AUTOIMMUNITY

CONTENTS

 DEFINITION
 HISTORY

 TOLERANCE

 MECHANISMS OF AUTOIMMUNITY

 AUTOIMMUNE DISEASES – CLASSIFICATION

 LABORATORY DIAGNOSIS OF AUTOIMMUNE DISEASES

 TREATMENT
DEFINITION

• Autoimmunity is
 breakdown of the immune system’s ability to discriminate between self and nonself.

 condition in which structural or functional damage is produced by the action of

immunologically competent cells or antibodies against the normal components
of the body.

 5-7 % of adults affected, 2/3rds involving women.

 > 40 human diseases, autoimmune in origin.
 1. The presence of an immune reaction
specific for some self antigen or self
3
REQUIREMENTS tissue.
TO CALL IT 2. Evidence that such a reaction is not
AUTOIMMUNITY :
secondary to tissue damage but is of
primary pathogenic significance.
3. The absence of another well-defined
cause of the disease.
 Serge Metalnikoff (1900) reported that
some animals were able to form
antibodies against their own
spermatozoa.

HISTORY Paul Ehrlich (1901) reported the concept

that an organism's immune system
could attack the organism's own tissue
calling it "horror autotoxicus".
Julius Donath and Karl Landsteiner
(1904)reported autoantibodies can
cause disease by showing that
autoantibodies (‘hemolysins’) caused
paroxysmal cold hemoglobinuria.
• Normally immune system does not
react to its own antigens due to a
protective mechanism called
tolerance .
• Breach in tolerance mechanisms
predisposes to several autoimmune
diseases.
 Autoimmunity results
from the loss of self-
tolerance.

TOLERANCE Types :

• Central Tolerance.
• Peripheral Tolerance.
CENTRAL TOLERANCE
•Immature T cells that recognize self
antigens in the central lymphoid organs,
some
• are killed by apoptosis - Negative
selection or deletion in Thymus.
• escape killing and differentiate into
regulatory T cells.

•In the B-cell lineage, some

• are killed by apoptosis.
•some of the self-reactive lymphocytes
switch to new antigen receptors that are
not self-reactive - Receptor editing in
Bone Marrow.
DELETION OF SELF-REACTIVE T CELLS IN THE
THYMUS:
•Many self antigens are processed and presented by thymic APCs in association
with self MHC.

•Any immature T cell that encounters a self antigen undergoes

deletion/negative selection and the T cells that complete their maturation are
thereby depleted of self-reactive cells

•AIRE (autoimmune regulator) is a regulator of gene transcription that stimulates

thymic expression of many self antigens which are largely restricted to peripheral
tissues .
• Mutations in AIRE gene gives rise
to Autoimmune Polyendocrine
Syndrome in which T cells
specific for multiple self
antigens escape deletion and
attack tissues expressing the self
antigens.
PERIPHERAL TOLERANCE

•Self-reactive T cells that escape negative selection in the thymus can

potentially become weak unless they are deleted
•Several mechanisms in the peripheral tissues occurs such potentially
autoreactive T cells :
• Anergy- functionally inactive .
or
• Suppression by regulatory T-lymphocytes.
or
• Deletion by activation induced cell death by
a. Activation of pro-apoptotic member of Bcl family.
b . Fas-Fas ligand system.
 Anergy -
functional If Signal 2 is not The T cell
inactivation of Activation of T delivered due to becomes
lymphocytes cells requires two weak expression anergic and
induced by signals: of costimulatory cannot respond
encounter with molecules, to the antigen.
antigens.

Signal 1 :
recognition of
Signal 2 :
peptide antigen
costimulatory
in association
signals provided
with self MHC
by the APCs.
molecules on
APCs.
Suppression by regulatory T
cells - The responses of T
Generated by self antigen
cells to self antigens may
recognition in the thymus or
be actively suppressed by
regulatory T cells.

Regulatory T cells are CD4+

cells that express high levels peripheral tissues
of CD25

Mutations in FoxP3 gene are

Generation requires a
the cause of a severe
transcription factor called
autoimmune disease in
Foxp3 gene.
humans.
• The factors that lead to a failure of
self-tolerance and the development
of autoimmunity include :
1. inheritance of
susceptibility - genes that
may disrupt different tolerance
pathways.
2. infections and tissue
alterations that may expose
self-antigens and activate
APCs and lymphocytes in the
tissues.
FACTORS
INFLUENCING
AUTOIMMUNIT
Y
ENVIRONMENTAL FACTORS

 Ultraviolet (UV) radiation causes cell death and may lead to the exposure of
nuclear antigens, which elicit pathologic immune responses in SLE.

