INJTRODUCTION

INTRODUCTION

The healthy human body is equipped with powerful set of tools to provide protection

from the diverse onslaught of microbes, foods and other ingested products

(Atkins, 2010). Unfortunately, this set ofmechanisms, known as the immune system,

sometimes goes awry and attacks the body itself. Anti-self responses are usually

generated in the process of mounting an immune response to foreign antigens, but

autoimmune disease results only if autoimmunity IS poorly regulated

(Schwartz, 2004). These breakdowns of immunological tolerance are referred to as

autoimmunity, which can be demonstrated by the presence of autoantibodies or T

lymphocytes reactive with host antigens (Abbas, 2004). Autoimmunity is present in

everyone to some extent. Autoimmune diseases are thus, defined when the

progression from benign autoimmunity to pathogenic autoimmunity occurs.

A combination of genetic predisposition and enviromnental factors contribute to the

development of autoimmune disease (Maniati, 2008) (Figure 1 ). Autoimmune disease

occurs when the autoimmune response to self constituents has damaging effects of a

structural or functional character. The concept of autoimmunity as the actual cause of

human illness (rather than a consequence or harmless accompaniment) can be used to

establish criteria that define a disease as an autoimmune disease (Alarcon-Segovia D,

2006).

Autoimmune disease is a clinical syndrome caused by the activation of autoreactive

T cells and B cells, or both, in the absence of an ongoing infection or other discernible

cause (Davidson, 2001). Activation-induced T cell death is cell cycle dependent

(Fotedar, 1995; Zhang, 2004). However, an accumulation of activated T cells resistant

to proliferation and apoptosis was also found in lupus conditions (Wither, J.E, 2000).

Autoimmune disease may arise either from defective elimination of autoreactive T or

B cells, resulting in tissue destruction, or from defective clearance of apoptotic cells

displaying autoantigens on their cell surface (Lauber, 2004). The self reactive T cell

expresses high levels of cyclin-dependent kinases inhibitors which induces a state of

"replicative senescence" and transcribes genes encoding the proinflammatory

cytokines in autoimmunity (Lawson BR, 2004).

f) \)

Immune regulation

Introduction

Figure 1. The influence of environmental, pathogens, genetic factors and

immunological factors can trigger autoimmunity in genetically predisposed individuals

under conditions of immune dysregulation (Fathman, 2005).

The cell cycle and apoptosis are opposing biological phenomena, however tolerance

play an important role in the development of autoimmunity by affecting apoptosis

(Mueller, 201 0). Mechanisms of self-tolerance, defined as a state of non­

responsiveness to self, can be divided into central and peripheral tolerance. In central

tolerance, immature lymphocytes in the bone marrow (B cells) and thymus (T cells)

that recognize self-antigens with high affinity die by apoptosis or programmed cell

death (Gregory, 2004). In peripheral tolerance, mature self-reactive lymphocytes are

inactivated, killed or turned off by regulatory mechanisms including functional

anergy, Ignorance and suppression by regulatory T cells (Schwartz, 2004). The

lymphocytes escaping the tolerance and defects in tolerance have a likelihood of

developing autoimmune diseases. Accumulation of activated T and B cells with

defective tolerance and resistant to proliferation and apoptosis is found in lupus

conditions (Wither, J.E, 2000).

Impaired apoptosis is also considered one of the key mechanisms in the pathogenesis

of autoimmune disease (Mount, 1994). Studies have elucidated several important

mechanisms underlying the maintenance of immune tolerance and defective apoptotic

pathways in autoimmune diseases (Maniati, 2008). An impaired apoptosis of PBMCs

2

Introduction

were seen in autoimmune patients suggesting a defective clearance and deregulated

expression of regulatory proteins in PBMCs (Harris, 2000; Peng, 2009). The changes

in the apoptotic cell death process, resulting in inappropriate cell death or survival or

disturbances in clearing apoptotic cells, are thought to be involved in the pathogenesis

of a number of autoimmune diseases such as rheumatoid arthritis, lupus and

Hashimoto's thyroiditis (Peng, 2005; Lleo, 2008). Evidence has been provided for

defective clearance of apoptotic cells in SLE patients (Ren, 2003 ), and persistently

circulating apoptotic debris was suggested to serve as immunogen for the induction of

autoreactive lymphocytes in these individuals (Maniati, 2008).

