 2001), rat
(Polfliet et al., 2006), and also dog, cattle, pig and monkey homologues (Calvert et al.,
2007; Sopp et al., 2007; Sanchez et al., 1999; Zwadlo-Klarwasser et al., 1992). As
previously mentioned expression of CD163 is restricted to cells of the
monoctye/macrophage lineage other white blood cells such as granulocytes and dendritic
cells do not express significant levels of CD163. CD163 expression is also dependant on
maturation stage of monocytes and macrophages.
There are many resident mature tissue macrophages that express high levels of CD163
such as red pulp macrophages in the spleen, Kupffer cells in the liver and interstitial and
alveolar macrophages in the lungs. Additionally, perifollicular macrophages in the
tonsils, medullary and cortical macrophages in the thymus, and perivascular and
meningeal macrophages in the central nervous system, and scattered macrophages within
various other tissues show the presence of CD163 (Table 3) (van den Heuvel et al.,
1999).
Table 3. Distribution of CD163 positive macrophages in most common studied tissues in human. Adapted from
Fabriek et al., 2005.
Tissue Macrophage subpopulation CD163
Spleen Red pulp macrophages +
Perifollicular macrophages -
Lymph nodes Medullary macrophages +
Perifollicular macrophages +
Thymus Medullary macrophages +
Cortical macrophages +
Liver Kupffer cells +
Brain Perivascular macrophages +
Meningeal macrophages +
Micrologia -
Lung Alveolar macrophages +
Interstitial macrophages +
Blood Monocytes +

26. 21
Macrophages expressing CD163are also found in tissues during the healing phase of
acute and chronic inflammation. They are also found in wound healing tissues, and in
highly inflamed tissues which suggest that CD163 has a role in the resolution of
inflammation (Fabriek et al., 2005). According to Radzun et al., 1987 and Moniuszko et
al., 2009 the highest expression of CD163 is found on CD14high CD16+ and the lowest
on CD14lowCD16+ cells. The subpopulation of CD14highCD16+ cells have a
predominant anti-inflammatory functions.
Expression of CD163 can be induced with the treatment of glucocorticoids (Buechler et
al., 2000). A number of pro- and anti-inflammatory mediators strongly affect CD163
expression, Table 4 shows the list of these mediators; Anti-inflammatory mediators such
as glucocorticoids and interleukin-10 (IL-10) up-regulates CD163 expression strongly.
As described by Williams et al., 2002, experiments that utilize gene-chip technology have
found that the response of CD163 to IL-10 was the strongest in all 19 of the up-regulated
genes. The up-regulation of monocyte/macrophage CD163 expression induced by
CD4+CD25+Foxp3+ T regulatory cells and up-regulation of monocye/macrophage
CD163 expression after shedding of the receptor in response to Toll like receptor (TLR)
stimulation is also caused by IL-10 (Tiemessen et al., 2007; Weaver et al., 2007). It is to
note that glucocorticoids are as potent inducers of CD163 expression, the magnitude of
CD163 expression will depend on the potency of the glucocorticoids where those that
have a high affinity for glucocorticoid receptors are most potent for up-regulation of
CD163 expression.
Endogenous pro-inflammatory cytokines and chemokines decrease CD163 expression
these are TNF-α, IL-1α, IL-1β, and CXCL-8 (IL-8). Also, exogenous pro-inflammatory
molecules like LPS decrease CD163 expression (Sulahian et al., 2000; Weaver et al.,
2007; Rassias et al., 2002). In a study by Buechler et al., 2000, dendritic cells that have
differentiated from monocytes via GM-CSF and IL-4 CD163 mRNA and protein are
suppressed. However, dendritic cells from a monocytic lineage can still express very low
levels of CD163 (Sulahian et al., 2000).
CD163 regulation by pro and anti-inflammatory mediators tells us that there is a link
between CD163 immune suppression and inflammatory resolution. The high level of

27. 22
CD163 expression found on mature tissue macrophages suggest that a role of CD163 is in
the recognition of pathogens and the innate immune system responses that follow.
Table 4. CD163 expression regulating factors. Adapted from Kowal et al., 2011.
Up-regulating factors of CD163 expression on
monocytes and macrophages
Down-regulating factors of CD163 expression on
monocytes and macrophages
Glucocorticoids Tumour necrosis factor alpha (TNF-α)
Interleukin-10 Interleukin-1 alpha (IL-α)
Interleukin-6 Interleukin-1 beta (IL-β)
Macrophage colony stimulating factor (M-CSF) Interleukin-4
Interleukin-13
CCL-3 (MIP-1a)
CXCL-4
CXCL-8 (IL-8)
Interferon-gamma (IFN-γ)
Transforming growth factor-beta (TGF-β)
Granulocyte macrophage colony stimulating factor
(GM-CSF)
Lipopolysaccharide (LPS)
Stimulation of Toll-like receptors TLR-2, TLR-4,
TLR-5
Oxidative stress
Hypoxia
Cross-linking of Fc-gamma receptor (FcγR)
8-iso-prostaglandin F2α
1.2.2.3 Functions
There are many roles and functions of CD163 that have been described. These functions
can be divided into two main headings such as homeostatic function and anti-
inflammatory response. Other functions have also been discussed.
1.2.2.3.1 Homeostatic function: Haemoglobin clearance by CD163
As previously discussed, CD163 has a role in clearing free haemoglobin in circulation.
Free haemoglobin is released into the blood by extra vascular and intravascular
haemolysis. Extra vascular haemolysis occurs when erythrocytes are phagocytosed by
macrophages in the bone marrow, liver, and spleen. At the same time alternatively to the
process of phagocytosis red blood cells suffer intravascular haemolysis in a few percent
(10% to 20%). As a result of these two mechanisms of haemolysis haemoglobin is