 Smoking is a risk factor for rheumatoid arthritis, perhaps because it leads to

chemical modification of self antigens.

 Strong gender bias, with many of these diseases being more common

in women > men due to hormones and other mechanisms.

GENETIC FACTORS

 There is increased expression of Class II HLA

antigens on tissues involved in autoimmunity.
 There is increased familial incidence of some
forms of the autoimmune disorders.
 There is higher incidence of autoimmune diseases in
twins (monozygotic > dizygotic) favouring genetic
basis.
 Most human autoimmune diseases are associated
with multiple genes.
  Polyclonal activation of B cells by stimuli such as infection
with microorganisms & their products bypasses T cell
tolerance.

 Generation of self-reacting B cell clones bypasses T cell

tolerance.
IMMUNOLOGICAL
FACTORS  Decreased T suppressor and increased T helper cell activity
lead to high levels of autoantibody production by B cells
contributing to autoimmunity.

 Sequestered ‘Self-antigens’ may act as ‘foreign-antigens’ if

introduced into the circulation later.
MECHANISMS • BREAK DOWN OF T-CELL ANERGY:
OF
• Normal cells that do not usually express
AUTOIMMUNITY
costimulatory molecules (B7) can be
induced to do so.
• Induction may occur in presence of tissue
necrosis and local inflammation .

• Multiple sclerosis.
• Rheumatoid arthritis.
• Psoriasis.
FAILURE OF AICD:
Failure of autoreactive activated T cells to undergo activation induced
cell death (AICD),
Apoptosis via Fas-Fas ligand can lead to autoimmunity.
• SLE
 Many self Ag Organ damage,
are found
Hidden Ag
in hidden
released ,
location
SEQUESTERED eg:Testes ,Eye Reaches blood
ANTIGENS: (CORNEA) stream,
certain self antigens are
present in closed Encounter Ag
systems and are not sensitive cells ,
accessible to immune Stimulate
apparatus autoimmunity
CROSS –REACTING FOREIGN ANTIGENS

Similarity between some foreign and self antigens is the

basis of the ‘cross-reacting antigen’ theory of
autoimmunity.

Organ specific antigens are present in several species.

Injection of heterologous organ specific antigens may

induce an immune response , damaging the particular
organ or tissue in the host.
• Streptococcal M proteins and the heart muscle shared antigenic
characteristics.
• Immune response induced by repeated streptococcal infection can
therefore damage the heart .
• Nephritogenic strains of streptococci possess antigens found in renal
glomeruli.
• Infection with such strains may lead to glomerulonephritis due to
antigenic sharing
 • Related type of autoimmunisation is
‘molecular mimicry’ which is due to the
presence in some infecting microorganisms
and self-antigens , of epitopes with identical
MOLECULAR peptide sequences.
MIMICRY
• Arthritogenic shigella flexneri and HLA-B27.
POLYCLONAL B CELL ACTIVATION

• Antigen generally activates only its corresponding B cell , certain

stimuli no-specifically turn on multiple B cell clones.
• Epstein Barr virus.

• Polyclonal antibodies are IgM in nature ,similar to the ‘natural

antibodies’ produced by CD5+ B cells.
AUTOIMMUNE DISEASES IN HUMANS

Classificati organ
systemic .
on specific
ORGAN SPECIFIC AUTOIMMUNE DISEASES

Disease Self antigen /Target Immune effector

gene
Addison's disease Adrenal cells Autoantibodies
Autoimmune haemolytic RBC membrane proteins Autoantibodies
anaemia
Goodpasture syndrome Renal and lung basement Autoantibodies
membrane
Graves disease Thyroid stimulating Autoantibody
hormone receptor
Type 1 diabetes mellitus Pancreatic beta cells TH1 cells ,Autoantibodies
Myasthenia gravis Acetyl choline receptors Autoantibody
Post streptococcal Kidney Antigen – antibody
glomerulonephritis complexes
SYSTEMIC AUTOIMMUNE DISEASES

Disease Target gene Immune effector

Ankylosing spondylitis Vertebrae Immune complexes
Multiple sclerosis Brain or white matter TH1 cells ,and Tc cells
Rheumatoid arthritis Connective tissue Autoantibodies ,immune
complexes
Sjogren's syndrome Salivary gland , liver, Autoantibodies
kidney, thyroid
SLE DNA , nuclear protein , Autoantibodies ,immune
RBC complexes

