Download unit-1-5 consise NOTES immunology - E

Gregory
NEHRU ARTS AND SCIENCE COLLEGE
IMMUNOLOGY
The immune system is the body’s natural defence in combating organisms.
Immunology has developed rapidly over the past decade owing to the refinements made
in the molecular tests employed in this area of research. Therefore, the keen reader is
encouraged to peruse the ophthalmic and immunological literature in order to keep
abreast of the latest developments in this field.
Owing to the complex nature of this subject, it is
far beyond the scope of this article to cover all
aspects of immunology. Rather, the aims of the
article are twofold: first to acquaint the busy
practitioner with the basic concepts of the
immune system; and second, to introduce the
reader to the more specific topic of ocular
immunology - the study of the ocular immune
system.
Finally, since it is envisaged that optometrists
will one day prescribe therapeutic agents, the
discussion is limited to the anterior segment and
anterior uvea.
Innate & adaptive immune systems
The immune system can be thought of as having
two “lines of defence”: the first, representing a
non-specific (no memory) response to antigen
(substance to which the body regards as foreign
or potentially harmful) known as the innate
immune system; and the second, the adaptive
immune system, which displays a high degree of
memory and specificity. The innate system
represents the first line of defence to an
intruding pathogen. The response evolved is
therefore rapid, and is unable to “memorise” the
same said pathogen should the body be exposed
to it in the future. Although the cells and
molecules of the adaptive system possess slower
temporal dynamics, they possess a high degree of
specificity and evoke a more potent response on
secondary exposure to the pathogen.
The adaptive immune system frequently
incorporates cells and molecules of the innate
system in its fight against harmful pathogens.
For example, complement (molecules of the
innate system - see later) may be activated by
antibodies (molecules of the adaptive system)
thus providing a useful addition to the adaptive
system’s armamentaria.
A comparison of the two systems can be seen
in Table 1.
Figure 1 The principle components of the immune system are listed, indicating which cells
produce which soluble mediators. Complement is made primarily by the liver, with some
synthesised by mononuclear phagocytes. Note that each cell only produces a particular set of
cytokines, mediators etc
Cells of the innate
immune system
Phagocytes
Although sub-divided into two main types,
namely neutrophils and macrophages, they
both share the same function - to engulf
microbes (phago - I eat, Latin).
Neutrophils
Microscopically, these cells possess a
characteristic, salient feature - a
multilobular nucleus (Figure 2). As such,
these cells have been referred to as
polymorphonuclear leukocytes (PMNs) and
have a pivotal role to play in the
development of acute inflammation. In
addition to being phagocytic, neutrophils
contain granules and can also be classed as
one of the granulocytes. The granules
contain acidic and alkaline phosphatases,
defensins and peroxidase - all of which
Table 1: Cells and molecules of the innate and adaptive immune systems
Immunity
Cells
Molecules
Innate
Natural killer (NK) cells
Mast cells
Dendritic cells
Phagocytes
Cytokines
Complement
Acute phase proteins
T and B cells
Cytokines
Adaptive
Antibodies
Components of the immune system can be seen in Figure 1.
Figure 2 Morphology of the neutrophil. This
shows a neutrophil with its characteristic
multilobed nucleus and neutrophilic granules
in the cytoplasm. Giemsa stain, x 1500
represent the requisite molecules required for
successful elimination of the unwanted
microbe(s).
Macrophages
Macrophages (termed monocytes when in the
blood stream) have a horseshoe-shaped nucleus
and are large cells. Properties of macrophages
include phagocytosis and antigen presentation
to T cells (see later). Unlike neutrophils (which
are short-lived cells), they are seen in chronic
inflammation as they are long-lived cells.
Mononuclear phagocytic system
The cells comprising the monocyte phagocytic
system are tissue bound and, as a result, are
further sub-divided depending on their location.
A list of the cells together with their
corresponding location can be found in Table 2.
Table 2: Examples of cells of the
mononuclear phagocytic system and their
respective locations
Cells
Location
Monocytes
Blood stream
Alveolar macrophages
Lungs
Sinus macrophages
Lymph nodes
and spleen
Liver
Kupffer cells
Phagocytosis - the process
Phagocytosis is the process by which cells engulf
microorganisms and particles (Figure 3). Firstly,
the phagocyte must move towards the microbe
under the influence of chemotactic signals, e.g.
complement (see later). For the process to
continue, the phagocyte must attach to the
microbe either by recognition of the microbial
sugar residues (e.g. mannose) on its surface or
complement/antibody, which is bound to the
pathogen. Following attachment, the
phagocyte’s cell surface invaginates and the
microbe becomes internalised into a
phagosome. The resultant phagosome fuses with
multiple vesicles containing O2 free radicals and
other toxic proteins known as lysosomes to form
a phagolysosome. The microbe is subsequently
destroyed.
Opsonisation (“to make tasty” - Greek)
Opsonins are molecules, which enhance the
efficiency of the phagocytic process by coating
the microbe and effectively marking them for
their destruction. Important opsonins are the
complement component C3b and antibodies.
Natural killer (NK) cells
NK cells are also known as “large granular
lymphocytes” (LGLs) and are mainly found in the
circulation. They comprise between 5-11% of the
total lymphocyte fraction. In addition to
possessing receptors for immunoglobulin type G
(IgG), they contain two unique cell surface
receptors known as killer activation receptor and
killer inhibition receptor. Activation of the
former initiates cytokine (“communication”)
molecules from the cell whilst activation of the
latter inhibits the aforesaid action.
NK cells serve an important role in attacking
virally-infected cells in addition to certain
tumour cells. Destruction of infected cells is
achieved through the release of perforins and
granyzymes from its granules, which induce
apoptosis (programmed cell death). NK cells are
also able to secrete interferon-γ (IFN-γ ). This
interferon serves two purposes: first, to prevent
healthy host cells from becoming infected by a
virus; and second, to augment the T cell
response to other virally infected cells
(see later).
Figure 3
Phagocytes arrive at a site of inflammation
by chemotaxis. They may then attach to
microorganisms via their non-specific cell
surface receptors. Alternatively, if the
organism is opsonised with a fragment of
the third complement component (C3b),
attachment will be through the phagocyte’s
receptors for C3b. If the phagocyte
membrane now becomes activated by the
infectious agent, it is taken into a
phagosome by pseudopodia extending
around it. Once inside, lysosomes fuse with
the phagosome to form a phagolysosome
and the infectious agent is killed. Undigested
microbial products may be released to the
outside
stain with a basic dye. Unlike mast cells, which are
present in close proximity to blood vessels in
connective tissue, basophils reside in the
circulation.
Both cell types are instrumental in initiating
the acute inflammatory response. Degranulation
is achieved either by binding to components of
the complement system or by cross-linking of
the IgE antibody which results in the release of
pro-inflammatory mediators including histamine
and various cytokines. The former induces
vasodilation and augments vascular permeability
whilst the latter are important in attracting
both neutrophils and eosinophils.
Dendritic cells
Dendritic cells consist of Langerhans’ and
interdigitating cells and form an important
bridge between innate and adaptive immunity, as
the cells present the antigenic peptide to the T
helper cell (adaptive immunity). Such cells are
therefore known as professional antigen
presenting cells (APCs). Table 3 illustrates the
various types of dendritic cells together with an
example of their location.
Eosinophils
Eosinophils (so called because their granules
stain with eosin - Figure 4) are granulocytes
that possess phagocytic properties. Despite the
fact that they represent only 2-5 % of the total
leukocyte population, they are instrumental in
the fight against parasites that are too big to be
phagocytosed.
Table 3: Dendric cells and location
Mast cells and basophils
Cells
Location
Morphologically, mast cells and basophils are
very similar in that both contain electron dense
granules in the cytoplasm. Basophils are
so-called owing to the fact that their granules
Langerhans cell
Limbus, skin
Interdigitating cell
T cell areas in
lymph nodes
Figure 4 Morphology of the eosinophil. The
multilobed nucleus is stained blue and the
cytoplasmic granules are stained red.