28. 23
released from ruptured erythrocytes, dissociates and dimerizes into two dimers αβ. These
αβ dimers are captured by plasma protein haptoglobin which form a haemoglobin-
haptoglobin (Hb-Hp) complex (Gravensen et al., 2002; Moestrup et al., 2004).
Haptoglobin (Hp) is a protein found in plasma where there are two common alleles
referred to as 1 and 2 found in humans. This protein is a haemoglobin binding protein
that is expressed due to genetic polymorphism. There are three distinct Hp phenotypes
found in humans: 1-1 (homozygous for allele 1), 2-1 (heterozygous, contains allele 1 and
allele 2), 2-2 (homozygous for allele 2) (Guetta et al., 2007; Langlois et al., 1996; Levy,
2004). CD163 can bind all phenotypes of haptoglobin, the complexes
haptoglobin/haemoglobin-2 subtype 2-2 form shows a higher affinity compared to
complexes haptoglobin/haemoglobin-1.This difference has no physiological role in
haemoglobin clearance as the 1-1 form has sufficient affinity for CD163 for being
endocytosed effectively. There is a difference in the cytokine response of which they
induce via this binding. CD163 binding of the haptoglobin-1 and the formation of
haptoglobin/haemoglobin complexes-1 stimulates the production of anti-inflammatory
cytokines such as IL-10. CD163 binding to the haptoglobin-2 and formed
haptoglobin/haemoglobin complexes-2 do not stimulate anti-inflammatory cytokine
production (Guetta et al., 2007; Strauss et al., 2008). The presence of calcium is required
for binding the complex haptoglobin/haemoglobin to CD163, calcium maintains the
proper tertiary structure of CD163 (Kristiansen et al., 2001; Moestrup et al., 2004).
The binding of Hb to Hp leads to the exposure of a neo-epitope causing a high affinity
interaction with the third SRCR domain of CD163 in a calcium dependent manner. Upon
binding to CD163 Hb-Hp complexes are internalized. After internalization the cargo is
delivered to early endosomes and CD163 is recycled to the plasma membrane to start a
new cycle of endocytosis. After Hb-Hp endocytosis the heme subunit of haemoglobin is
acted upon and degraded by heme-oxygenases (HO) enzymes located in lysozymes. HO
is a potent anti-inflammatory and anti-oxidative enzyme, it has three isoforms, HO-3 is
nearly devoid of catalytic activity, HO-2 which is constitutively present and HO-1 which
is inducible by anti-inflammatory stimuli (Fabriek et al., 2005; Philippidis et al., 2004).
This breakdown of the heme subunit produces biliverdin, free iron, and carbon monoxide

29. 24
which have anti-inflammatory effects (Wagener et al., 2003) and also cryoprotective
effects.
The removal of haptoglobin/haemoglobin complexes is necessary to remove free
haemoglobin from plasma. This removal overcomes oxidative damage, prevents the
glomeruli from haemoglobin filtration and heme intoxication of kidneys by free heme
and iron accumulation because of oxidative and toxic properties of heme and iron. This
mechanism also avoids oxidative stress, reactive oxygen species, and cell injury
(Kristiansen et al., 2001; Moestrup et al., 2004).
Figure 11. Representation of free haemoglobin elimination by CD163 positive macrophage. Adapted from Onofre et
al., 2009.
1.2.2.3.2 Regulation of erythropoiesis by CD163
Erythropoiesis is a complex developmental process that starts in the bone marrow and
end at the production of erythrocytes or red blood cells. The erythroblastic island is the
functional unit for this process found in the bone barrow. It is a multi-cellular structure
that is composed of a macrophage surrounded by erythroblasts at different stages of
differentiation. This contact between erythroblasts and macrophage support the growth,
survival and differentiation, and allow for phagocytosis of the erythroid nucleus after
being removed. On a macrophage there are four adhesion molecules for erythroblasts

30. 25
these are vascular cell adhesion molecule-1, av integrin, erythroblast-macrophage protein,
and sialoadhesin (Chasis, 2006).
CD163 interacts with erythroblast cells. In the second SRCR domain of CD163 is a 13
amino acid motif that mediate the binding between the erythroblast and CD163. This
interaction was found to encourage the growth and survival of erythroblasts according to
Polfliet et al., 2007. This would suggest that CD163 has a regulatory role in the process
of erythropoiesis. The tissues that have CD163+ macrophages such as the liver and the
spleen can have two important functions which is to mediate the clearance of Hb and to
promote erythropoiesis. The link between the clearance of Hb and erythropoiesis shows
an efficient mechanism for iron recycling for erythroblast development. CD163 binding
site for Hb-Hp complexes is different from CD163 erythroblast binding site which leads
to the coordination of Hb-Hp and erythroblast binding by CD163 in erythroblastic islands
(Akila et al., 2012).
1.2.2.3.3 Tweek scavenging function of CD163
Tumour necrosis factor (TNF) and tumour necrosis factor receptor (TNFR) has roles in
the development and regulation of the immune system and are involved in processes
such as apoptosis, cell proliferation, and bone remodelling (Locksley et al., 2001).
According to Raplan et al., 2002, TWEAK is TNF-like weak inducer of apoptosis via a
non-death domain-dependent mechanism which mediates angiogenesis and inflammation.
Fibroblast growth factor inducible 14/TweakR is reported to control proliferation of
endothelial cells and angiogenesis associated with TWEAK (Wiley et al., 2001).
TWEAK mediates signal transduction and linear differentiation of monocytes and
macrophages that lack TweakR. CD163 was found to be a TWEAK binding protein that
has 44 putative interaction sites located in eight out of the nine SRCR domains of CD163.
Inhibition of TWEAK binding to CD163 was found when Hb-Hp complexes and
antibodies recognize CD163 binding sites. TWEAK is internalized after binding to
CD163 molecules on macrophages and is degraded. CD163 can act as a TWEAK
scavenger or act as a TWEAK receptor for cells that lack TweakR (Polek et al., 2003;
Bover et al., 2007).

31. 26
1.2.2.3.4 CD163 as bacterial receptor
CD163 is thought to be involved in host defence. CD163 that are expressed on cells or as
an immobilized protein can support binding of both gram-positive and gram-negative
bacteria. Within the second SRCR domain of CD163 is a peptide motif that mediates
erythroblast binding and demonstrates bacterial binding that suggests a binding site for
cellular and bacterial adhesion is located in said domain. Monocytic CD163+ cells
promote bacterial induced pro-inflammatory cytokine production such as TNF-α. Other
inflammatory mediators and cytokines can be produced due to CD163-mediated bacterial
recognition and signalling which generate local inflammatory response to eliminate
bacterial infection. However, more studies are needed to determine the bacterial ligands
involved, intracellular signal pathways used, and the role of CD163 during a bacterial
infection in vivo (van Bruggen et al., 2009).
1.2.2.3.5 CD163 as a molecule with an Immunoregulatory function
It is said from the studies of Zwadlo et al., 1987 and Schaer et al., 2001 that CD163
receptor activity is linked to an anti-inflammatory response. This is based on
macrophages that express CD163 is present in large numbers during the resolution of an
inflammatory response and that CD163 is induced by anti-inflammatory mediators such
as glucocorticoids, IL-10 and IL-6 that result in a cell population called ‘alternatively
activated macrophages’. IL-10 is produced by alternatively activated macrophages inhibit
T lymphocyte proliferation which implicates them during wound healing, angiogenesis,
and protection from inflammatory response. This means that alternatively activated
macrophages has a regulatory and recovery role.
As previously discussed, macrophages expressing CD163 can internalize Hb-Hp
complexes via endocytosis and allow the heme subunit to be degraded by HO enzymes.
These HO enzymes are potent anti-oxidative and anti-inflammatory enzymes. The
products which are formed during heme degradation, carbon monoxide (CO), biliverdin,
and bilirubin, have strong anti-oxidative and anti-inflammatory effects. At low
concentration in vivo CO can inhibit expression of lipopolysaccharide-induced pro-
inflammatory cytokines TNF-α, IL-1β and macrophage-inflammatory protein-1β and