Immune dysregulation Loss of Foxp3 gene Missing regulatory T cells

polyendocrinopathy
enteropathy x-linked
 • Criteria for diagnosis of autoimmune diseases includes:
1.Presence and documentation of relevant
autoantibodies in the serum.
2. Demonstration of T cell reactivity to self
antigen.
LABORATORY 3. Lymphocytic infiltrate in the pathologic lesion.
DIAGNOSIS 4. Production of cytokines by helper T cells e.g.
interferon,IL4
5. Transfer of an autoimmune disease to an
experimental animal by administration of
autoantibodies.
LABORATORY INVESTIGATION OF
AUTOIMMUNE DISEASE
• Routine investigations - Urine examination
• Haematological investigations -routine & special
• Biochemical tests – Liver Function Tests , Renal Function Tests ,Thyroid
Function Tests.
• Demonstration of autoantibodies
• Serological tests .
• More specific serological tests like Rose-Waaler method, Latex
Agglutination, turbidometry , nephelometry , ELISA ,
Immunofluorescence
URINE EXAMINATION

• Proteinuria -glomerular filter malfunctioning .

• Active urine sediments - active glomerular damage- SLE , Vasculitis.
• Haemoglobinuria - acute exacerbation of haemolysis in AIHA.
• Oliguria /anuria- lupus nephritis.
HEMATOLOGICAL INVESTIGATIONS:

• RBC indices
• Normocytic Normochromic- chronic disease
• Macrocytic – pernicious anaemia.
• Features of AIHA –
• Autoagglutination
• Microspherocytes
• Polychromasia , Schistocytes , Tear drop cells .
• WBC –
• Eosinophilia - Churg strauss Syndrome
• Leucopoenia is very common in active SLE.
• Platelet count:
• Decreased in – ITP, SLE, RA , Scleroderma .
• Increased in - Wegner granulomatosis.
LE CELL

• ANA is clearly responsible for the characteristic LE bodies and LE cells.

• Although the auto antibodies cannot penetrate the healthy cells , they can
attack the nuclei of damaged cells.
• LE factor is an IgG antibody directed against deoxyribonucleoprotein .
• This antibody acts on the damaged nucleus producing swelling &
homogenisation of nuclear material-LE bodies.
• LE bodies phagocytosed by neutrophils – Known as LE cell.
• LE cells are demonstrated in SLE, chronic hepatitis, rheumatoid arthritis and
other collagen disorders.
LE CELL
• 1.Autoimmune haemolytic anaemia – diagnosed by coombs test.
Red cells are incubated with an anti-human IgG antiserum.
If IgG auto-antibodies are present on red blood cells ,the cells
are agglutinated by the antiserum.
BIOCHEMICAL TESTS

• LFT:
• Increased Alkaline Phosphatase , Increased Aminotransferase-
sclerosing cholangitis , biliary cirrhosis.
• RFT :
• Urine R & M-to detect protein , sugar, ketones & casts in SLE &
vasculitis.
SEROLOGICAL
TESTS
RHEUMATOID FACTOR

• RF are autoantibodies ( usually IgM )directed against the Fc portion of

the IgG molecule.
• IgG & IgA RF also detected. IgA RF related with more severe disease &
erosion.
• Sensitivity- 80%.
• Specificity is compromised(72%).
• Other conditions associated with RF are :-SLE , Vasculitis, Sjogren's
Syndrome, Chronic Liver Disease , Hepatitis B , Hepatitis C , Sarcoidosis
.
• Techniques for estimating RF :
• Rose Waaler method ( sheep RBC agglutination)-reported as titre eg. 1:32 , 1: 64 .
• Slide agglutination method for qualitative and quantitative detection of RF in
human serum
• Latex agglutination .
• Turbidometry .
• Nephelometry.
• ELISA.
• RA is associated with several autoantibodies, which can serve as
diagnostic and prognostic markers other than RF:
• a) Antikeratin antibodies (AKA)
• b) Antiperinuclear factor (APF)
• c) anti-CCP [anti-cyclic-citrullinated-peptide])
• d) Anti-Sa antibodies, targeting citrullinated vimentin
• Anti-CCP antibodies can be detected in 50% of patients with early RA,
at a time when RF is negative, allowing for improved diagnosis and
early specific treatment .
ANTI NUCLEAR ANTIBODY