Leishman stain, x 1800
Molecules of the innate
immune system
Figure 5
When host cells become infected
by virus, they may produce
interferon. Different cell types
produce interferon-α (IFN-α )
or interferon-β (IFN-β );
interferon-γ
There are many molecules, which work in concert
with the cells of the innate immune system and
which also foster close functional links with their
adaptive counterpart. The three major molecules
are:
• Complement
• Acute phase proteins (APP)
• Interferons (IFNs)
(IFN-γ ) is produced by some types
of lymphocyte (TH) after activation
by antigen. Interferons act on other
host cells to induce a state of
resistance to viral infection. IFNγ
has many other effects as well
Complement
The complement system represents a large group
of independent proteins (denoted by the letter C
and followed by a number), secreted by both
hepatocytes (liver cells) and monocytes.
Although these proteins maybe activated by both
the adaptive immune system (classical pathway)
or innate immune system (alternative pathway),
the nomenclature is derived from the fact that
the proteins help (“complement”) the antibody
response.
Activation of complement via the microbe
itself is known as the alternative pathway. The
classical pathway requires the interaction of
antibody with specific antigen. The C3
component is the pivotal serum protein of the
complement system. Binding of the antigen to
C3 results in two possible sequelae. In either
case, C3 component becomes enzymatically
converted to C3b. The bacterial cell wall can
either remain bound to C3b and become
opsonised (since phagocytes have receptors for
C3b) or act as a focus for other complement
proteins (namely C5, 6, 7, 8 and 9). The latter
form the membrane attack complex (MAC), which
induces cellular lysis.
The functions of the complement system may
be summarised as follows:
• Opsonisation
• Lysis (destruction of cells through damage/
rupture of plasma membrane)
• Chemotaxis (directed migration of immune
cells)
• Initiation of active inflammation via direct
activation of mast cells
It is important that complement is regulated to
protect host cells from damage and/or their total
destruction. This is achieved by a series of
regulatory proteins, which are expressed on the
host cells themselves.
Acute phase proteins
These serum proteins are synthesised by
hepatocytes and are produced in high numbers
in response to cytokines released from
macrophages.
Interferons (IFNs)
IFNs are a group of molecules, which limit the
spread of viral infections (Figure 5). There are
two categories of IFNs, namely type I and type II.
Type I IFNs maybe sub-divided further into IFN-α
and β . IFN-γ is the sole type II interferon. Type
I
IFNs are induced by viruses, pro-inflammatory
cytokines and endotoxins from gram negative
bacterial cell walls. Their presence remains vital
Table 4:
Primary and secondary lymphoid organs
Primary lymphoid organs
Secondary lymphoid organs
Bone marrow
Lymph nodes
MALT - mucosa associated lymphoid
tissue (includes bronchus, gut, nasal and
conjunctival associated mucosal tissues)
Spleen
for the successful eradication of an invading
virus by the innate immune system.
Type II IFN, IFN-γ , is produced by T Helper
cells and NK cells and is able to augment both
the antigen presenting properties together with
the phagocytic properties of the APCs (e.g.
macrophages and dentritic cells).
Adaptive immunity
As mentioned previously, there is a great deal of
synergy between the adaptive immune system
and its innate counterpart. The adaptive immune
system comprises two main types of leukocyte
known as B and T lymphocytes. Before describing
these important cell types, it is necessary to
acquaint the reader with both the primary and
secondary lymphoid organs and tissues in the
body. These are summarised in Table 4.
The bone marrow represents the dominant site
for haemopoiesis (production of blood cells and
platelets). Although most of the haemopoietic
cells maturate in this region, T lymphocytes do
so in the thymus. In the thymus, premature T
cells undergo a process of positive and negative
selection whereby the former are allowed to
progress to maturity whilst the latter are marked
for termination via apoptosis (see central
tolerance).
Lymphocytes
Morphologically, there are three types of
lymphocytes: T, B and NK cells. However, only T
and B lymphocytes exhibit memory and
specificity and, as such, are responsible for
the
unique quality of the adaptive immune system.
Resting B lymphocytes are able to react with
free antigen directly when it binds to their cell
surface immunoglobins which act as receptors. T
lymphocytes do not react with free antigen and
instead make use of APCs to phagocytose the
antigen and then to express its component
proteins on the cell surface adjacent to special
host proteins called major histocompatibility
complex (MHC) class II molecules. As discussed,
antigen presenting cells which express MHC class
II molecules include dendritic cells and
macrophages. This “afferent” phase must occur
in order for the T cell to recognise the antigen.
The “efferent” phase occurs when activated
lymphocytes enter the tissue and meet antigen
again. This results in multiplication and secretion
of cytokines or immunoglobins in order to
destroy the antigen.
T cells
T cells can be broadly divided into both T helper
(TH) and cytotoxic T cells (Tc). Furthermore, TH
cells may be sub-divided into TH1 and TH2. The
former are pro-inflammatory T cells and
stimulate macrophages whilst the latter
orchestrate B cell differentiation and maturation
and hence are involved in the production of
humoral immunity (antibody mediated). T cells
express cell surface proteins, described by cluster
determination (CD) numbers. TH cells express CD4
molecules on their cell surface, which enable the
lymphocyte to bind to a MHC class II molecule.
The T cell receptor is unique in that it is only
able to identify antigen when it is associated
with a MHC molecule on the surface of the cell.
Cytotoxic T cells are primarily involved in the
destruction of infected cells, notably viruses.
Unlike TH cells, cytotoxic cells possess CD8 cell
surface markers, which bind to antigenic
peptides expressed on MHC class I molecules.
B cells and antibodies
(immunoglobulins - Ig)
B cells are lymphocytes that produce antibodies
(immunoglobulins) and can recognise free
antigen directly. They are produced in the bone
marrow and migrate to secondary lymphoid
organs. B cells are responsible for the
Table 5:
Antibody isotypes and corresponding functions
Antibody
IgG
IgM
Figure 6
When a microorganism lacks the
inherent ability to activate
complement or bind to
phagocytes, the body provides
antibodies as flexible adaptor
molecules. The body can make
several million different antibodies
able to recognise a wide variety of
infectious agents. Thus the
antibody illustrated binds microbe
1, but not microbe 2, by its
‘antigen-binding protein’ (Fab).
The ‘Fc portion’ may activate
complement or bind to Fc
receptors on host cells,
particularly phagocytes.
Characteristics
Crosses placenta thus providing newborn with useful humoral immunity
High affinity
Predominant antibody in blood and tissue fluid
Large pentameric structure in circulation
Present in monometric form on B cell surface
Secreted form is predominant antibody in early immune
response against antigen
Reaches 75% of adult levels at 12 months of age
IgA
Exists in both a monometric and dimeric form
Secretory IgA (dimeric form) represents 1st line of defence against
microbes invading the mucosal surface, e.g. tears
IgE
Low levels in circulation
Increased levels in worm infections
Fc region has high affinity for mast cell thus involved in allergy
IgD
Antigen receptor on B cells
Absent from memory cells
Table 6:
Various cytokines, their sources and functions
Cytokine
IL-1
Source
Macrophages
Function
1. T, B cell activation
2. Mobilisation of PMNs
3. Induction of acute phase proteins
IL-2
T cells
Proliferation of T and NK cells
Il-4
Th2 cells
Mast cells
B cell activation
IgE response
IL-8
Macrophages
T cells
Fibroblasts
Keratinocytes
Chemotaxis of PMNs
IL-10
T cells
Inhibits other cytokines
Macrophages
Inflammation
TH2 cells
Macrophages
B cell activation
Suppres s macrophages
B cells Stimulate
TH1 TH2Inhibit
IL-12
TGF β
(transforming
growth
factor)
TNF α
(tumour necrosis factor)
development of antibody mediated immunity
known as humoral mediated immunity.
When activated by foreign antigen, B cells
undergo proliferation and mature into antibody
secreting plasma cells. The latter are rich in
organelles such as rough endoplasmic reticulum
and mitochondria, which confer their ability to
secrete soluble proteins (antibodies). Not all
proliferating B cells develop into plasma cells.
Indeed, a significant proportion remain as
memory B cells through a process known as
clonal selection. This process is vital in
eliminating the antigen should the body become
re-exposed to it in the future. T cells are also
clonally selected and this confers to the
production of T memory cells.
Although T and B cells behave differently,
both are able to recirculate around the body
migrating from blood to tissue and vice versa.
The ability to recirculate obviously increases the
efficiency with which cells of the immune system
can home onto the invading antigen.
Antibodies
Antibodies have two roles to play - the first is to
bind antigen and the second is to interact with
host tissues and effector systems in order to
ensure removal of the antigen
(Figure 6).