32. 27
increase LPS-induced expression of anti-inflammatory cytokine IL-10 (Otterbein et al.,
2000). Interleukin-6 (IL-6) can induce synthesis and expression of HO-1, haptoglobin,
and CD163 where it can be said that IL-6 co-regulates Hp, CD163, and HO-1. CD163+
macrophages degrade heme, and exert anti-inflammatory effects with the release of heme
metabolites and IL-10. CD163 signalling also implicates pro-inflammatory cytokine
production. CD163-specific antibodies cross-linked with CD163 can induce secretion of
nitric oxide (NO), IL-1β, IL-6, and TNF-α. Where, as previously mentioned, TNF-α and
IL-1β are pro-inflammatory cytokines, and NO and IL-6 can have pro- and anti-
inflammatory response. CD163 is an immunomodulator that can stimulate or suppress the
immune response (Dijkstra et al., 2006).
1.3 Soluble CD163 (sCD163)
CD163 is expressed as a membrane bound protein and is the only scavenger receptor that
is actively shed from the cell surface. This soluble variant of CD163 can be present in
plasma, cerebrospinal, synovial, or ascitic fluid. Healthy individuals can have a range of
1-3mg/L with a median value of 1.9 mg/L of sCD163 in the plasma (Moller et al., 2002;
Hintz et al., 2001).
This soluble form of CD163 is a biomarker used with a number of conditions; it is
relevant to coronary artery disease detection, transplantation, atherosclerosis, rheumatoid
arthritis, lysomal Gaucher storage diseases, and cancer (Fabriek et al., 2005; Kolackova
et al., 2008). Inflammatory conditions characterised by monocytic infiltration is
characterized by the presence of sCD163. Conditions such as inflammation, systemic
inflammatory response syndrome, sepsis, bacteraemia, mononucleosis, leishmaniasis,
Crohn’s disease, coeliac sprue/coeliac disease, spondylarthropathy, synovitis, sclerosis,
hepatitis and fulminant hepatic failure can also be detected with the use of sCD163
(Rassias et al., 2002; Moestrup et al., 2004; Pioli et al., 2004). In Immunohaematological
diseases, such as lymphoma, reactive haemophagocytic syndrome, histiocystic neoplasm,
myeloproliferative diseases, and lye-lomonocytic leukaemia, sCD163 is also present.
Additionally, sCD163 levels are increased in the resolution of inflammatory diseases
(Bachli et al., 2006; Gravensen et al., 2002).

33. 28
The patients of most of these diseases are exposed to very high amounts of free heme
from intravascular haemolysis and tissue damage. Toxic and pro inflammatory
haemoglobin can be removed efficiently by CD163 from inflamed sites and also from
circulation; Thus, CD163 can be used as a marker for many diseases listed and mainly in
inflammation (Kolackova et al., 2008).
The shedding of CD163 from the plasma membrane is caused by metalloproteinases, it
has been suggested that a member of the A Disintegrin and Metalloprotease family is
responsible. At least two enzymes are involved in the shedding process of CD163: matrix
metalloproteinase-9 and tumour necrosis alpha-converting enzyme (Rassias et al., 2002;
Etzerodt et al., 2010). Matrix metalloproteinase-9 is found to be involved in the
regulation of CD163 shedding in vivo (Vloet et al., 2007). CD163 can be shed from
glucocorticoid-stimulated monocytes after an inflammatory stimulus. Treatment with
phorbol 12-myristate 13-acetate, stimulation from the cross-linking of immunoglobulin G
with FcγR, and LPS, can induce rapid shedding of CD163 (Droste et al., 1999; Wardwell
et al., 2004; Rassias et al., 2002). Physiological activators, such as oxidative stress or 8-
isoprostaglandin F (2α), of CD163 shedding in inflammatory conditions was identified
(Timmermann et al., 2005).This shedding is protein kinase C dependent and is blocked
by protease inhibitors (Droste et al., 1999). Additionally, metalloproteinases tissue
inhibitors like TIMP-3 prevent CD163 shedding. CD163 cleavage occurs near the
plasma membrane (Moller et al., 2002) and is thought to be catalyzed by membrane-
bound metalloproteinases.
Proteolytic shedding of CD163 can account for CD163 found in plasma in healthy
individuals. As discussed earlier, sCD163 concentrations are increased in individuals that
have certain diseases, for example, Gaucher diseases are disease with proliferation of
cells of myelo-monocytic origin; other conditions such as sepsis, and myeloid leukaemia
also have high sCD163 (Aerts et al., 2004). According to Gronbaek et al., 2002, sCD163
do not reflect acute inflammatory response. In this condition, there is an inverse
relationship for membrane-bound CD163 and sCD163where sCD163 is derived from
circulating monocytes and tissue macrophages. It is to note that, although there is a high
amount of sCD163 in the plasma and also in affected tissue, functional relevance of

34. 29
sCD163 is unknown. It is clear that proteolytic cleavage can act as a feedback mechanism
to lower CD163 levels on macrophages; this would be helpful if CD163 was contributing
to cytokine production (Davis & Zarev et al., 2005).
sCD163 has the potential to be used as a marker molecule for macrophage activation in
diseases such as liver disease, multiple sclerosis, and malaria. Standardized and sensitive
diagnostic kits and tests are available to detect sCD163. In fluids such as plasma, serum,
and many others sCD163 can be detected using ELISA (enzyme linked immunosorbent
assay) method. Methods such as flow cytometry, immunochemistry,
immunoprecipitation, and western blotting can also be used for sCD163 detection.
2 Methodology
2.1 ELISA – Sandwich
As described by Sulahian et al. 2001, a solid phase sandwich enzyme linked
immunosorbent assay (ELISA) was developed in order to measure soluble CD163 in
biological fluids such as plasma and serum.
The enzyme-linked immunosorbent assay is a rapid, high-throughput, quantitative
immunoassay for the selective detection of target antigens. The general principle of
ELISA is antibody-mediated capture and detection of the target antigen with a
quantitative substrate. This method is utilized in many diagnostic and screening
applications; such applications that use ELISA include screening of antibody markers in
coeliac disease, screening for HIV antibodies in blood, detection of hepatitis B markers in
serum, and detection of microorganisms and toxins in faeces.
A sandwich ELISA utilizes two antibodies to detect the antigen; a primary antibody is
bound to the surface of the well, the target antigen is applied to the primary antibody,
then a secondary antibody which can be enzyme linked is applied which binds to the
antigen, with this binding the detection is done by finding a coloured product.
As previously stated CD163 is expressed as a membrane bound protein and is actively
shed from the cell surface. Soluble CD163 is present in plasma. In this ELISA two