Subthreshold • levels of .t•' ...

novel structure

~ Normal clearance of apoptotic material

NORMAL

lN Drug

~ g ;usA CTL

V-. Supra threshold

~ levelsof novel structure

Impaired clearance of apoptolic material e.g. C1 q deficiency

AUTOIMMUNE

Figure 2. The influence of environmental, pathogens, physiological factors and

immunological factors influence on apoptotic cells, which is responsible prerequisite

for triggering an autoimmune response. Clustering of autoantigens in apoptotic

surface structures (Rosen et al, 1999)

The combined action of apoptosis and anergy are results in a state of peripheral

tolerance are altered in autoimmune progression (Mueller, 201 0). Autoimmune

disease may occur only when defective clearance is linked to a modified (aberrant)

response of the macrophage to the unengulfed debris. In contrast, ingestion and

presentation of self-antigen from apoptotic cell corpses under steady-state conditions

may serve to induce immune tolerance (Gregory, 2004). The most common feature of

all autoimmune diseases is the presence of autoantibodies and inflammation,

including mononuclear phagocytes, autoreactive T lymphocytes and plasma cells

3

Introduction

(autoantibody producing B cells) (Fairweather, 2008). The inference that a systemic

disease is autoimmune is based on the presence of autoantibodies and their

localization in diseased tissue of antibody, complement, and T-lyrnphocytes (Rosen,

1999; Fadeel, 2005) (Figure 2).

History and dimensions of autoimmunity

This concept of autoimmunity as the cause of human illness is relatively new, and it

was not accepted into the mainstream of medical thinking until the 1950s and 1960s.

Ehrlich and Morgenroth (1898) proposed that the consequences of the formation of

self-antibodies were so severe "horror autotoxicus" that the immune system

stringently prohibited its occurrence. Although investigators observed a clear

evidence of anti-self-agglutinins, it was not until the 1950s that the general

acceptance of the concept that autoantibodies could cause immune injury (Silverstein,

2001). Autoimmune diseases affect approximately 8% of population out of which

75-78% are women population (Jacobson DL, 1997). Women are known to respond to

infection, vaccination, and trauma with increased antibody production and a more T

helper (Th) 2-predominant immune response, whereas a Th1 response and

inflammation are usually more severe in men (Fairweather, 2008). Autoimmune

diseases that are more prevalent in males usually manifest clinically before age 50 and

are characterized by acute inflammation, the appearance of autoantibodies, and a

proinflammatory Thl immune response. In contrast, female-predominant autoimmune

diseases that manifest during the acute phase, such as Graves' disease and systemic

lupus erythematosus, are diseases with a known antibody-mediated pathology

(Fairweather, 2008).

Autoimmune diseases are among the ten leading causes of death among women in all

age groups up to 65. The bar graph shows the prevalence of the top 10 autoimmune

diseases in the United States in 1996 (Jacobson DL, 1997) (Figure 3)

4

GIOYBS disease

Rheumat>ld crthlltls

Hashfnot>' 5 thl~CidlfS Vrtiligo

Type I dk:l b«es Pemido.ls anemia

Muliple scfelosls Gomerulalephtitls

S~k: Lupus E

Sjoglen Sldnd!ome

Introduction

Figure 3. The rate of particular autoimmune disorders with progression among the

population of developed countries, in which female were much affected as compared

to male population. (Showing the prevalance of top 10 autoimmune disorder in US, 1996,

(Adapted from Jacobson DL, Clin lmmunollmmunopathol, 1997)

Classification and types of human systemic autoimmune disorders

Autoimmune diseases can be classified as organ-specific or non-organ specific

depending on whether the autoimmune response is directed against a particular tissue

like the thyroid in Hashimoto's thyroiditis, or against widespread antigens such as cell

nuclear antigens in lupus (Peng, 2005; Fairweather, 2007). Systemic autoimmune

diseases include SLE, Sjogren's syndrome, scleroderma, rheumatoid arthritis, and

dermatomyositis conditions which tends to be associated with generation of

autoantibodies to antigens which are not tissue specific (Takehara, 2005).

A feature of autoimmune disease is usually referred to as "overlap syndrome". The

concept of "overlap syndromes" arises when autoimmunity comprises various forms

of disease and the development of two or more autoimmune rheumatic diseases in one

patient, the so-called overlap syndromes (Rodriguez-Reyna, 2005). The genetic and

environmental factors that led to these phenomena interact in a complex fashion and

influence the distinct phenotypic characteristics of each patient (Alarc6n-Segovia D,

2006) and some patients have an autoimmune disease sometimes termed "mixed

connective tissue disease," which has features of SLE, scleroderma, and polymyositis

(Maddison, 2000).

5

Introduction

However, systemic lupus erythematosus is an autoimmune connective-tissue disorder

with a wide range of clinical features, which predominantly affects women

(D'Cruz, 2007). A number of environmental triggers and a number of genetic

susceptibilities roles in causing autoimmunity (Fathman, 2005). SLE is polygenic

where multiple genes appear to influence a person's chance of developing lupus when

triggered by environmental factors. HLA region on chromosome 6 mutations may

occur randomly or may be inherited. HLA class I, class II, and class III are associated

with SLE, but only class I and class II contribute independently to increased risk of

SLE (Martens, 2009).

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus

erythematosus (SLE) and unique markers of the immunological disturbances critical

to disease pathogenesis (Pisetsky, 2009). SLE is best known for its array of

antinuclear antibodies, antibodies to many other self-components are well described.