There are five different types (known as
isotypes) of antibody in the human immune
system - namely IgM, IgG, IgA, IgE and IgD. In
addition, there are four sub classes of IgG
(IgG1-4). The basic antibody unit consists of a
glycosylated protein consisting of two heavy and
two light, polypeptide chains. The region which
binds to the antigen is known as the Fab region,
while the constant region, Fc, not only
determines the isotype but is the region
responsible for evoking effector systems, e.g.
mast cell activation. The term immune complex
refers to the combination of antigen and
antibody and will be discussed later in the article
(see type III hypersensitivity).
The antibody isotypes together with their
corresponding function are illustrated in Table 5.
MHC
Major histocompatability complex (MHC) are cell
surface proteins classified as class I (also
termed
human leucocytic antigen [HLA] A, B and C),
found on all nucleated cells and class II (termed
HLA, DP, DQ and DR), found on all antigen
presenting cells (APCs). MHC molecules are the
sine qua non of T cell induced immunity.
Clinically, there is a strong association
between HLA and certain systemic and ocular
diseases (see later).
Cytokines
Cytokines (also termed interleukins [IL] meaning
“between white blood cells”) are small molecules
that act as a signal between cells and have a
variety of roles including chemotaxis, cellular
growth and cytotoxicity. Owing to their ability to
control immune activity, they have been
described as the “hormones” of the immune
system.
Table 6 summarises some of the cytokines
pertinent to ocular immunology, their functions
and their progenitors. Since interferons have
been discussed earlier in the article, they have
been omitted from the table.
endothelial cells and the presence of junctional
complexes linking retinal pigment epithelial
cells4.
Lymphatic role
Since various cells of the immune system are
capable of reacting with self-antigens, it is
therefore essential that the human body has
mechanisms to suppress/eliminate autoreactive
cells. Failure to do so, can, in some cases, lead
to the development of autoimmune diseases
(see later).
The fact that skin allografts were not rejected
following lymph node removal5 led investigators
to hypothesise that immune privilege was solely
due to the absence of the same said system at
a
particular anatomical site. However, although
certain immune privileged sites do indeed lack
lymphatic drainage, others such as the testes6
and eye7 do possess such a system. It appears
that a proportion of the aqueous humour drains
via the uveoscleral pathway into the lymphatic
vessels in the head and neck.
Central tolerance
The eye, APCs & MHCs
Central and peripheral
tolerance: nature’s way of
containing the immune
response
Central tolerance refers to the process whereby
both immature B and T cell lymphocytes, which
react against normal, healthy cells
(self-antigens), are eliminated via apoptosis.
Peripheral tolerance
This involves the removal of mature lymphocytes,
which are not tolerant to healthy cells.
Ocular immune privilege
There are numerous sites in the body whereby
tissue may be grafted with minimal risk of
rejection. Such regions include, inter alia,
the testis, thyroid lens, anterior chamber,
cornea, iris and ciliary body1,2.
It is important that immune privilege is not
simple interpreted as the host’s inability to
initiate an immune response to a transplanted
tissue. Rather, it is an area of the body in which
there exists a paucity of various elements of the
human immune system in response to an
antigen.
Factors
The factors purported by investigators that
contribute to the phenomenon of ocular immune
privilege include:
• Isolation from a vascular supply
• Isolation from a lymphatic supply
• Presence of a vascular barrier
• Ability to suppress the immune response
• Anterior chamber associated immune
deviation (ACAID)
Vascular supply
The healthy cornea is a good example of an
ocular site devoid of a vascular network. The
evidence to support the role a vascular network
has to play in the mechanism of graft rejection is
unequivocal since the risk of failure correlates
positively with the degree of host
vascularisation3.
Vascular barrier
There is a plethora of evidence in the
ophthalmic literature to support the existence of
a blood-ocular barrier. Furthermore, the same
said barrier encompasses different elements
including tight junctions between retinal
As mentioned previously, APCs, through their
ability to express MHC class II molecules, are
potent progenitors of the immune response.
Moreover, such cells are capable of activating T
cells within the tissue itself. It is therefore not
unreasonable to assume that a paucity of APCs
may play an important role in immune
privilege. In addition, failure to express MHC
class I molecule would make a tissue immune
against the lytic action of the cytotoxic T cells.
Although the aforementioned mechanisms are
theoretically plausible, cells expressing both
MHC class I and II molecules have been
detected in the eye. Table 7 illustrates the
relationship between histocompatability class
and ocular cell type.
It is noteworthy that the epithelial cells of
the crystalline lens are devoid of class I
expression14 and that the Langerhans’ cells
(class II expression) are absent from the central
cornea15.
It is interesting that not all cells, which
express MHC class II act as professional APCs in
the eye. Indeed, it has been shown that such
cells reside in the iris and ciliary body and not
only fail to present alloantigens to T cells, but
have the ability to suppress mixed lymphocyte
reactions16.
The failure to incite the inflammatory
response has attracted a great deal of interest
amongst ophthalmologists and immunologists
alike. It appears such suppression is achieved by
various factors present in the aqueous humour
(e.g. transforming growth factor - β ).
Anterior chamber associated immune
deviation (ACAID)
As a result of experiments with rats,
investigators discovered that antigens placed in
the anterior chamber resulted in systemic
inhibition of delayed type hypersensitivity (DTH
or type IV hypersensitivity) reactions to the
same said antigens17. This phenomenon has been
coined anterior chamber associated immune
deviation (ACAID). The anterior chamber is thus
able to suppress delayed type hypersensitivity
reactions and inhibit the production of
complement fixing antibodies18,19. However, it
has no inhibitory effect on cytotoxic T cell
activity and has a minimal influence on the
production of non-complement fixing
antibodies.
With respect to the endothelium, two
adaptations prevent it from immunological
injury: first, avoidance of cytotoxic T lymphocyte
(CTL)-mediated lysis; and second, inhibition of
DTH responses in the anterior chamber22. It
achieves the former through the cells inability
to express MHC class I molecules21. The corollary
of this, however, is that virally infected cells
may persist in this region. The râison d’etre of
ACAID is to protect the eye from the DTH
response to pernicious antigens. As a result of its
location in relation to the anterior chamber,
the corneal endothelium seems well placed to
reap the benefits of ACAID.
ACAID is beneficial in reducing the incidence
of stromal keratitis in herpes simplex virus
infection. It therefore seems reasonable to
assume that such an unwanted corneal sideeffect occurs as a result of a DTH response
rather than the toxic effect of the virus per se22.
Corneal graft rejection may be due, in part,
to failure to invoke ACAID. Streilein at al23 not
only discovered that the immunosuppressive
effects of the cornea were abolished in corneas
Table 7:
MHC and ocular cell type8-13
MHC class I
Ocular cell type
Corneal epithelium
•
Corneal stroma
•
Corneal endothelium
•
Trabecular meshwork
•
Pigmented and non pigmented
cells of ciliary body
•
Anterior iris
•
RPE cells
•
MHC class II
Corneal limbus
•
Iris and ciliary body
•
Uveoscleral pathway
Ora serrata
•
that had ACAID removed via cauterisation or
keratoplasty but also abolished in corneas that
had been denervated.
Ocular immunology
Anterior segment immunology may be
sub-divided into the following aspects:
• Tear film
• Corneal immunology
• Conjunctival immunology
• Scleral immunology
• Uveal immunology
Tear film
a) Mucous layer
This layer is produced both by the conjunctival
goblet and epithelial cells. The glycocalyx
synthesised by the corneal epithelial cells serves
to attach the mucous layer and in doing so binds
to immunoglobulin in the aqueous24. It has been
suggested that the latter immunological sign may
have antiviral effects. The evidence to support
this is that certain intestinal mucosa, which have
similar binding properties to those seen in the
eye, are able to exert an inhibitory action against
viral replication25.
b) Aqueous layer
The aqueous layer contains the following
antimicrobial factors:
1. Lactoferrin
2. Lysozyme
3. IgA
4. Miscellaneous proteins
1. Lactoferrin
Lactoferrin is produced via the acinar cells of the
lacrimal gland. Its main function is to bind iron,
which is required for bacterial growth. It
therefore possesses both bacteriostatic and
bacteriocidal properties. Furthermore, it is able
to enhance the effects of certain
immunoglobulins.