35. 30
specific monoclonal antibodies (mAbs) (Mac 2-158 and RM3/1) are used to measure the
immunoreactivity of sCD163 in plasma, serum and in cell culture supernatant.
In 2006, Daly and colleagues measured soluble CD163 in serum samples of individuals
that were normal healthy subjects, a group of celiac patients, and a group of patients who
has Crohn’s disease. The methodology used in this experiment is adapted from their
study.
Nunc maxisorb 96-well microtitre plates were coated overnight with mouse monoclonal
anti-CD163 (Mac 2-158) at 4 degrees Celsius at a 1ug/ml concentration. Plates were then
washed four times with wash buffer (phosphate buffered saline (PBS) 0.05% Tween 20
pH 7.3). Sites where no reaction occurred were blocked with PBS/ human serum albumin
0.25% for 30 minutes at room temperature. This blocking step is done to prevent non-
specific binding from occurring. Plates were washed four times and serum was added to
duplicate wells and is incubated for two hours at room temperature, the serum can be
diluted with blocking buffer. Bound sCD163 was detected by the addition of mouse
monoclonal antibody which is biotinylated and is specific to CD163 (RM3/1) incubated
at room temperature for one hour. Plates are washed four times with wash buffer and
streptavidin alkaline-phosphatase was added. After 30 minutes of incubation the plates
were washed four times with washing buffer and a developing solution (Othophenylene
diamine, DAKO Cytomation) in distilled water containing 30% hydrogen peroxide was
added. Colour development was allowed to occur and after sufficient colour development
the reaction was stopped by adding 100uL/well of 0.5N hydrogen sulphite (H2SO4).

36. 31
Figure 12. Soluble CD163 ELISA assay. Steps and diagram of a sandwich ELISA assay which measures sCD163 in a
serum sample.
2.2 Statistics
The Mann-Whitney test is used for statistical anaysis for unpaired data where P < 0.05 is
considered significant. The Ordinary one-way ANOVA test is used to compare a group of
result to another different group, P < 0.05 is considered significant. An online diagnostic
test evaluation tool (Medcalc.net) is used in order to calculate various values such as the
Positive Predictive Value (probability that the disease is present when the test is positive),
Specificity (probability that the test result will be negative when the disease is not
present, true negative rate), and Sensitivity (probability that a test result will be positive
when the disease is present, true positive rate) of the test. % Coefficient Variation (%CV)
was calculated to determine reliability of the triplicate results. GraphPad Prism 6.0
software was used to perform statistical analysis on simulated result data and to generate
graphs of the analysed data.

37. 32
3 Results
The data received was from a simulated set of triplicate results. These results were
analyzed and interpreted. In the determination of sCD163 concentrations using ELISA, a
reference range of 0-133 AU was established from normal healthy control values. Figure
13, shows sCD163 serum levels from three cohorts of individuals. It can be seen that
levels of CD163 in coeliac patients (median 256.7 AU) were significantly elevated when
compared to normal healthy controls (median 122.6AU), and to Crohn’s disease patients
(median 146.9AU) (P value < 0.0001, in each instance, Mann-Whitney test).
sCD163 levels of Coeliac disease patients were analyzed with normal healthy controls
and it was found that Coeliac disease patients have significant elevated sCD163 levels
than the control group (P < 0.0001, Mann-Whitney test). When sCD163 levels of Coeliac
disease patients was analyzed with Crohn’s Disease patients it was found that Coeliac
disease patients have a significantly higher amount of sCD163 than Crohn’s disease
patients (P < 0.0001, Mann-Whitney test).

38. 33
C o n tr o ls C o e lia c D is e a s e C r o h n 's D is e a s e
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
S e ru m le v e ls o f s o lu b le C D 1 6 3 in c o e lia c p a tie n ts ,
C ro h n 's d is e a s e p a tie n ts , a n d n o rm a l h e a lty c o n tro ls
sCD163concentration(AU)
* **
Figure 13 Serum levels of soluble CD163 in healthy controls, Coeliac disease patients, and Crohn's disease patients The broken line represents the cut off point of
133AU, where any value above said cut off point is considered to be elevated. *When compared to normal healthy controls Coeliac disease patients have significant
elevated sCD163 levels (P < 0.0001). Crohn’s disease patients have significantly elevated serum sCD163 levels (P < 0.0001) compared to control group. **Although
both diseases show elevated sCD163 levels there is a significant difference between Coeliac disease sCD163 levels and Crohn’s disease sCD163 levels (P<0.0001)
(Mann-Whitney test).

39. 34
0 1 2 3
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
S e ru m le v e ls o f s o lu b le C D 1 6 3 , a c c o rd in g to M a rs h s c o re o f m u c o s a l le s io n .
M a rs h s c o re s v a rie d fro m 0 to 3 , T o ta l 9 4 c o e lia c p a tie n ts
In fla m m a to ry le s io n (M a rs h s c o re )
sCD163concentration(AU) * **
Figure 14. Serum levels of soluble CD163, according to Marsh score of mucosal lesion. Marsh scores varied from 0
to 3, Total 94 coeliac disease patients. *There is a significant difference of sCD163 levels of Coeliac disease patients
that have a Marsh score 0 when analyzed with patients that have Marsh score 1 (P < 0.0001). **It can also be said that
there is also a significant difference of sCD163 levels between patients that have Marsh score 1 and patients that have
Marsh score 2 (P < 0.0001).
In Figure 14, the results in 94 coeliac patients are shown according to their histological
status; It was observed that patients that have Marsh grade 3 lesions have significant
elevated sCD163 levels when compared to patients that have grade 1 (P < 0.0001) lesions
and grade 0 lesions (P < 0.0001). Patients with Marsh grade 2 lesions have significant
high levels of sCD163 than patients with Marsh grade 1 lesions (P < 0.0001) and with
Marsh grade 0 lesions (P < 0.0001).
A positive predictive value analysis was done. In this analysis it was found that when the
Control group and Crohn’s disease group were analyzed, the test result was negative
(Positive Predictive Value of 36%) with Sensitivity of 53% and Specificity of 57%.
95%CI of Sensitivity was from 26% to 79% and 95% CI of Specificity was from 39% to
74%. When Control group and Coeliac disease group were analyzed, the test result gave a
positive result (Positive Predictive Value of 87%), with Sensitivity of 99% and