In the case of IgG and clotting factors, it is possible that the true autoantibody target is

cell bound in the form of immune complexes or of activated clotting factors

(Kavai, 2008). In the form of immune complexes, anti-DNA autoantibodies can

deposit in the tissue to incite inflammation and damage; in addition, these complexes

can induce cytokine production, most prominently, type 1 interferon (Pisetsky, 2009).

The lymphoproliferative and autoimmune disorder occurs in a primary form, not

associated with a rheumatic disease, and as a complication of rheumatoid arthritis,

SLE, or scleroderma. Patients develop infiltration of exocrine glands, mostly salivary

and lachrymal glands, with activated polyclonal CD4 + T cells, together with

hypergammaglobulinemia, autoantibodies, and sometimes vasculitis (Price EJ, 1995).

Multiple sclerosis (MS) is an autoimmune disorder characterized by multifocallesions

of the CNS myelin and accumulating clinical signs due to axonal damage

(Keegan, 2002). Myelin is damaged due to an immune attack consisted of several

pathways and molecules, leading to impaired nerve function (Grigoriadis, 2006).

Autoantibodies and autoreactive T cells activated against myelin antigens myelin

basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte

glycoprotein (MOG), have been detected in MS patients (Garren, 1998). It is

considered that MS as a CD4+T-helper 1 (Thl)-mediated inflammatory demyelinating

disease (Hafler, 2004).

6

Introduction

Primary Sjogren's syndrome (SS) is an autoimmune disorder associated with

lymphocytic infiltration of salivary and lachrymal glands and systemic production of

autoantibodies (Tucci, 2005). The presented autoantigens are mainly SSA/Ro,

SSB/La, a.-fodrin and ~-fodrin, or cholinergic muscarinic receptors. Help from

CD4 cells leads to production of specific autoantibodies. Anti-SSA/Ro antibodies

may be detected alone, whereas anti-SSB/La antibodies are always found in

conjunction with anti-SSA/Ro, suggesting a spreading of the autoantibody response

(Gottenberg, 2003).

Rheumatoid arthritis (RA) is a common chronic inflammatory polyarthritis of

worldwide distribution, with a female predominance of 3:1 and a peak onset in the

fourth decade of life. Intense inflammation occurs in synovial joints, so that the

normally delicate synovial "membrane" becomes infiltrated with mononuclear

phagocytes, lymphocytes, and neutrophils. An essential feature of the rheumatoid

nodule, as with the joint lesion, is tissue destruction (Highton, 2007). Autoimmune

diseases of joints are marked by inflammation, followed by generation of increased

amounts of collagen in skin and viscera, accompanied by certain antinuclear

antibodies (Clements PJ, 2000). In rheumatic disease, probing the transcriptome of

circulating immune cells has shed light on mechanisms underlying the pathogenesis

of complex diseases, such as systemic lupus erythematosus (Bauer JW, 2009).

Analysis of relevant signaling pathways at the protein level is an important step

towards understanding the functional consequences of aberrant gene expression.

Scleroderma is characterized by marked vascular abnormalities, the most dramatic of

which is the episodic reduction in peripheral arterial perfusion (often provoked by

cold temperatures) known as Raynaud's phenomenon which causes an impairment of

circulation which can lead to pain, infections, and frequent ischemic amputation of the

distal fmgertips (Anderson, 2007).

Apoptosis

Apoptosis is a highly regulated process that faci litates deletion of unwanted or

damaged cells in various circumstances of stress and plays a fundamental role in the

maintenance of tissue homeostasis (Fadeel, 2005). Apoptosis elicits specific cell

surface changes, such as the exposure of phosphatidylserine, present on the

7

Introduction

intracellular aspect of the cell membrane, resulting in the swift uptake and degradation

of apoptotic cells either by local resident cells or infiltrating phagocytes

(Stuart, 2002). This process is very rapid such that apoptotic cells are conspicuously

absent in normal tissues. Furthermore, cell deletion by apoptosis leading to clearance

by professional phagocytes such as macrophages is not associated with

proinflammatory mediator release but rather causes release of anti-inflammatory

agents (Green DR. 2000). The mechanisms whereby macrophages and semi­

professional phagocytes (including mesangial cells) recognize and ingest apoptotic

cells are complicated and may involve numerous molecules including the vitronectin

receptor (rr.Jh integrin), CD36, thrombospondin, the phosphatidylserine receptor, the

first component of complement Clq, jJ2 thrombomodulin, class A scavenger

receptors, etc (Savill, 2002 ; Platt N, 2000).

Phases of apoptotic death

Apoptotic cell death can be divided into a triggering phase (ligation of "dedicated

death receptor" such as Fas, or withdrawal of growth/survival factors), a signaling

phase (protein kinase cascades that include MAPK family, JNK and p38), an

execution phase (activation of caspases and nucleases), and a burial phase

(phagocytosis of dying cells by neighboring cells) (Utz PJ, 1998).