2. Lyszome
Produced by type A cells in the lacrimal gland,
lysozome constitutes up to 25% of the total
tear
protein. It is surprising somewhat that despite
being effective at lysing gram positive bacterial
cell walls, staphylococcus aureus appears
recalcitrant to its action26. However, it is
instrumental in enhancing IgA against gram
negative bacteria.
3. IgA
This is the predominant isotype found in the
human tears in the non-inflamed eye. Very little
is present in serum with the majority secreted
through epithelial cells of structures such as the
lacrimal gland and the lactating breast. The IgA
present in the tears is produced by plasma cells
situated underneath the secretory cells lining the
acini in the lacrimal gland.
IgA is very effective at binding microbes and,
as such, prevents the microbe from adhering to
the mucosal surface. The antibody achieves this
either by interfering with the binding site
directly or by agglutination. Successful binding of
the microorganism enables the tears to wash
them away. IgA possesses other functions
including opsonisation and inactivation of
various bacterial enzymes and toxins. In
addition, it may be involved in antibody
dependent cell-mediated cytotoxicity. It must be
emphasised, however, that IgA is unable to bind
to complement and, as such, is not involved in the
classical pathway27.
4. Miscellaneous proteins
β -Lysin, a bacteriocidal cationic protein, is
present both in the tears and aqueous humour28.
Its bacteriocidal properties are conferred
through their ability to disrupt the microorganisms’ walls. Various components of
complement are present in varying degrees
depending on whether the eye is closed or not.
Closed eye vs open eye
Open eye
In addition to the components of complement
mentioned above, the tears are rich in lysozyme,
lactoferrin and lipocalin (tear pre albumin)
together with low levels of IgA29,30.
Closed eye
In the closed eye environment, levels of IgA and
complement components are increased. In
addition, neutrophils are also recruited. The eye
can be interpreted as being in a state of
subclinical inflammation. However, the eye is
protected from damage induced by either
complement or neutrophils by DAF (Decayed
Accelerating Factor [an inhibitory component of
the complement system])31 and α 1 antitrypsin33.
Corneal immunology
Paradoxically, the cornea, an immune privileged
site, is capable of evoking an immunological
response evidenced by the presence of
subepithelial infiltrates, keratic precipitates,
epithelial and stromal keratitis under certain
clinical conditions.
Cytokine release
Numerous cytokines are synthesised in the
cornea including IL-1, IL-6, IL-8, TNF-α , IFN-γ ,
C5a and prostaglandins33. Phagocytosis of certain
antigens via corneal epithelial cells initiate the
release of IL-1 which, in turn, gives rise to the
recruitment of Langerhans’ cells from the limbus
to the central cornea. It is worthy of note that
the same said APC may be recruited centrally in
response to chemical or localised damage34,35.
Investigators have discovered that stromal
fibroblasts synthesise
IL-8 in response to infection by the herpes
simplex virus36. Furthermore, it seems plausible
that the latter cytokine release may be
responsible for the neutrophilic infiltration
observed in cases of herpetic keratitis.
Immunoglobulins
The three isotypes detected in the cornea are
IgM, G and A. IgG predominates in the central
cornea37. By contrast, IgM predominates in the
limbal region. The latter antibody is restricted
from the central cornea owing to its large size.
Antibodies in the cornea are found in the
stroma since they are cationically charged. Due
to their positive ionic charge, they bind to
proteoglycans and the anionic
glycosaminoglycans38.
Antigen/antibody complexes may sometimes
be observed in the corneal stroma in a variety
of pathological conditions (e.g. herpes simplex
keratitis), and because of their ringed shaped
appearance are known as “Wessley rings”.
Complement
The elements of complement found in the
cornea include C1-739 in addition to the
regulatory proteins H and I and C1 inhibitor40.
Interestingly, C1 is present in high
concentrations in the limbus and is restricted
from the central cornea. This finding is
significant since the limbal region is susceptible
to ulceration following complement activation
and immune complex deposition. It has been
suggested that C1 is produced by corneal
fibroblasts whereas the remaining components
detected in the cornea appear to be derived
from the plasma via linked vessels.
Conjunctival immunology
The conjunctival associated lymphoid tissue
(CALT) is part of the more general mucosa
associated lymphoid tissue (MALT). Langerhans’
cells and lymphocytes exist within the
conjunctival epithelial layer. In the substantia
propria, neutrophils, lymphocytes, IgA and IgG,
dendritic cells and mast cells all reside. It is
noteworthy that eosinophils and basophils are
not present in the healthy conjunctiva.
Langerhans’ cells and dendritic cells present
the antigenic peptide to the conjunctival T
helper cells. Following antigenic presentation,
the T cells secrete the cytokine IFN-γ which
serves to promote antigen elimination by
macrophages. This is the delayed-type
hypersensitivity (DTH) response (see later) and
is characteristic of conjunctival pathology such
as phlyctenulosis.
Scleral immunology
There exists only a small number of immune
cells in the sclera compared to the conjunctiva
since it is relatively avascular. In its resting
state, IgG appears to be present in large
amounts41. However, a sclera under stress, may
become immunologically active as a result of
migrating cells from the overlying episcleral and
underlying choroidal vasculature42,43.
Uveal immunology
The uveal tract is an important site from an
immunological perspective for two reasons:
first, it is highly vascular in nature; and
second,
the majority of vessels are highly fenestrated
which facilitates the recruitment of leukocytes
during the inflammatory cascade. The uvea
contains numerous cellular components of the
immune system including macrophages, mast
cells, lymphocytes and plasma cells in addition
Table 8:
Hypersensitivity reactions
Hypersensitivity Appearance
type
time
Mediators
Mechanism
I. Immediate
2-30 mins
IgE
Mast cell response
II. Cytotoxic
5-8 hours
IgM and IgG
III. Immune
complex
2-8 hours
IgM and IgG
immune complexes
24-72 hours
CD4 and CD8 T cells,
APCs
T cell mediated
Macrophages activated
Include granulomatous reactions
Autoantibodies
Autoantibodies against hormone
(enhance acute inflammation)
IV. Delayed type
V. Stimulatory
to an appreciable number of immune factors.
Although IgG and IgM have been detected in
both the choroid and iris, they exist in greater
numbers in the former structure. The reason for
such disparate levels is the dearth of anionic
antibody binding sites on the iris surface44. By
contrast, the ciliary body harbours tremendous
amounts of IgG by virtue of its anionic tissue
sites.
Immunopathology and the eye
Immunopathology encompasses both pathology
as a result of an over-active immune system
(hypersensitivity and autoimmunity) together
with that acquired through an individuals
inability to fight off infection - namely
immunodeficiency. Unfortunately, it is far
beyond the scope of this article to describe
the latter and its ophthalmic correlates.
In this section, the classification system
pertaining to hypersensitivity reactions will be
described and each subtype with an anterior
segment manifestation will be discussed. The
association between HLA and systemic and
ophthalmic disease will be illustrated together
with a brief overview of the concepts pertaining
to autoimmunity.
Hypersensitivity
The term hypersensitivity refers to the process
whereby the adaptive immune response overreacts to a variety of infectious and inert
antigens resulting in damage to the host
tissue. Five types of hypersensitivity reactions
exist
all of which vary in their timing following
contact with the antigen (Table 8).
• Type I hypersensitivity: allergy
Allergies may affect approximately 17% of the
population45. The term atopic is used to describe
those individuals who possess a genetic
predisposition to allergy. Allergies may occur to
otherwise innocuous antigens (known as
allergens) and infectious agents, e.g. worms.
Type I hypersensitivity exists in two phases, the
sensitisation and effector phases.
Firstly, a harmless allergen causes production
of IgE antibody on first exposure. This IgE
Antibody and complement
Antibody/antigen
complexes
diffuses throughout the body until it comes into
contact with mast cells and basophils. Both
these cell types have receptors for IgE antibody.
Although the patient experiences no symptoms
after the initial binding, reintroduction of the
antigen/allergen induces the production of
more IgE and, furthermore, increase the
likelihood of cross-linking with existing
antibodies on the mast cell surface. Such crosslinking induces the mast cell to degranulate and
release a host of inflammatory mediators such
as histamine, prostaglandins and bradykinin.
Histamine characteristically causes the itchy
symptoms experienced by patients and as a
result of binding to H1 receptors in the eye,
induces vasodilation and enhances mucous
secretion by the goblet cells. Bradykinin
augments vascular permeability, decreases
blood pressure and contracts smooth muscle.
Prostaglandins are also powerful inflammatory
mediators.