40. 35
Specificity of 58%; 95% CI of Sensitivity was from 94% to 99% and 95% CI of
Specificity was from 39% to 75%. From this analysis we can say that our test works.
C o n tr o ls C o e lia c d is e a s e C r o h n 's D is e a s e
0
5
1 0
1 5
2 0
% C o e ffic ie n t v a ria tio n
CoefficientVariation(%)
Figure 15. Percent coefficient variation of Normal control, Coeliac disease, and Crohn’s disease samples. Line
represents the cut off point of 5%, where any test that resulted in >5% should be repeated, and any that are <5% are
accepted. It was found that 30%, 11%, and 13% of control, Coeliac disease, and Crohn’s disease cohorts have 5%CV,
this means that it is recommended that the test should be repeated.
In Figure 15, Coefficient variation (CV%) of normal control samples, Coeliac disease
patients, and Crohn’s disease patients are shown, the line represents 5% CV mark where,
any test that resulted with a value >5% should be repeated and any test that resulted in a
value <5% is accepted. 10 out of 33 normal healthy controls which is approximately 30%
have >5% CV and the rest of the control cohort have <5%CV. With over 70% of the
cohort have <5% CV we can accept sCD163 values to be fairly reliable. In Crohn’s
disease patients and in Coeliac disease patients it was found that 2 out of 15 and 11 out
of 94 have >5% CV, respectively. It is recommended to repeat tests with these patients to
get more accurate results and to address any problems with handling of the liquid sample.

41. 36
4 Discussion
Crohn’s disease is a relapsing systemic inflammatory disease that affects the
gastrointestinal tract (GIT) with manifestations outside GIT and associated immune
conditions. As the cause of Crohn’s disease is not known it is thought that genetics and
environmental triggers play a part in the disease, when triggered the disease cause a
disturbance in the immune response of the individual where normal micro biota and flora
found in the GIT are attacked by the immune system and cause inflammation (Baumgart
& Sandborn, 2012). Diagnosis of Crohn’s disease rely on the gold standard of an
endoscopy, and other procedures such as CT and MRI enterography, ultrasound, and use
biomarkers such as C-reactive protein and faecal granulocyte proteins lactoferrin and
calprotectin. Coeliac disease (CD), as discussed previously, is a gluten-sensitive
enteropathy triggered by gluten exposure which causes a range of gastrointestinal and
extra intestinal problems where its’ diagnosis and detection rely on histological biopsies
of the small intestine. CD is diagnosed by using serological tests and small bowel biopsy
where histological biopsies are the gold standard. As biopsy is the gold standard of
diagnosing CD it is important to research and investigate non-invasive methods of
diagnosis. This is where a biomarker molecule, specific to CD, found in serum that can
be detected and quantified is a vital discovery to which it can change diagnostic methods
of CD.
In this study, sCD163 was measured in serum samples from patients that have coeliac
disease to determine if the amount of sCD163 correlates with inflammatory lesion in
coeliac disease in which sCD163 can be used as a biomarker for Coeliac disease. CD163
was found to be significantly elevated in individuals with CD compared to normal
healthy controls and with patients that have Crohn’s disease (Figure 13).
sCD163 levels of coeliac disease patients were also compared with levels from patients
that have Crohn’s disease to determine if there is a significant level of difference in both
diseases as CD163 expression in these diseases are elevated. It was found from the
Results that there was a notable difference between sCD163 levels of patients with CD
patients and Crohn’s disease patients which indicates that CD patients have much higher

42. 37
levels of CD163 than patients with Crohn’s disease. With this significant difference it is
possible to use sCD163 as a biomarker molecule for CD patients.
The amount of sCD163 is analyzed with the Marsh score of CD patients (Figure 14); it
was found that CD patients that have Marsh grade 3 lesions has a significant higher
amount of sCD163 compared with patients that have Marsh grade 1 and 0 (P < 0.0001).
Thus, the level of soluble CD163 reflects the extent of inflammatory lesion (Marsh grade)
in coeliac disease. With this finding, sCD163 can be useful as a biomarker for the
disease; also, response to the strict gluten free diet in CD patients can be indicated by
sCD163.
With Positive Predictive Value Analysis, Crohn’s disease group results were analyzed
against Control group and Coeliac disease group were analyzed with Control group. In
this analysis it was found that the test did not work in the first analysis of Crohn’s disease
group and Control group as Positive Predictive Value was 36% which is low when
compared to the Positive Predictive Value of Coeliac disease group and Control group
which is 87% where the test was positive. In this analysis Specificity and Sensitivity of
the tests was also given. In the first analysis, Crohn’s disease and Control group,
Sensitivity was 53% and Specificity was 57%; With this result it can be argued that the
test was not very good as Sensitivity and Specificity of test was low, the probability of
the test result being positive is 53% when disease is present and probability of our test
result being negative is 57% when the disease is absent. In the second analysis, Coeliac
disease and Control group, Sensitivity was 99% and Specificity was 58%; These results
show that the probability of our test result being positive is 99% when the disease is
present and that the probability of our test result being negative will be 58% when the
disease is not present.
Coefficient Variation was calculated for all the triplicate samples taken. This was
calculated in order to determine reliability of the results where if %CV is >5% the test
should be repeated and if %CV is < 5% the test results are accepted. It was found that
small percentages in each cohort have %CV > 5, therefore it is recommended that these
tests should be repeated and reanalysed where samples should be handled carefully to
avoid discrepancies in the triplicate results.

43. 38
As a CD patient begins a strict gluten free diet it was observed that the levels of
serological markers fall. Antibody levels (endomysial, gliadin, and tissue
transglutaminase) fall in response of a GFD diet, however, it does not mean that the
resulting lesions are healing and also that the GFD does not improve or improvement of
inflammatory lesions can take up to one year in patients (Ciacci et al., 2002).
In a study of Daly et al., 2006, they investigated if levels of sCD163 in coeliac disease
correlate with the inflammatory process found in the disease. They have found that
sCD163 can be a useful method of monitoring inflammatory lesion in coeliac disease. It
was found that macrophages that expressed CD163 were found in the lamina propria of
the small intestine both in normal healthy controls and in coeliac disease patients (Daly et
al., 2006). In the control cohort the most prominent area where these cells were present
was in the villi and in coeliac disease they were present away from enterocytes and
scattered in the lamina propria. Also in their study, it was found that there is no apparent
difference of positive cell numbers between normal and coeliac mucosa which suggest
that shedding of CD163 is increased.
Though the mechanism for increasing soluble CD163 in coeliac disease is currently not
known it is possible that cytokines, such as IFN-γ and TNF-α, responsible for
inflammatory lesions of CD163 are involved in the cleavage of CD163.
If sCD163 is used as a biomarker or used as a diagnostic tool to diagnose CD it would
greatly benefit patients where invasive procedures such as biopsies can be avoided.
Improvement of inflammatory lesions with strict adherence to GFD can also be
monitored with the use of sCD163.