Apoptotic impairment in autoimmunity

Autoimmune disease arise either from defective elimination of autoreactive T or B

cells, resulting in tissue destruction, or from defective clearance of apoptotic cells

displaying autoantigens on their cell surface (Lauber, 2004) . However, the immune

homeostasis and immune tolerance are dependent on apoptosis induction and the

rapid clearance of cell corpses, in peripheral and central lymphoid organs

(Siegel, 2000). The removal of apoptotic cells in organism by macrophages and

nonprofessional phagocytes receptors promote phagocytosis by enhancing binding to

the apoptotic cell. Phagocytic defects have also been linked to autoimmune

conditions; macrophages from patients with systemic lupus erythematosus (SLE)

show defects in clearance of apoptotic cells (Tas, 2006; Maniati, 2008).

A major direction in apoptosis research related to autoimmunity concerns aberrations

in the disposition, clearance of apoptotic debris (Gaipl US, 2005). A large numbers of

8

Introduction

apoptotic cells are generated continuously which are efficiently engulfed by

macrophages that recognize them via a battery of receptors. These receptors includes

CD14, PS, PS-R, together with the integrin ocv133, CD36/vitronectin, a receptor for

phosphatidylserine, class A scavenger receptor SR-A, the ATP-binding cassette

transporter, ABC-1 , MER and the oc-2-macroglobulin receptor, CD91. Additional

intermediate factors including thrombospondin, C 1 q, C-reactive protein which may

opsonise apoptotic cells or function in bridging macrophage and apoptotic-cell

surfaces (Savill, 2002; Devitt, 2003). (Figure 4)

Mer

ICAM3

c RecoenHIOn of non-dNctqlll

N~ Rmew. ftmmunolog;

Figure 4. Receptors that are expressed by the apoptotic cells and the phagocytes for

the purpose of clearance mechanisms.

A. Innate recognition of non-self involves phagocyte CD14, i3 2-integrins (which bind the

opsonic complement fragment inactivated C3b, iC3b) and the CD91-calreticulin complex

(which can bind the first component of complement, C1q, and mannose-binding lectin,

MBL, which recognizes pathogen-like apoptotic-cell-associated molecular patterns)

B. Recognition of altered-self involves an array of scavenger receptors, including the class­

A scavenger receptor (SRA), CD68, LOX1 (oxidised low-density lipoprotein receptor 1)

and C036, which recognize oxidised sites on apoptotic cells that mimic oxidised low­

density lipoprotein (oxLDL).

9

Introduction

C. Recognition of non-detaching self involves disabling the detachment signals that are

conferred by apoptotic-cell CD31 and, possibly, similar alterations in another

immunoglobulin-superfamily member, intercellular adhesion molecule 3 (ICAM3). Disabled

apoptotic-cell CD31 binds tightly to phagocyte CD31 , which may promote binding of the

bridging protein thrombospondin-1 (TSP1) by phagocyte integrins. (Savill, 2002)

Defects in clearance lead to exposure of autoantigens, which subsequently results in

autoimmune diseases, however the engulfment process is a potential therapeutic

target. The process of corpse removal occurs via a series of recognizable steps.

Initially, a phagocyte binds to the apoptotic cell, leading to specific recognition of the

target, phagocytic cup formation and sustained signaling that induces actin

rearrangement and phagocytosis. Ultimately, the apoptotic cell is completely

internalized, and its corpse is processed for degradation (Kinchen, 2007).

Immunization of normal mice with large amounts of apoptotic cells leads to mild and

short-lived autoimmunity, indicating the potential immunogenicity of apoptotic debris

(Mohan, 2001 ). Complement is important in the clearance of apoptotic debris

(Navratil, 2000). The absence ofCR2 converts the mildly autoimmune B6/lpr strain to

a strain with severe lupus-like disease, probably reflecting impaired handling of

apoptotic debris as do B6/lpr mice lacking C4 (Einav, 2002). Humans with Clq

deficiency develop SLE almost uniformly, as do mice in which the Clq gene is

deleted (Botto M., 2001 ). The well-known association between SLE and complement

deficiency may reflect the role of the classical pathway in disposing of apoptotic

corpses (Waldport MJ, 2001). A number of signals distinguish apoptotic cells from

normal cells and a significant number of phagocyte receptors have been identified to

be involved in sensing the presence and the uptake of apoptotic cells (Savill, 2002;

Henson PM, 2006)

Apoptotic impaired cells express potential autoantigens on their cell

surface

Apoptosis acts to preserve peripheral T cell homeostasis, participating m the

elimination of both immature thymocytes during thymic development and mature

peripheral T cells following antigen stimulation (Janssen, 2000; Zandman-Goddard,

2002). The combined action of apoptosis and anergy are results in a state of peripheral

tolerance but this balance is altered in autoimmune progression (Abbas, 2004).