Figure 7:
Corneal thinning and vascularisation in AKC,
excess mucous is present
Figure 8:
Epithelial macroerosion in VKC
Ocular correlates:
The following anterior segment conditions are
due, if not only in part, to type I
hypersensitivity:
• Seasonal allergic conjunctivitis (type I)
• Giant papillary conjunctivitis (types I and IV)
• Vernal keratoconjunctivitis (VKC)
(type I and IV)
• Atopic keratoconjunctivitis (AKC)
(types I and IV) (Figure 7)
Seasonal allergic conjunctivitis can be easily
recognised by chemosis and hyperaemia of the
conjunctiva, lid swelling and excessive
lacrimation. The cornea is not affected.
GPC is well known to optometrists as an
allergic response to contact lenses,
prosthetic
lenses, protruding corneal sutures and scleral
buckles. Histological examination of eyes
suffering from GPC have revealed degranulated
mast cells (type I hypersensitivity)46 and CD4+ T
cells (type IV)47, thus corroborating the theory
that such a condition is mediated by both types
of hypersensitivity.
Vernal conjunctivitis can present either in a
palpebral form characteristically exhibiting
Figure 9:
Corneal plaque in VKC
giant cobblestone papillae, or in a limbal form
with gelatinous deposits known as Trantas’-dots,
which represent degenerating epithelial cells and
eosinophils. The first corneal change is a
punctate epithelial keratitis, which if
left
unchecked develops into a macroerosion
(Figure 8) and finally a corneal plaque develops
(Figure 9). The conjunctiva itself is
characteristically oedematous and contains an
array of immunological cells including
lymphocytes and mast cells. Evidence of type I
involvement includes the histological detection
of, inter alia, degranulated mast cells,
eosinophils and increased levels of IgE in
affected eyes49. The detection of CD4+ T cells and
macrophages is indicative of a delayed
inflammatory component49.
Type II hypersensitivity: cytotoxic
This classification of hypersensitivity involves
either IgG or IgM antibodies, which may induce
cellular lysis due to the involvement of the
classical complement pathway (as seen in blood
transfusion reactions) or recruit and activate
inflammatory cells via complement. The
components of complement include the C5a,
which serves to attract inflammatory cells to the
site of interest. The hypersensitivity reaction is a
result of the excessive amount of extracellular
mediators released by the inflammatory cells to
antigens that are too big to be completely
phagocytosed.
Antibodies to self-antigens, such as the
acetylcholine receptor in myasthenia gravis, is
not only another example of type II
hypersensitivity but also an example of
autoimmune disease.
Ocular manifestations
• Mooren’s ulcer
• Cicatrical pemphigoid
Mooren’s ulcer is a rare peripheral ulcerative
keratitis that exists either as a unilateral,
non-progressive form which has a predilection
for elderly patients or as a more severe,
progressive form affecting both eyes of relatively
young individuals. The signs range from a small
patch of grey infiltrate near the margin to frank
ulceration involving the entire corneal
circumference and, in some cases, the central
region as well. The healing process results in a
thin, vascularised, opaque cornea. Investigators
have identified a significant number of
lymphocytes, neutrophils and plasma cells50 in the
cornea, thus providing unequivocal evidence to
support the theory that this condition has an
immunopathological aetiology.
Cicatrical pemphigoid is a chronic, blistering
disease, which has a predilection for both the
ocular and oral mucous membranes. Unlike its
self-limiting counterpart, pemphigus vulgaris,
cicatrical pemphigoid rarely affects the skin.
Ocular cicatrical pemphigoid is a serious,
bilateral condition that represents
effective
shrinkage of the conjunctiva. Although the
initial presentation may be subacute and
non-specific, it frequently progresses to
symblepharon, entropion with secondary
trichiasis, dry eye, ankyloblepharon and
conjunctival fornix shortening. There is evidence
to support the presence of IgG antibodies
directed against self-antigen in the basement
membrane of both the skin and eye51. Binding of
the aforementioned antibodies may activate
complement with subsequent recruitment of
inflammatory cells into the area. The process of
cicatrisation is achieved through the secretion of
collagen via fibroblasts as a result of stimulation
via cytokines released from the invading
inflammatory cells.
Type III hypersensitivity:
immune complex
Large pathogens with multiple antigenic sites
have several antibodies bound to them forming
immune complexes. Normally, these complexes
are removed by the mononuclear phagocytes in
the liver and spleen with no adverse sequelae.
However, persistence of immune complexes does
occur in certain individuals leading to their
deposition in tissue. As a consequence of the
latter action, complement may be activated thus
paving the way for inflammatory cells to enter
the deposition site. Since blood vessels (which
filter plasma at high pressure and exhibit a great
deal of tortuosity) are more susceptible for
immune complex deposition, the ciliary body is
particularly vulnerable to this type of
hypersensitivity reaction.
Ocular manifestations
• Uveitis (Crohn’s disease)
• Peripheral corneal lesions associated with
rheumatoid arthritis
• Stevens Johnson syndrome
• Sjögren’s syndrome
The signs and symptoms of the above will be
described in future articles in the series.
Type IV hypersensitivity: delayed-type
hypersensitivity (DTH)
The term DTH has been used to describe such a
reaction owing to its prolonged time-scale
relative to the other hypersensitivity types.
Although DTH can be transferred by T cells that
have been previously sensitised by an antigen, it
cannot be transferred in serum.
The sequence of events leading to DTH begins
with initial presentation of the antigen peptide
to T cells by APCs (e.g. Langerhans’ cells). The
primed T cell migrates to the site of antigenic
entry whereby it releases pro-inflammatory
mediators such as TNF. The release of these
cytokines facilitates blood flow and extravasation
of plasma contents to the area. The activation of
CD4 T helper and CD8 cells results in the release
of IFN-γ and, as a consequence, enhances
macrophage activity in that area. Resolution of
DTH is dependent on the efficacy with which
such phagocytes can remove the offending
antigen.
Recalcitrant infectious agents result in a
chronic DTH that causes the chronically activated
macrophages to fuse together and form
multinucleated giant cells. In an attempt to
contain the infectious agent, macrophages may
undergo further inter connections to resemble an
epithelial layer. Owing to the similarity to this
layer they are referred to as epitheloid cells.
Both epitheloid and multinucleated giant cells
secrete factors that induce fibrosis resulting in
granuloma formation. Thus granulomata are the
hallmark of chronic inflammation. Damage and
loss of function of the neighbouring tissues
frequently ensues until the agent is removed
either chemically or surgically.
Ocular manifestations
• Ocular allergies (VKC, AKC GPC)
• Idiopathic uveitis
• Sympathetic ophthalmia
• Phlyctenulosis
Type V hypersensitivity
This relatively new category encompasses the
concept of autoantibodies binding to hormone
receptors that mimic the hormone itself. This
results in stimulation of the target cells.
Examples include thyrotoxicosis.
Autoimmunity
The ability to react against self-antigens is
known as autoimmunity. However, a significant
number of people exist who harbour autoantibodies and yet remain asymptomatic. The
corollary of this is that the presence of
autoreactive cells per se is not sufficient to
trigger autoimmune disease. In fact,
autoimmune disease is a result of breakdown of
one of the immunoregulatory mechanisms.
Furthermore, autoimmune disease may be
classified as either being organ specific (e.g.
insulin dependent diabetes mellitus, Grave’s
disease) or non-organ specific (e.g. Sjögren’s
syndrome, ankylosing spondylitis).
It is important to realise that the causes of
autoimmune diseases are multifactorial. The
main predisposing factors are age, gender,
infection and genetics. However, one of the
most important factors of interest to
immunologists and clinicians alike is the
association between HLA and autoimmune
disease. When determining the likelihood of
contracting a disease both epidemiologists and
clinicians refer to the relative risk. In the case of
HLA antigen, the relative risk compares the
chance of a person who has a particular HLA
antigen acquiring a disease to those individuals
who do not have such an antigen. The
association is exemplified by the relative risk of
suffering from ankylosing spondylitis (AS) in
individuals who possess HLA-B27. In patients
suffering from AS, the prevalence of HLA-B27 is
90% and this figure rises to 95% in patients
who suffer both with the disease and acute
iritis. Indeed, in the UK approximately 45% of
patients who present with acute iritis will
harbour HLA-B2752.
It is therefore important that patients who
present with anterior uveitis are screened for
HLA-B27 because although a positive result may
not necessarily be diagnostic, its presence will
certainly improve the sensitivity of further
radiological tests.