44. 39
5 Concluding remarks
CD163 is a member of scavenger receptor cysteine rich super family class B and is only
expressed in macrophages of monocytic origin. There are many functions of CD163, it
has been found that it functions as a Hb-Hp complex receptor which eliminates free Hb
from circulation that prevents oxidative damage of tissue. Hb-Hp complex binding to
CD163 triggers a signalling cascade that will produce anti-inflammatory molecules.
Other functions of CD163 are as follows: CD163 is involved in the regulation of
erythropoiesis by interaction of CD163+ macrophages with erythroblasts which help in
the maturation of erythroblasts; CD163 can act as a TWEAK binding protein molecule;
CD163 support binding of Gram-positive and Gram-negative bacteria which promoted
production of cytokines and other inflammatory mediators; CD163 has a role as a two
faced immunomodulator that can either stimulate or suppress immune response. More
investigation and research is needed to identify all other biological functions of CD163.
sCD163 can be detected in a range of inflammatory diseases and in
immunohaematological diseases. In this study, soluble CD163 molecule was investigated
as a potential biomarker molecule for Coeliac disease, sCD163 was measured using
ELISA, Sandwich method. It was found that there is potential for sCD163 molecule to be
used for detection and diagnosis of Coeliac disease. The availability of a convenient and
non-invasive method for coeliac disease patients has many advantages such as
convenience, comfort for Coeliac disease patients where small intestinal biopsy is not as
needed, and a readily quick and easy detection and quantification method for measuring
soluble CD163 in serum samples is greatly advantageous if available. More research and
development into this subject would be encouraged to fine-tune and test the method to
verify and support the results obtained in this investigation.

45. 40
Bibliography
Aerts, H., Fost, M., Moller, H., Hollak, C. and Moestrup, S. (2004). Plasma level of the macrophage-derived soluble CD163 is
increased and positively correlates with severity in Gaucher's disease. European Journal of Haematology, 72(2), pp.135-139.
Akila, P., Prashant, V., Suma, M., Prashant, S. and Chaitra, T. (2012). RETRACTED: CD163 and its expanding functional
repertoire. Clinica Chimica Acta, 413(7-8), pp.669-674.
Aruffo, A., Bowen, M., Patel, D., Haynes, B., Starling, G., Gebe, J. and Bajorath, J. (1997). CD6—ligand interactions: a paradigm for
SRCR domain function?. Immunology Today, 18(10), pp.498-504.
Bachli, E., Schaer, D. and Walter, R. (2006). Functional expression of the CD163 scavenger receptor on acute myeloid leukemia cells
of monocytic lineage. Journal of Leukocyte Biology, 79(2), pp.312-318.
Baumgart, D. and Sandborn, W. (2012). Crohn's disease. The Lancet, 380(9853), pp.1590-1605.
Bover, L., Cardo-Vila, M., Kuniyasu, A., Sun, J., Rangel, R., Takeya, M., Aggarwal, B., Arap, W. and Pasqualini, R. (2007). A
Previously Unrecognized Protein-Protein Interaction between TWEAK and CD163: Potential Biological Implications. The Journal
of Immunology, 178(12), pp.8183-8194.
Briani, C., Samaroo, D. and Alaedini, A. (2008). Celiac disease: From gluten to autoimmunity. Autoimmunity Reviews, 7(8), pp.644-
650.
Bruce, H. and Zarev, P. (2005). Human monocyte CD163 expression inversely correlates with soluble CD163 plasma
levels. Cytometry Part B: Clinical Cytometry, 63B(1), pp.16-22.
Buechler, C., Ritter, M., Orso, E., Langmann, T., Klucken, J. and Schmitz, G. (2000). Regulation of scavenger receptor CD163
expression in human monocytes and macrophages by pro- and anti-inflammatory stimuli. J. Leukoc. Biol, 67, pp.97-103.
Chasis, J. (2006). Erythroblastic islands: specialized microenvironmental niches for erythropoiesis. Current Opinion in Hematology,
13(3), pp.137-141.
Ciclitira, P., Evans, D., Fagg, N., Lennox, E. and Dowling, R. (1984). Clinical testing of gliadin fractions in coeliac patients. Clinical
Science (London), (66), pp.357-364.
Cooper, S., Kennedy, N., Mohamed, B., Abuzakouk, M., Dunne, J., Byrne, G., McDonald, G., Davies, A., Edwards, C., Kelly, J. and
Feighery, C. (2013). Immunological indicators of coeliac disease activity are not altered by long-term oats challenge. Clin Exp
Immunol, 171(3), pp.313-318.
Daly, A., Walsh, C., Feighery, C., O'Shea, U., Jackson, J. and Whelan, A. (2006). Serum levels of soluble CD163 correlate with the
inflammatory process in coeliac disease. Aliment Pharmacol Ther, 24(3), pp.553-559.
Davis, B. and Zarev, P. (2004). Human monocyte CD163 expression inversely correlates with soluble CD163 plasma
levels. Cytometry Part B: Clinical Cytometry, 63B(1), pp.16-22.
Dijkstra, C., Fabriek, B., Daniëls, W., Polfliet, M. and van den Berg, T. (2006). The rat macrophage scavenger receptor CD163:
Expression, regulation and role in inflammatory mediator production. Immunobiology, 211(6-8), pp.419-425.
Di Sabatino, A. and Corazza, G. (2009). Coeliac disease. The Lancet, 373(9673), pp.1480-1493.
Droste, A., Sorg, C. and Högger, P. (1999). Shedding of CD163, a Novel Regulatory Mechanism for a Member of the Scavenger
Receptor Cysteine-Rich Family. Biochemical and Biophysical Research Communications, 256(1), pp.110-113.
Etzerodt, A., Maniecki, M., Moller, K., Moller, H. and Moestrup, S. (2010). Tumor necrosis factor -converting enzyme
(TACE/ADAM17) mediates ectodomain shedding of the scavenger receptor CD163. Journal of Leukocyte Biology, 88(6), pp.1201-
1205.
Fabriek, B., Dijkstra, C. and van den Berg, T. (2005). The macrophage scavenger receptor CD163.Immunobiology, 210(2-4), pp.153-
160.
Fabriek, B., Polfliet, M., Vloet, R., van der Schors, R., Ligtenberg, A., Weaver, L., Geest, C., Matsuno, K., Moestrup, S., Dijkstra, C.
and van den Berg, T. (2007). The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor. Blood, 109(12),
pp.5223-5229.
Fasano, A. and Catassi, C. (2012). Celiac Disease. New England Journal of Medicine, 367(25), pp.2419-2426.