10

Introduction

Apoptosis contributes to autoimmune phenomena via the failure to terminate immune

responses and to control autoreactive lymphocytes (Fadeel, 2005). A significant number

of investigations have linked the failure to achieve programmed cell death or to clear

apoptotic cells induces autoimmunity (Fadeel, 2005; Maniati, 2008). Although defects

in apoptosis propagate autoimmunity and disease susceptibility, however a breakdown

of multiple immunoregulatory mechanisms is required for full disease penetrance. The

disease phenotype generated by targeted deletion of the Clq gene and impaired

clearance played an important role in apoptotic cell clearance (Figure 5).

•· NllriiWII conditions

b. Autoimmunity/Mer chfldenqt

Low flux

High flux

Mor

SR-A

CDl6

• Robvit production of antl·

.............. lnfl•-•tory

......,.. cytollnH

•Tcall•lcall toleranu

• LowerptVduction ofantl· lnfl<tmmotory cytotlnes

• T call I I cell tolerance

• Pro-Immune .,_..,tatioft of self· antigen

• Autoimmunity

Figure 5. Impaired clearance of apoptotic cells cause autoantigens to initiate systemic

autoimmunity. (Rosen eta/., 2001, Nature Med Reviews)

In autoimmunity, a buildup of non-phagocytosed apoptotic cells was occurs and

consequently undergoes modification of antigen structure. Thus a pro-immune

presentation of the self-antigen induces an autoimmune response (Rosen, 2001,

Mitchell DA, 2002).

11

Introduction

The ongm of the autoantibodies typically present in the sera of patients with

autoimmune conditions such as SLE (often directed towards intracellular antigens

such as DNA, ribonucleoproteins and nucleosomes) has been perplexing as these

autoantigens are normally invisible to the immune system because of their localization

within the cell (Stuart, 2002). Cells undergoing apoptosis exposes potential

autoantigens upon their cell surface which make them available for interaction with

immunologically competent cells (Maniati, 2008). The autoantigens expressed by

apoptotic cells are immunogenic and altered self motifs developed by undergoing

various post-translational modifications (Eggleton, 2008). Cleavage by granzyme B

(GrB) is predictive for autoantigenicity of self-proteins, thereby further implicating

CTL-induced death in the initiation and propagation of autoimmunity (Darrah, 2010).

However, unique modification of potential intracellular autoantigens raises the

possibility that cytotoxic lymphocyte-mediated death of target cells may play a

specific role in the development of autoimmunity (Niland B, 20 I 0).

A number of protein autoantigens have been shown to have undergone

post-translational modification (Doyle, 2001 ). Rheumatoid arthritis patients frequently

have antibodies to citrullinated proteins, which can be demonstrated in inflamed

synovium (Baeten, 2001). Many SLE-associated autoantigens undergo cleavage

during apoptosis, and it has been argued that this process and also phosphorylation

render these proteins immunogenic (Utz, 2000). Autoimmunity may reflect epitope

spreading, that is, the recruitment ofT cells reactive to additional epitopes on the

autoantigen (Anderton, 2002).

Tolerance usually does not extend to all epitopes, however a mechanism capable of

enlisting progressively more autoreactive T cells might amplify any initial breakage

of tolerance (Ho, 2006). B cells expressing antibody cross-reactive with self­

determinants could then selectively take up autoantigens, process them, and express

autoantigenic peptides, including cryptic epitopes (Farris, 2000).

12

Introduction

Figure 6. The sequestration of various autoantigens from the apoptotic cells which

have the potential to induce an inflammatory response due to dysfunctional clearance

mechanisms. A cell that is undergoing apoptosis releases debris containing molecules that

have localized in surface blebs. Abundant evidence indicates that these molecules travel to

where they can stimulate the formation of autoantibodies. Other evidence shows that certain

structural features of these molecules, such as coiled-coils, contribute to the probability that

they will induce an immune response (Plotz, 2003).

Defective clearance of apoptotic cells induces autoimmunity

Defects of apoptotic pathways in T cells promote the survival of potentially

autoreactive, proinflammatory cells (Pamaik R, 2000). Failure to eliminate activated

cells can result in prolonged effector functions, such as CD40 ligand for B cells,

inappropriate survival of primed autoantibody-producing B cells, or cytokine release

by macrophages (Maniati E, 2008).

A common feature of autoimmune diseases is the breakdown of tolerance of self

antigens, a consequence of which is the production of autoantibodies reactive with

multiple self proteins. Apoptosis acts as a source of immunogens (Nagata 2010,

Maniati E, 2008). It has been speculated that highly accelerated rates and/or abnormal

sites or abnormal processing of apoptotic cells could lead to autoantibody production.

The elucidation of the biochemical pathways and specific proteins that regulate

apoptosis provide a remarkable opportunity to manipulate the fate of the cell

(Stuart L et a!., 2002).