Table 9 compares the HLA associations with
both ophthalmic disorders together with those
systemic disorders relevant to the ophthalmic
practitioner.
Conclusion
This article has only broached the fascinating
subject of ocular immunology. A basic
understanding of immunology is required if
practitioners are to therapeutically manage their
patients. Further articles in the series will help
to reinforce the concepts of this challenging
subject.
.
Disease
Table 9:
HLA and ophthalmic disease
HLA
association
Relative risk 54
Ocular
manifestation
Ankylosing spondylitis
B27
90
Anterior uveitis
Reiter’s disease
B27
33
Mucopurulent conjunctivitis
Anterior uveitis
Keratitis
Rheumatoid
DR4
7
Keratoconjunctivitis sicca
Keratitis
Scleritis
DR3, DR5
10
Keratoconjunctivitis sicca
DR3
Not known
Anterior uveitis (acute and chronic)
Dacryoadentis
Retinal vasculitis,
neovascularisation,
Optic nerve granulomata
DQw7
Not known
Shrinkage of conjunctiva
B5
3
Sympathetic
ophthalmia
DR4, A11,
B40
Not known
Systemic lupus
erythematosus
DR2, DR3
3
Primary Sjögrens’ syndrome
Sarcoidosis
Cicatrical pemphigoid
Behcet’s disease
Multiple choice questions - Basic immunology
1. Which one of the following is not part of
the innate immune system?
a. Mast cells
b. Complement
c. Phagocytes
d. T cells
2. Which one of the following statements is
correct regarding the innate immune system?
a. It is specific
b. It evokes a more potent response on
secondary exposure
c. It represents the first line of defence
d. It is able to memorise pathogens on
subsequent exposures
3. Which one of the following statements
is correct regarding the cells of the innate
system?
a. Basophils are important phagocytes
b. During phagocytosis the pathogen becomes
initially internalised as a phago-lysosome
c. Eosinophils play an important role in
combating virally-infected cells
d. Langerhans’ cells form a bridge between
innate and adaptive immunity
4. Which one of the following statements
is correct regarding the adaptive immune
system?
a. It consists of all types of lymphocytes
b. T cells produce antibodies
c. T cells maturate in the thymus
d. B cells are produced in the spleen
5. Which one of the following statements
is correct regarding T cells?
a. T cells can be subdivided into TH1 and TH2
subtypes only
b. T cells alone can identify any type of antigen
c. T cells express cell surface proteins denoted by
cluster determinant (CD) numbers
d. All T cells are involved in initiating the
inflammatory response
6. Which one of the following statements
is incorrect?
a. B cells have antibodies as their cell surface
receptor
b. There are five types of antibody
c. IgE is an important antibody in allergies
d. All B cells differentiate into plasma cells
7. Which one of the following statements is
correct regarding ocular immune privilege?
a. There is an absence of Langerhans’ cells in the
central cornea
b. Aqueous humour has no role
c. Abundant vascular supply is vital
d. All ocular cells express MHC class II
8. Which one of the following statements
concerning ocular immunology is incorrect?
a. Levels of IgA and complement
increase when the eyes are closed
b. IgA is the predominant antibody
in blood and tissue fluid
c. IgM, IgG and IgA antibody isotypes have been
identified in the cornea
d. The sclera contains a smaller number
of immune cells than the conjunctiva
Anterior uveitis
Retinitis, periphlebitis,
retinal oedema
Panuveitis
Punctate epithelial keratopathy
Keratopathy
Necrotising scleritis
Retinal lesions (cotton wool spots)
Autoimmune optic neuropathy
Please note there is only one correct an
9.
In a patient suffering from vernal
conjunctivitis, which one of the following
statements is correct?
a. Trantas’-dots represent infiltrating T cells
b. Affected eyes have increased levels of IgD
c. Histologically, mast cells, lymphocytes and
macrophages have been identified
d. Is due to type III Hypersensitivity
10. Which one of the following statements
is incorrect?
a. HLA-B27 is a risk factor for both anterior
uveitis
and ankylosing spondylitis
b. Granulomas are present in type IV
hypersensitivity reactions
c. Histamine is an important vasoconstrictor
d. IgE mediated hypersensitivity is of rapid onset
11. What proportion of patients with acute iritis will
harbour HLA-B27?
a. 15%
b. 25%
c. 45%
d. 65%
12. Which one of the following statements is correct
regarding a type I hypersensitivity reaction?
a. It always occurs in isolation
b. It is characterised by the presence of
macrophages
c. It is associated with myasthenia gravis
d. It involves the degranulation of mast cells
following the cross-linking of IgE bound to its
cell surface
POLYCLONAL SERUM
Most antigens offer multiple epitopes and therefore induce proliferation
and differentiation of a variety of B-cell clones, each derived from a B cell that recognizesa
particular epitope. The resulting serum antibodies are heterogeneous, comprising a mixture of
antibodies, each specific for one epitope. Such a polyclonal antibody response facilitates the
localization, phagocytosis, and complement-mediated lysis of antigen; it thus has clear
advantages for the organism in vivo. Unfortunately, the antibody heterogeneity that increases
immune protection in vivo often reduces the efficacy of an antiserum for various in vitro
uses.
Polyclonal antibodies are a minor component in a complex mixture of
serum proteins and are a heterogeneous mixture of molecules with a wide range of binding
affinities. Therefore such antisera lack the degree of definition required for many of the
current molecular techniques, where an increase in assay sensitivity is often counteracted by
a decrease in serological specificity. Nonetheless, the fact that polyclonal antibodies can bind
a particular antigen from so many different perspectives is of great technical advantage and
they are therefore excellent for: (a) routine affinity purification of the native antigen; and (b)
relating an expressed partial gene sequence to the mature gene product. However, they have
the disadvantage that non-specific or crossreactive binding reactions can be a serious problem
when antisera are used to identify or quantify antigens, e.g. the study of differentiation or
tumour antigens, or in the clinical laboratory for immunodiagnosis of disease. This is because
of other components in an antiserum.
MONOCLONAL ANTIBODIES
Most antigens offer multiple epitopes and therefore induce proliferation and
differentiation of a variety of B-cell clones, each derived from a B cell that recognizesa
particular epitope. The resulting serum antibodies are heterogeneous, comprising a mixture of
antibodies, each specific for one epitope. Such a polyclonal antibody response facilitates the
localization, phagocytosis, and complement-mediated lysis of antigen; it thus has clear
advantages for the organism in vivo. Unfortunately, the antibody heterogeneity that increases
immune protection in vivo often reduces the efficacy of an antiserum for various in vitro
uses. For most research, diagnostic, and therapeutic purposes, monoclonal antibodies,
derived from a single clone and thus specific for a single epitope, are preferable. Direct
biochemical purification of a monoclonal antibody from a polyclonal antibody preparation is
not feasible. In 1975, Georges Köhler and Cesar Milstein devised a method for preparing
monoclonal antibody, which quickly became one of immunology’s key technologies. By
fusing a normal activated, antibody-producing B cell with a myeloma cell (a cancerous
plasma cell), they were able to generate a hybrid cell, called a hybridoma, that possessed the
immortal growth properties of the myeloma cell and secreted the antibody produced by the B
cell . The resulting clones of hybridoma cells, which secrete large quantities of monoclonal
antibody, can be cultured indefinitely. B-cell hybridomas that secrete antibody with a single
antigenic specificity, called monoclonal antibody, in reference to its derivation from a single
clone, have revolutionized not only immunology but biomedical research as well as the
clinical laboratory.
Basis of fusion and selection:
Spleen cells, prepared from immunized mice or rats, are induced to fuse with
myeloma cells using polyethylene glycol. Many cells show cytoplasmic fusion; a lower
proportion complete the nuclear fusion. This procedure results in a heterogeneous mixture of
fused and unfused cells, although there is a preferential association of ontogenetically similar
cells: myeloma cells tend to ‘rescue’ large, recently activated B lymphocytes. After
dispensing into culture wells, the cell mixture is cultured in a selective medium that
positively selects for fusion hybrids. Culture supernatants are tested for antibody activity after
1–3 weeks. Positive cultures are cloned by conventional cell cloning techniques.