46. 41
Franco, L. and completo, V. (2014). VIRCHOW'S EYE: MARSH CLASSIFICATION - CELIAC DISEASE. [Online]
Virchowseye.blogspot.ie. Available at: http://virchowseye.blogspot.ie/2010/04/marsh-classification-celiac-disease.html [Accessed
Oct. 2014].
Gordon, S., Clarke, S., Greaves, D. and Doyle, A. (1995). Molecular immunobiology of macrophages: recent progress. Current
Opinion in Immunology, 7(1), pp.24-33.
Gosink, J. (2012). Laboratory diagnostics for celiac disease. [Online] Www2.mlo-online.com. Available at: http://www2.mlo-
online.com/features/201203/special-feature/laboratory-diagnostics-for-celiac-disease.aspx [Accessed Oct. 2014].
Graversen, J., Madsen, M. and Moestrup, S. (2002). CD163: a signal receptor scavenging haptoglobin–hemoglobin complexes from
plasma. The International Journal of Biochemistry & Cell Biology, 34(4), pp.309-314.
Grøbæk, H., Møller, H., Aerts, H., Peterslund, N., Petersen, P., Hornung, N., Renjmark, L., Jabbarpour, E. and Moestrup, S. (2002).
Soluble CD163: a marker molecule for monocyte/macrophage activity in disease. Scandinavian Journal of Clinical & Laboratory
Investigation, 62(7), pp.29-33.
Guandalini, S. and Assiri, A. (2014). Celiac Disease. JAMA Pediatrics, 168(3), p.272.
Guetta, J., Strauss, M., Levy, N., Fahoum, L. and Levy, A. (2007). Haptoglobin genotype modulates the balance of Th1/Th2 cytokines
produced by macrophages exposed to free hemoglobin. Atherosclerosis, 191(1), pp.48-53.
Gujral, N. (2012). Celiac disease: Prevalence, diagnosis, pathogenesis and treatment. WJG, 18(42), p.6036.
Hall, A. and Yates, C. (2010). Immunology. New York: Oxford University Press Inc., pp.127-136.
Heap, G. and van Heel, D. (2009). Genetics and pathogenesis of coeliac disease. Seminars in Immunology, 21(6), pp.346-354.
Howdle, P., Ciclitira, P., Simpson, F. and Losowsky, M. (1984). Are all gliadins toxic in coeliac disease? An in vitro study of alpha,
beta, gamma, and w gliadins. Scand J Gastroenterol, (19), pp.41-47.
Jabri, B., Kasarda, D. and Green, P. (2005). Innate and adaptive immunity: the Yin and Yang of celiac disease. Immunol Rev, 206(1),
pp.219-231.
Kaplan, M., Lewis, E., Shelden, E., Somers, E., Pavlic, R., McCune, W. and Richardson, B. (2002). The Apoptotic Ligands TRAIL,
TWEAK, and Fas Ligand Mediate Monocyte Death Induced by Autologous Lupus T Cells. The Journal of Immunology, 169(10),
pp.6020-6029.
Kolackova, M., Kudlova, M., Kunes, P., Lonsky, V., Mandak, J., Andrys, C., Jankovicova, K. and Krejsek, J. (2008). Early
Expression of Fc γ RI (CD64) on Monocytes of Cardiac Surgical Patients and Higher Density of Monocyte Anti-Inflammatory
Scavenger CD163 Receptor in “On-Pump” Patients. Mediators of Inflammation, 2008, pp.1-6.
Kowal, K., Silver, R., Sławińska, E., Bielecki, M., Chyczewski, L. and Kowal-Bielecka, O. (2011). CD163 and its role in
inflammation. Folia Histochem Cytobiol., 49(3), pp.365-374.
Kristiansen, M., Graversen, J. and Jacobsen, C. (2001). Identification of the haemoglobin scavenger receptor. Nature, 209, pp.198-
201.
Langlois, M. and Delangue, J. (1996). Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem, 42,
pp.1589-600.
Law, S., Micklem, K., Shaw, J., Zhang, X., Dong, Y., Willis, A. and Mason, D. (1993). A new macrophage differentiation antigen
which is a member of the scavenger receptor superfamily. European Journal of Immunology, 23(9), pp.2320-2325.
Leon, F., Sanchez, L., Camarero, C. and Roy, G. (2005). Immunopathogenesis of celiac disease. Immunology, 24(3), pp.313-325.
Levy, A. (2004). Haptoglobin: a major susceptibility gene for diabetic cardiovascular disease. Israel Med Assoc J, 6(5), pp.308-10.
Locksley, R., Killeen, N. and Lenardo, M. (2001). The TNF and TNF Receptor Superfamily: integrating mammalian biology. Cell,
104(4), pp.487-501
Marsh, M. (1992). Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to
the spectrum of gluten sensitivity ('celiac sprue'). Gastroenterology, (102), pp.330-354.
Matysiak-Budnik, T., Moura, I., Arcos-Fajardo, M., Lebreton, C., Menard, S., Candalh, C., Ben-Khalifa, K., Dugave, C., Tamouza,
H., van Niel, G., Bouhnik, Y., Lamarque, D., Chaussade, S., Malamut, G., Cellier, C., Cerf-Bensussan, N., Monteiro, R. and
Heyman, M. (2008). Secretory IgA mediates retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease.
Journal of Experimental Medicine, 205(1), pp.143-154.