13

Introduction

Diminished clearance of apoptotic

' cells e.g. C1 q Autoantigen deficiency presentation by dendritic cells

' t Autoimmune

/ Disease

Facilitation of autoimmune

Diminished

/' responses apoptosis of autoreactive lymphocytes e.g. Fas mutation

Stuart L et a/ , 2002

Figure 7. The mechanisms responsible for autoimmune disorder (Stuart eta/., 2002)

Figure 8. This figure shows the possibility that the encounter leads also to the entry of

the tRNA synthetase into the immature DC and the maturation of the immature DC into

a mature APC, probably with the help of other local cytokine signals. The synthesis of

autoantibodies, shown on the right, might follow conventional T-cell-dependent or -

independent pathways. APRIL, a proliferation-inducing ligand; BAFF, B-cell-activating

factor of the tumour-necrosis factor family; BAFFR, BAFF receptor; PRR, pattern­

recognition receptor; TLR, Toll-like receptor (Plotz, 2003).

14

Introduction

The molecules that elicit an autoantibody response are likely to do so in the same

manner as does foreign antigen. Presumably, this involves interaction with the surface

of an immature dendritic cell (DC) or other antigen-presenting cell (APC).

Autoantigens have shown chemoattractive for immature DCs are histidyl-transfer

RNA synthetase (His-RS), through interaction with CC-chemokine receptor 5

(CCR5); and asparaginyl-tRNA synthetase (Asn-RS), through interaction with CCR3

(Plotz, 2003).

The failure to achieve programmed cell death or to clear apoptotic cells causes the

autoimmune phenomena via two ways. The failure to terminate immune responses

and to control autoreactive lymphocytes and other is the exposure of self-antigens

in an inflammatory context that can initiate immune responses against

them (Fadeel, 2005). Although, defects in apoptosis propagate autoimmunity

and significantly contribute to disease susceptibility, a breakdown of

multiple immunoregulatory mechanisms is required for full disease penetrance

(Maniati, 2008). Apoptotic cells have been proposed as a possible source of nuclear

SLE autoantigens (Plotz PH, 2003). A defect in the clearance machinery and a

subsequent overload of apoptotic cells is a potential mechanism for the breakdown of

self-tolerance in SLE (Potter PK, 2003). The generation of the autoimmune response

is that non-ingested apoptotic cells undergo secondary necrosis, favoring responses

against self-antigens (Fadeel, 2005).

Tumour suppressor protein "p53"

In order to elucidate the mechanism responsible for apoptotic impairment, p53 gene

was studied, as p53 is the apoptosis inducing gene and p53 triggers the apoptotic

phenomenon. A significant number of p53 inducible genes are under expressed and a

number of p53 repressible genes are over expressed in autoimmune PBMCs

(Aune et al., 2004). The p53 tumor suppressor gene, a nuclear transcription factor,

plays an important role in the control of cell proliferation and apoptosis. The pivotal

· role of p53 in maintaining genomic integrity has earned it the nickname "guardian of

the genome" (Vogelstein, 2000). The consensus DNA-binding sequence for p53

consists of two repeats of the 10 bp motif 50-PuPuPuC(A/T)(A/T)GPyPyPy-30

separated by 0-13 bp ( elDiery, 1992 ). Activation of p53 by cellular stresses such as

DNA damage, hypoxia and oncogene activation leads to the induction of cell cycle

15

Introduction

arrest or apoptosis. Transactivation of the p53 target genes p21, GADD45 and 14-3-3

is involved in mediating cell cycle arrest by p53 ( elDeiry, 1995; Kastan 1992;

Hermeking, 1997; Chan 2000). Transcriptional repression by p53 has been shown be

important for the promotion of apoptosis (Ho, 2003). Targets of p53 mediated

repression include cyclin B1, b-tubulin, Map4, survivin, AFP and POLD1 genes

(Innocente, 2005; Li, 2001).

Mutational inactivation of p53

Once p53 dependent mechanisms are broken, conditions for the rapid accumulation of

genetic changes are established. The most common changes to the p53 gene are

missense point mutations within the coding regions. Over 80% of point mutations are

localized within the specific DNA binding domain of the p53 protein (Blagosklonny,

2000). A large number of p53 point mutations are have a dominant negative effect,

that transform the cells by acting as inhibitory proteins competing with and blocking

the activity of endogenous wild type p53 protein. Dominant negative p53 mutants

possesses an incorrect protein conformations and produce inactive mixed tetramers

with wild type p53 (Chan et al., 2004). Inactivation of wild-type p53 by mutant p53 is

generally thought to inhibit p53 binding to DNA (Nicholls et al., 2002; Willis et al.,

2004). Besides losing p53 function, some mutant p53 proteins acquire a novel

function that contributes to transformation and oncogenic potential by mode of "gain

of function" mutants (Dittmer et al., 1993; Blagosklonny, 2000).

Non-mutational inactivation of p53

Whenever wild type p53 does not retains its active pathways and any mutation if not

found, then there is always a possibility of its inactivation either by non-mutational

pathways. A range of various posttranslational modifications like phosphorylation,

acetylation, ubiquitination etc also decides the functionality as well as inactivation of

p53 protein (Coutts, 2009). Some of the normal cellular functions of p53 can be

modulated and sometimes inhibited by interaction with viral oncoproteins like SV 40

large T antigen, adenovirus ElB, HPV E6 protein or with the cellular protein E3

ligase Mdm2. Inactivation of p53 also includes ARF through gene deletion, promoter

methylation and aberrant subcellular localization of p53 (Puisieux et al., 2006).