This is done by the use of a myeloma cell line deficient in the enzyme
responsible for incorporation of hypoxanthine into DNA. Briefly, cells can synthesize DNA
in two ways, either by de novo synthesis or via the so-called ‘salvage’ pathway using
exogenous or endogenous sources of preformed bases, as summarized in. If myeloma cells
are grown in the presence of a purine analogue, for example 8-azaguanine or 6-thioguanine,
the hypoxanthine guanine phosphoribosyltransferase (HGPRT) enzyme catalyses the
incorporation of the purine analogue into DNA where it interferes with normal protein
synthesis and so the cells die. Gene coding for the HGPRT enzyme is on the X chromosome,
so only a single copy per cell is expressed. Cells will arise that are deficient in the HGPRT
gene and therefore do not incorporate the purine analogue, i.e. HGPRT-deficient cells which
are unable to utilize hypoxanthine and therefore can only synthesize ribonucleotides by de
novo synthesis. A selective medium containing aminopterin (or amethopterin, methotrexate),
hypoxanthine and thymidine (HAT medium) is used. Aminopterin (analogue of folic acid)
binds folic acid reductase and blocks the coenzymes required for de novo synthesis of DNA.
To grow in this medium a cell must make DNA via the ‘salvage’ pathway. If HGPRT-
deficient plasmacytoma cells are fused with normal lymphoid cells and then placed in HAT
medium, only the hybrids between plasmacytoma and normal cells will grow (plasmacytoma
cell provides immortality; plasma cell provides the HGPRT enzyme.
SCREENING:
The initial screen for antibody activity should be carried out as soon as growth
of hybrid cells is seen under the microscope or when the pH indicator dye has become
yellow. Although cells are diluted to limit the number of independent hybrid cells per well,
several hybrids may grow, perhaps at different rates, each producing their own clone of cells.
This might affect the screening assay in two ways:
(i) A positive clone (secreting the desired antibody) may be detected soon after fusion, but
then
might be lost by overgrowth of a negative or other positive clones.
(ii) No activity may be detected during the first assay due to the cells of a positive clone
being in a minority. Once antibody activity has been detected in any particular well, it is
essential to clone and retest the cells as soon as possible. The type of assay used to detect
antibody is determined by the nature of the antigen and the type of antibody desired.
Antibody-secreting hybrid cells from positive culture wells must be cloned to ensure that the
antibody is homogeneous and monospecific.
CLONING:
Cloning is necessary to ensure that non-producers, arising either in the original fusion wells
or as spontaneous variants, do not outgrow the antibody-secreting hybrids. If continuous
growth of a hybrid line is required, it will be necessary to repeat the cloning and positive
selection procedure at regular intervals.
USES:
ORGANS OF THE IMMUNE SYSTEM
A number of morphologically and functionally diverse organsand tissues have various
functions in the development of immune responses. These can be distinguished by function
as the primary and secondary lymphoid organs (Figure 2-13). The thymus and bone
marrow are the primary (or central) lymphoid organs, where maturation of lymphocytes takes
place. The lymph nodes, spleen, and various mucosalassociated lymphoid tissues (MALT)
such as gut-associated lymphoid tissue (GALT) are the secondary (or peripheral) lymphoid
organs, which trap antigen and provide sites for mature lymphocytes to interact with that
antigen. Once mature lymphocytes have been generated in the primary lymphoid organs, they
circulate in the blood and lymphatic system, a network of vessels that collect fluid that has
escaped into the tissues from capillaries of the circulatory system and ultimately return it to
the blood.
PRIMARY LYMPHOID ORGANS
Immature lymphocytes generated in hematopoiesis mature and become committed to a
particular antigenic specificity within the primary lymphoid organs. Only after a lymphocyte
has matured within a primary lymphoid organ is the cell immunocompetent (capable of
mounting an immune response). T cells arise in the thymus, and in many mammals—humans
and mice for example—B cells originate in bone marrow.
THYMUS:
The thymus is the site of T-cell development and maturation. It is a flat, bilobed organ
situated above the heart. Each lobe is surrounded by a capsule and is divided into lobules,
which are separated from each other by strands of connective tissue called trabeculae. Each
lobule is organized into two compartments: the outer compartment, or cortex, is densely
packed with immature T cells, called thymocytes, whereas the inner compartment, or
medulla, is sparsely populated with thymocytes. Both the cortex and medulla of the thymus
are crisscrossed by a three-dimensional stromal-cell network composed of epithelial cells,
dendritic cells, and macrophages, which make up the framework of the organ and contribute
to the growth and maturation of thymocytes. Many of these stromal cells interact physically
with the developing thymocytes (Figure 2-14). Some thymic epithelial cells in the outer
cortex, called nurse cells, have long membrane extensions that surround as many as 50
thymocytes, forming large multicellular complexes.
THE THYMUS AND IMMUNE FUNCTION:
The role of the thymus in immune function can be studied in mice by examining the effects
of neonatal thymectomy, a procedure in which the thymus is surgically removed from
newborn mice. These thymectomized mice show a dramatic decrease in circulating
lymphocytes of the T cell lineage and an absence of cell-mediated immunity. Other evidence
of the importance of the thymus comes from studies of a congenital birth defect in humans
(DiGeorge’s syndrome) and in certain mice (nude mice) in which the thymus fails to
develop. In both cases, there is an absence of circulating T cells and of cell-mediated
immunity and an increase in infectious disease. Aging is accompanied by a decline in thymic
function. This decline may play some role in the decline in immune function during aging in
humans and mice. The thymus reaches its maximal size at puberty and then atrophies, with a
significant decrease in both cortical and medullary cells.
BONE MARROW
In humans and mice, bone marrow is the site of B-cell origin and development. Its having
two regions1) Vascular region2) Hematopoietic region. The bone marrow weighs about 3 Kg
in adult. The vascular region supplies nutrients and removes the waste materials during
hematopoiesis. The hematopoietic region is also called as yellow marrow. Arising from
lymphoid progenitors, immature B cells proliferate and differentiate within the bone marrow,
and stromal cells within the bone marrow interact directly with the B cells and secrete various
cytokines that are required for development. Bone marrow is not the site of B-cell
development in all species. In birds, a lymphoid organ called the bursa of Fabricius, a
lymphoidtissue associated with the gut, is the primary site of B-cell maturation. In mammals
such as primates and rodents, there is no bursa and no single counterpart to it as a primary
lymphoid organ. In cattle and sheep, the primary lymphoid tissue hosting the maturation,
proliferation, and diversification of B cells early in gestation is the fetal spleen.
SECONDARY LYMPHOID ORGAN
Lymph nodes and the spleen are the most highly organized of the secondary
lymphoid organs; they comprise not only lymphoid follicles, but additional distinct regions of
Tcell and B-cell activity, and they are surrounded by a fibrous capsule. Less-organized
lymphoid tissue, collectively called mucosal-associated lymphoid tissue (MALT), is found in
various body sites. MALT includes Peyer’s patches (in the small intestine), the tonsils, and
the appendix, as well as numerous lymphoid follicles within the lamina propria of the
intestines and in the mucous membranes lining the upper airways, bronchi, and genital tract.
As blood circulates under pressure, its fluid component (plasma) seeps through the thin wall
of the capillaries into the surrounding tissue.Much of this fluid, called interstitial fluid,
returns to the blood through the capillary membranes. The remainder of the interstitial fluid,
now called lymph, flows from the spaces in connective tissue into a network of tiny open
lymphatic capillaries and then into a series of progressively larger collecting vessels called
lymphatic vesselsWhen a foreign antigen gains entrance to the tissues, it is picked up by the
lymphatic system (which drains all the tissues of the body) and is carried to various organized
lymphoid tissues such as lymph nodes, which trap the foreign antigen. As lymph passes from
the tissues to lymphatic vessels, it becomes progressively enriched in lymphocytes. Thus, the
lymphatic system also serves as a means of transporting lymphocytes and antigen from the
connective tissues to organized lymphoid tissues where the lymphocytes may interact with
the trapped antigen and undergo activation.