• Antinuclear antibodies are antibodies that are directed against several

nuclear antigens and can be grouped into four categories-
• Antibodies to DNA .
• Antibodies to histone .
• Antibodies to nonhistone proteins bound to RNA .
• Antibodies to nucleolar antigens
• Antinuclear antibody (ANA) test a marker of the autoimmune process that is
positive with a variety of autoimmune diseases. It may be positive in
• systemic lupus erythematosus,
• Sjogren's syndrome.
• Rheumatoid arthritis.
• Autoimmune hepatitis.
• Primary biliary cirrhosis.
• Alcohol-related liver disease.
METHODS FOR DETECTION OF AUTOANTIBODIES TO
NUCLEAR AND INTRACELLULAR ANTIGENS

Method antigen Source sensitivity

Iimmunofluorescent Tissue sections, cell lines Sensitive assay-screening
microscopy
Double immunodiffusion Tissue and cell extracts High specificity
Counter- Tissue and cell extracts Increased sensitivity
immunoelectrophoresis
Line immunoassays (dot Recombinant antigens Average sensitivity
blot , linear blot)
ELISA Recombinant antigens High sensitive
INDIRECT IMMUNOFLORESCENCE

• Hep2 Cells ( immortalized, derived from human larynx carcinoma (human

epithelioma type 2 - clone ATCC CCl 23) fixed on glass slides.
• Incubated with diluted patients' sera with a titter of 1 / 80 in pH 7.2 phosphate
buffered saline (PBS) for 30 minutes in a moist chamber at room temperature.
• Then, the plates are washed twice for 10 minutes in PBS and incubated for 30
minutes with secondary antibody conjugated with antigamaglobulina human
fluorescein isothiocyanate (FITC) in the dark at room temperature.
• After incubation, slides are washed in PBS and mounted with buffered
glycerine and cover slip.
• Reading is done with a fluorescence microscope,
• The extent of staining is determined by dilution of serum (1:100, 1:400
and 1:1600) in order to establish the endpoint.
• The antibody patterns and the titres (ie the dilution at which the
staining was still visible) are reported.
• Pattern of nuclear fluorescence suggests the type of antibody present
in the patients serum
FOUR BASIC PATTERNS

• (1)Homogenous or Diffuse nuclear staining pattern - commonly

indicates antibodies to chromatin, histones and occasionally double
stranded DNA .
• (2)Rim or Peripheral staining pattern - commonly indicates antibodies
to dsDNA.
• (3)Speckled pattern - presence of uniform or variable sized speckles.
• This is the most commonly observed pattern of fluorescence and
therefore the least specific. It indicates the presence of antibodies to
non-DNA nuclear constituent eg.Sm antigen, ribonucleoprotein, SS-
A,SS-B
• 4)Nucleolar pattern -presence of a few discrete spots of fluorescence in
the nucleus and represents antibodies to nucleolar RNA.
• This pattern is reported most often in patients of systemic sclerosis.
SPECKLED” PATTERN OF STAINING IS MORE CHARACTERISTIC
OF THE PRESENCE OF AUTOANTIBODIES
THE “NUCLEOLAR PATTERN” OF STAINING IN WHICH THE BRIGHT
FLUORESCENCE IS SEEN WITHIN THE NUCLEOLI OF THE HEP2 CELLS-
PROGRESSIVE SYSTEMIC SCLEROSIS
RIM PATTERN-SLE
 Current therapies to treat autoimmune
diseases fall into two categories :
TREATMENT
1.immunosuppressive treatments
2.more recent mechanisms - cell
type specific strategies.
IMMUNOMODULATORS

• Drugs which modify immune response generally categorized as

immunomodulators:
• Immunosuppressants
• Immunostimulants.
IMMUNOSUPPRESSANTS

• Glucocorticoids .
• Calcineurin inhibitors
1.cyclosporine
2tacrolimus
• Antiproliferative/ antimetabolite agents
1.Azathioprine
2. Methotrexate
IMMUNOSTIMULANTS

• Levamisole -RA
• Thalidomide –Multiple myeloma
• Recombinant cytokines
1. Interferons-chronic hepatitis B, Kaposis sarcoma
2. Interleukin-2 –Malignant melanoma
Name Mechanism of action Target disease
Zanolimumab Human anti-CD4 Rheumatoid arthritis
mAb ,partially depleting
Rituximab Chimeric anti-CD20 mAb Rheumatoid arthritis

Fingolimod Sphingosine 1Phosphate Relapsing multiple

receptor antagonist sclerosis
Cyclosporine A Calcineurin inhibitor Severe active RA
Tacrolimus Calcineurin inhibitor RA , Myasthenia gravis

Abatacept Fc fusion protein with RA , lupus nephritis

extracellular domain of
CTLA-4 blocks
CYCLOSPORINE MOA