16

Introduction

p53 ubiquitination

In normal cells, p53 is present at extremely low levels because the protein is very

rapidly degraded following synthesis. p53 is subject to a variety of post-translational

modifications, including phosphorylation, sumoylation, acetylation, methylation and

ubiquitination, which all impact significantly on p53 activity (J.P. Kruse, 2008). A

pictorial representation of p53 protein showing the domains and amino acids involved

with ubiquitination.

However, p53 can be modified by several E3 ubiquitin ligases, including MDM2,

CHIP, Pirh2, COPl , ARF binding protein and E6AP (Marine, 2010; Tripathi et al,

2007; Brooks, 2006; Yang, 2004).

p53

1-100 101-300 301-393

Transactivation DNA binding Neg

Ill rv

~p60 Mdm2

P300/CBP

Mdmx

Figure 9. Showing the domains of mdm2 and p53 involved with protein-protein

interactions and sites of p53 ubiquitination (Coutts, 2009).

One of the important components of the p53 degradation pathway is Mdm2. Mdm2

tightly and affects its cellular localization, stability and activity through a variety of

mechanisms (Marine, 2010). Mdm2 is an E3 ligase of p53 and itself the product of

p53 inducible gene (Honda, 2000; Fang, 2000; Yang, 2004). It binds toN-terminus of

p53 and inhibits p53 transactivation function as well as induces its degradation by

ubiquitination. Mdm2 monoubiquitinates six lysine residues (370, 372, 373, 381 , 382,

386) at C-terminal region ofp53 (Lai Z, 2001). Ubiquitination ofp53 has emerged as

a mechanism of p53 control which plays important roles in regulating its stability and

activity (Coutts, 2009).

17

Introduction

Relevance of Biomarkers in autoimmunity

The goal of studies of autoimmune disease biomarkers is to identity markers that

fluctuate with disease development and severity, and then normalize following

successful therapy (Casiano, 1998). The development of genomic and proteomic

technologies provides an unprecedented ability to identify novel biomarkers to

diagnose, classify, and guide therapeutic decision making in patients with

autoimmune disease. Peripheral blood is a convenient and rich source of potential

biomarkers, but surveying purified cell populations and target tissues can also

enhance our understanding of disease states (Olsen, 2004).

A biomarker indicates a change in expression or state of a protein that correlates with

the risk or progression of a disease, or with the susceptibility of the disease to a given

treatment. Biomarkers are characteristic biological properties that can be detected and

measured in parts of the body fluids and tissues. They may indicate either normal or

diseased processes in the body. Biomarkers can be specific cells, molecules, or genes,

gene products, enzymes, or hormones (Gibson, 2010). The structural modification

during cytotoxic granule-induced cell death is a frequent and striking feature of

autoantigens, and may be an important principle driving disease. Such autoimmune­

disease-specific arrays include self-antigens, viral proteins and peptides, and bacterial

antigens with complex carbohydrates and recombinant proteins (Fathman, 2005).

The perfect marker could serve as a diagnostic tool, as well as a monitoring device for

therapeutic drug efficacy. Current biomarker discovery efforts are focused on three

groups of proteins reflective ofthe autoimmune disease process:

( 1) Degradation products arising from destruction of affected tissues,

(2) Enzymes that play a role in tissue degradation,

(3) Cytokines and other proteins associated with immune activation (Prince, 2005).

A list showing the serum biomarkers that are overexpressed during autoimmune

condition development and progression and induces an immune response against the

secreted proteins is shown below (Table 1 ).

18

Introduction

Tablet. List of serum biomarker proteins expressed in various

autoimmune disorder conditions.

Autoimmune Disease Biomarker Antigen Humoral response Reference SLE IFN-alpha Autoantibodies Kirou, 2005

Pro-inflammatory mediators

SLE Nephrities components of Autoantibodies Rovin, 2007

complement (C3 , C4), to C3 and C4,

double stranded DNA anti-double stranded DNA antibodies (ADNA).

SLE andMCTD 70 kDA, Ribonucleo Auto-antibodies Salmhofer , conditions protein 2007

Hypocomplementemic Cl q Anti-C1q Mok, 2010 urticarial vasculitis, non- autoantibodies SLE active glomerulonephritis

TP53 SLE, Rheumatoid TBP Anti-p53 Chauhan et arthritis, organ-specific autoantibodies al. , 2004 autoimmune diseases, Anti-TBP MCTD/OS autoantibodies

serum C-type Inflammation Olewicz-

Systemic Sclerosis natriuretic peptide Anti -centromere,

Gawlik , (CNP), 2010

anti-Sci 70 antibodies, anti-

Doran, 2008 RNA polymerase III, anti-fibrillarin

Willebrand factor, Endothelial cell Tinazzi, vascular endothelial damage and skin 2010 growth factor, fibrosis intracellular adhesion molecule- I, monocyte chemotactic protein-!