47. 42
Moestrup, S. and Møller, H. (2004). CD163: a regulated hemoglobin scavenger receptor with a role in the anti‐inflammatory
response. Annals of Medicine, 36(5), pp.347-354.
Moller, H. (2002). Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma. Blood, 99(1),
pp.378-380.
Moniuszko, M., Bodzenta-Lukaszyk, A., Kowal, K., Lenczewska, D. and Dabrowska, M. (2009). Enhanced frequencies of
CD14++CD16+, but not CD14+CD16+, peripheral blood monocytes in severe asthmatic patients. Clinical Immunology, 130(3),
pp.338-346.
Onofre, G., Kolackova, M., Jankovicova, K. and Krejsek, J. (2009). Scavenger Receptor CD163 and Its Biological Functions. ACTA
MEDICA, 52(2), pp.57-61.
Otterbein, L., Bach, F. and Alam, J. (2000). Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein
kinase pathway. Nat Med, 6, pp.422-8.
Philippidis, P., Mason, J. and Evans, B. (2004). Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and heme
oxygenase-1 synthesis. Circ Res, 94, pp.119-26.
Pioli, P. (2004). TGF- regulation of human macrophage scavenger receptor CD163 is Smad3-dependent.Journal of Leukocyte
Biology, 76(2), pp.500-508.
Polek, T., Talpaz, M., Darnay, B. and Spivak-Kroizman, T. (2003). TWEAK Mediates Signal Transduction and Differentiation of
RAW264.7 Cells in the Absence of Fn14/TweakR: EVIDENCE FOR A SECOND TWEAK RECEPTOR. Journal of Biological
Chemistry, 278(34), pp.32317-32323.
Radzrun, H., Kriepe, H., Bodewadt, S., Hansmann, M., Barth, J. and Parwaresch, M. (1987). Ki-M8 monoclonal antibody reactive
with intracytoplasmic antigen of monocyte/macrophage lineage. Blood, 69, pp.1320-1327.
Rassias, J., Hintz, K. and Wardwell, K. (2002). Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-
regulation of the monocytes hemoglobin scavenger receptor CD163.Leukoc Biol, 72, pp.711-17.
Sarrias, M., Gronlund, J., Padilla, O., Madsen, J., Holmskov, U. and Lozano, F. (2004). The Scavenger Receptor Cysteine-Rich
(SRCR) Domain: An Ancient and Highly Conserved Protein Module of the Innate Immune System. Critical Reviews in Immunology,
24(1), pp.1-38.
Schaer, D., Boretti, F. and Hongegger, A. (2001). Molecular cloning and characterization of the mouse CD163 homologue a highly
glucocorticoid-inducible member of the scavenger receptor cysteine-rich family. Immunogenetics, 53, pp.170-7.
Schuppan, D., Esslinger, B. and Dieterich, W. (2003). Innate immunity and coeliac disease. The Lancet, 362(9377), pp.3-4.
Strauss, M. and Levy, A. (2008). Regulation of CD163 associated casein kinase II activity is haptoglobin genotype dependent. Mol
Cell Biochem, 317(1-2), pp.131-135.
Sulahian, T., Hintz, K., Wardwell, K. and Guyre, P. (2001). Development of an ELISA to measure soluble CD163 in biological
fluids. Journal of Immunological Methods, 252(1-2), pp.25-31.
Sulahian, T., Högger, P., Wahner, A., Wardwell, K., Goulding, N., Sorg, C., Droste, A., Stehling, M., Wallace, P., Morganelli, P. and
Guyre, P. (2000). HUMAN MONOCYTES EXPRESS CD163, WHICH IS UPREGULATED BY IL-10 AND IDENTICAL TO
p155. Cytokine, 12(9), pp.1312-1321.
Tiemessen, M., Jagger, A., Evans, H., van Herwijnen, M., John, S. and Taams, L. (2007). CD4+CD25+Foxp3+ regulatory T cells
induce alternative activation of human monocytes/macrophages. Proceedings of the National Academy of Sciences, 104(49),
pp.19446-19451.
Timmermann, M. and Hogger, P. (2005). Oxidative stress and 8-iso-prostaglandin F induce ectodomain shedding of CD163 and
release of tumor necrosis factor-α from human monocytes. Free Radical Biology and Medicine, 39(1), pp.98-107.
Thom, S., Longo, B., Running, A. and Ashley, J. (2009). Celiac Disease. The Journal for Nurse Practitioners, 5(4), pp.244-253.
University of Maryland Medical Center, (2010). Researchers Find Increased Zonulin Levels Among Celiac Disease Patients. [Online]
Available at: http://umm.edu/news-and-events/news-releases/2000/researchers-find-increased-zonulin-levels-among-celiac-disease-
patients [Accessed Oct. 2014].
van Bruggen, R., Fabriek, B., Deng, D., Ligtenberg, A., Nazmi, K., Schornagel, K., Vloet, R., Dijkstra, C. and van den Berg, T.
(2009). The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria. Blood, 113(4), pp.887-892.

48. 43
Van den Heuvel, M., Tensen, C. and van As, J. (1999). Regulation of CD163 on human macrophages: cross-linking of CD163 induces
signalling and activation. J Leukoc Biol, 66, pp.858-866.
Van Gorp, H., Delputte, P. and Nauwynck, H. (2010). Scavenger receptor CD163, a Jack-of-all-trades and potential target for cell-
directed therapy. Molecular Immunology, 47(7-8), pp.1650-1660.
Vila, J., Padilla, O. and Arman, M. (2000). The scavenger receptor cysteine-rich super-family (SRCR-SF). Structure and function of
group B members,. Immunologia, 19(4), pp.105-21.
Vloet, R., Møller, H., Fabriek, B., van Winsen, L., Hanemaaijer, R., Teunissen, C., Uitdehaag, B., van den Berg, T. and Dijkstra, C.
(2007). Proteolytic shedding of the macrophage scavenger receptor CD163 in multiple sclerosis. Journal of Neuroimmunology,
187(1-2), pp.179-186.
Wagener, F. (2003). Different Faces of the Heme-Heme Oxygenase System in Inflammation.Pharmacological Reviews, 55(3),
pp.551-571.
Wardwell, K., Sulahian, T., Pioli, P. and Guyre, P. (2004). Cross-linking of Fc R triggers shedding of the hemoglobin-haptoglobin
scavenger receptor CD163. Journal of Leukocyte Biology, 76(1), pp.271-277.
Weaver, L., Pioli, P., Wardwell, K., Vogel, S. and Guyre, P. (2007). Up-regulation of human monocyte CD163 upon activation of
cell-surface Toll-like receptors. Journal of Leukocyte Biology, 81(3), pp.663-671.
Weinstein, M. and Segal, H. (1984). Uptake of free hemoglobin by rat liver parenchymal cells. Biochemical and Biophysical Research
Communications, 123(2), pp.489-496.
Wiley, S., Cassiano, L., Lofton, T., Davis-Smith, T., Winkles, J., Lindner, V., Liu, H., Daniel, T., Smith, C. and Fanslow, W. (2001).
A Novel TNF Receptor Family Member Binds TWEAK and Is Implicated in Angiogenesis. Immunity, 15(5), pp.837-846.
Williams, L., Jarai, G., Smith, A. and Finan, P. (2002). IL-10 expressing profiling in human monocytes. J Leukoc Biol, 72, pp.800-
809.
Zwadlo, G., Voege, R., Osthoff, K. and Sorg, C. (1987). A Monoclonal Antibody to a Novel Differentiation Antigen on Human
Macrophages Associated with the Down-Regulatory Phase of the Inflammatory Process. Expl Cell Biol, 55(6), pp.295-304.