RNA Pol III anti-RNA polymerase III Nihtyanova, antibodies 2010

CD31 was used as a reduction in the Akhmetshina pan-endothelial cell number of , 2010

19

Introduction

marker lymphatic capillaries and lymphatics vessels

CCR5 Delta32 Autoantibodies Martens,

Rheumatoid arthritis 2009

Impaired Tolluso, ZAP-70 ofB cells apoptosis, 2009

immune activation Prolonged Survival

citrullinated alpha- Autoantibodies Lundberg, enolase 2008

circulating TNF- Autoantibodies Corona-alpha and E-selectin and Tissues Sanchez , (sE-selectin) damages 2009

Multiple Sclerosis VLA -4 integrin neuroinflammato Brahamachar ry cascade in i, 2010 CNS

Relapsing-remitting Interferon beta Inflammatory Gandhi, 201 0 (IFNbeta) Signalling

Multiple sclerosis Prolyl oligopeptidase N erurodegenerati Tenorio-(RRMS). (POP) on and Laranga ,

Inflammatory 2010 responses

aquaporin 4 anti -aquaporin 4- Min, 2010

Sjogren's syndrome antibody

Auto-antibodies Tobon, 2010 tyrosine kinase3- mediated chronic ligand damages of

lymphoid tissues

Wegener's membrane-bound anti-neutrophil Westra, 2009 Granulomatosis PR3 (mPR3) cytoplasmic

autoantibodies (AN CAs)

Autoimmune amylase a-2A amylase a-2A JWWiley, pancreatitis (AlP) autoantibodies 2009

Biomarker protein arrays have been constructed and validated for over a number of

autoimmune diseases, including connective-tissue diseases (such as SLE, scleroderma

and myositis), primary biliary cirrhosis, experimental autoimmune encephalomyelitis

and multiple sclerosis, rheumatoid arthritis, diabetes, Crohn's disease, and sclerosing

cholangitis (Fathman, 2005).

20

~ CV) '--~ '-

I

~

Introduction

The systemic autoimmune diseases are a complex group of disorders characterized by

elaboration of high titer autoantibodies and immune-mediated damage of tissues. The

presence of a immune effector pathways drive the generation/release of autoantigens,

which in tum fuel the immune response (Rosen, 1999, 2001; Fadok, 2001). Protein

arrays have been shown to have applications in profiling the antibody repertoire in

healthy individuals and subjects with autoimmune diseases (Cahill, 2001). A humoral

immune response is generated to disease associated antigens during the development

of autoimmune diseases and rendering serum autoantibody detection suitable for

early-stage diagnosis of autoimmune diseases. Analysis of relevant signaling

pathways at the protein level is an important step towards understanding the

functional consequences of aberrant gene expression in autoimmunity.

Autoantigen

Autoantibodies

Tcell Beef!

Figure 10. Discrete components of the autoimmune process from the presentation of MHC-peptide complexes to T cells to the production of autoantibodies by plasma cells analyzed using various genomic and proteomic technologies. The numbers in the figure indicate the technologies that have demonstrated potential in the analysis of the corresponding components. (1) Peptide-MHC tetramer arrays. (2) Reverse-phase protein analysis (3) Multiparameter flow cytometry for intracellular antigens. (4) eDNA analysis and oligonucleotide microarrays. (5) Antibody profiling (6) Bead-based multiplex assays. (7) Autoantigen analysis and profiling (8) Whole­proteome analysis by 20-PAGE. Both mass spectrometry and 2D gel electrophoresis can be used to analyze complex mixtures of proteins and/or peptides (Fathman CF, 2005).

21

Introduction

In this study, the objectives were to identify novel biomarkers which are

overexpressed during autoimmunity. The delineation of such targets are important for

the understanding of mechanisms responsible for autoimmune disorder. By finding

the biomarkers of the diseases, therapy can be elucidated in order to minimize the

pathological severity and diseases spreading. We have implemented 2D-PAGE

proteomics based strategies to find the autoantigens profiles. Direct evidences have

shown defective apoptotic machinery with the development of autoimmune disorder,

however genetic evidences also had shown that defects in individual cell-death genes

can lead to autoimmune disorders. To detect the whether p53 function was impaired

in autoimmune condition, we will investigate the factors responsible for p53

inactivation. Further, to elucidate the mechanisms responsible for apoptotic

impairment in PBMCs of autoimmune patients. The role of overexpressed co­

activators or transcription factors in autoimmunity and their cross-talk with p53

inactivation is of our interest. In concise biomarker protein analyses in autoimmune

disorder and their role in context to inactivation of p53 protein and apoptosis will be

described in the following thesis. We presented here experimental evidence and

mechanisms for autoimmune disorder phenomenon due to aberration of the apoptosis

and p53 inactivation. In view of this, following were the main aims and objectives of

the study undertaken:

22