LYMPH NODES
Lymph nodes are the sites where immune responses are mounted to antigens in lymph. They
are encapsulated beanshaped structures containing a reticular network packed with
lymphocytes, macrophages, and dendritic cells. Clustered at junctions of the lymphatic
vessels, lymph nodes are the first organized lymphoid structure to encounter antigens that
enter the tissue spaces. As lymph percolates through a node, any particulate antigen that is
brought in with the lymph will be trapped by the cellular network of phagocytic cells and
dendritic cells (follicular and interdigitating). The overall architecture of a lymph node
supports an ideal microenvironment for lymphocytes to effectively encounter and respond to
trapped antigens. Morphologically, a lymph node can be divided into three roughly
concentric regions: the cortex, the paracortex, and the medulla, each of which supports a
distinct microenvironment (Figure 2-18). The outermost layer, the cortex, contains
lymphocytes (mostly B cells), macro-phages, and follicular dendritic cells arranged in
primary follicles. After antigenic challenge, the primary follicles enlarge into secondary
follicles, each containing a germinal center. In children with B-cell deficiencies, the cortex
lacks primary follicles and germinal centers. Beneath the cortex is the paracortex, which is
populated largely by T lymphocytes and also contains interdigitating dendritic cells thought
to have migrated from tissues to the node. These interdigitating dendritic cells express high
levels of class II MHC molecules,which are necessary for presenting antigen to TH cells. The
innermost layer of a lymph node, the medulla, is more sparsely populated with lymphoidlineage cells; of those present, many are plasma cells actively secreting antibody molecules.
As antigen is carried into a regional node by the lymph, it is trapped, processed, and
presented together with class II MHC molecules by interdigitating dendritic cells in the
paracortex, resulting in the activation of TH cells. The initial activation of B cells is also
thought to take place within the T-cell-rich paracortex. Once activated, TH and B cells form
small foci consisting largely of proliferating B cells at the edges of the paracortex. Some B
cells within the foci differentiate into plasma cells secreting IgM and IgG. These foci reach
maximum size within 4–6 days of antigen challenge. Within 4–7 days of antigen challenge, a
few B cells and TH cells migrate to the primary follicles of the cortex. It is not known what
causes this migration.Within a primary follicle, cellular interactions between follicular
dendritic cells, B cells, and TH cells take place, leading to development of a secondary
follicle with a central germinal center. Some of the plasma cells generated in the germinal
center move to the medullary areas of the lymph node, and many migrate to bone marrow.
Lymph coming from the tissues percolates slowly inward through the cortex, paracortex, and
medulla, allowing phagocytic cells and dendritic cells to trap any bacteria or particulate
material (e.g., antigen-antibody complexes) carried by the lymph.
SPLEEN
The spleen plays a major role in mounting immune responses to antigens in the blood stream.
It is a large, ovoid secondary lymphoid organ situated high in the left abdominal cavity.
While lymph nodes are specialized for trapping antigen from local tissues, the spleen
specializes in filtering blood and trapping blood-borne antigens; thus, it can respond to
systemic infections. Unlike the lymph nodes, the spleen is not supplied by lymphatic vessels.
Instead, bloodborne antigens and lymphocytes are carried into the spleen through the splenic
artery. Experiments with radioactively labeled lymphocytes show that more recirculating
lymphocytes pass daily through the spleen than through all the lymph nodes combined. The
spleen is surrounded by a capsule that extends a number of projections (trabeculae) into the
interior to form a compartmentalized structure. The compartments are of two types, the red
pulp and white pulp, which are separated by a diffuse marginal zone (Figure 2-19). The
splenic red pulp consists of a network of sinusoids populated by macrophages and numerous
red blood cells (erythrocytes) and few lymphocytes; it is the site where old and defective red
blood cells are destroyed and removed.Many of the macrophages within the red pulp contain
engulfed red blood cells or iron pigments from degraded hemoglobin. The splenic white pulp
surrounds the branches of the splenic artery, forming a periarteriolar lymphoid sheath
(PALS) populated mainly by T lymphocytes. Primary lymphoid follicles are attached to
thePALS. These follicles are rich in B cells and some of them contain germinal centers. The
marginal zone, located peripheral to the PALS, is populated by lymphocytes and
macrophages. Blood-borne antigens and lymphocytes enter the spleen through the splenic
artery, which empties into the marginal zone. In the marginal zone, antigen is trapped by
interdigitating dendritic cells, which carry it to the PALS. Lymphocytes in the blood also
enter sinuses in the marginal zone and migrate to the PALS. The initial activation of B and T
cells takes place in the Tcell- rich PALS. Here interdigitating dendritic cells capture antigen
and present it combined with class II MHC molecules to TH cells. Once activated, these TH
cells can then activate B cells. The activated B cells, together with some TH cells, then
migrate to primary follicles in the marginal zone. Upon antigenic challenge, these primary
follicles develop into characteristic secondary follicles containing germinal centers (like
those in the lymph nodes), where rapidly dividing B cells (centroblasts) and plasma cells are
surrounded by dense clusters of concentrically arranged lymphocytes. The effects of
splenectomy on the immune response depends on the age at which the spleen is removed. In
children, splenectomy often leads to an increased incidence of bacterial sepsis caused
primarily by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae.
Splenectomy in adults has less adverse effects, although it leads to some increase in bloodborne bacterial infections (bacteremia).
LYMPHOCYTE TRAFFIC AND REGULATION
Lymphocytes are capable of a remarkable level of recirculation, continually moving
through the blood and lymph to the various lymphoid organs (Figure 15-1). After a brief
transit time of approximately 30 min in the bloodstream, nearly 45% of all lymphocytes are
carried from the blood directly to the spleen, where they reside for approximately 5 h. Almost
equal numbers (42%) of lymphocytes exit from the blood into various peripheral lymph
nodes, where they reside for about 12 h. A smaller number of lymphocytes (10%) migrate to
tertiary extralymphoid tissues by crossing between endothelial cells that line the capillaries.
An individual lymphocyte may make a complete circuit from the blood to the tissues and
lymph and back again as often as 1–2 times per day.
Cell-Adhesion Molecules:
The vascular endothelium serves as an important “gatekeeper,” regulating the movement of
blood-borne molecules.A number of endothelial and leukocyte CAMs have been cloned and
characterized, providing new details about the extravasation process. Most of these CAMs
belong to four families of proteins: the selectin family, the mucin-like family, the integrin
family, and the immunoglobulin (Ig) superfamily.
Lymphocyte Extravasation:
Various subsets of lymphocytes exhibit directed extravasation at inflammatory sites and
secondary lymphoid organs. The recirculation of lymphocytes thus is carefully controlled to
ensure that appropriate populations of B and T cells are recruited into different tissues.
Extravasation of lymphocytes involves interactions among a number of cell-adhesion
molecules (Table 15-1). The overall process is similar to what happens during neutrophil
extravasation and comprises the same four stages of contact and rolling, activation, arrest and
adhesion, and, finally, transendothelial migration.
High-Endothelial Venules Are Sites of Lymphocyte Extravasation:
Some regions of vascular endothelium in postcapillary venules of various lymphoid organs
are composed of specialized cells with a plump, cuboidal (“high”) shape; such regions are
called high-endothelial venules, or HEVs (Figure 15-4a, b). Their cells contrast sharply in
appearance with the flattened endothelial cells that line the rest of the capillary. Each of the
secondary lymphoid organs, with the exception of the spleen, contains HEVs The general
process of lymphocyte extravasation is similar to neutrophil extravasation. An important
feature distinguishing the two processes is that different subsets of lymphocytes migrate
differentially into different tissues. This process is called trafficking, or homing. The
different trafficking patterns of lymphocyte subsets are mediated by unique combi- nations of
adhesion molecules and chemokines; receptors that direct the circulation of various
populations of lymphocytes to particular lymphoid and inflammatory tissues are called
homing receptors.Researchers have identified a number of lymphocyte and endothelial celladhesion molecules that participate in the interaction of lymphocytes with HEVs and with
endothelium at tertiary sites or sites of inflammation. chemokines, play a major role in
determining the heterogeneity of lymphocyte circulation patterns.
Adhesion-Molecule Interactions Play Critical Roles in Extravasation:
The extravasation of lymphocytes into secondary lymphoid tissue or regions of
inflammation is a multistep process involving a cascade of adhesion-molecule interactions
similar to those involved in neutrophil emigration from the bloodstream. Figure 15-7 depicts
the typical interactions in extravasation of naive T cells across HEVs into lymph nodes. The
first step is usually a selectin-carbohydrate interaction similar to that seen with neutrophil
adhesion. Naive lymphocytes initially bind to HEVs by L-selectin, which serves as a homing
receptor that directs the lymphocytes to particular tissues expressing a corresponding mucinlike vascular addressin such as CD34 or GlyCAM-1. Lymphocyte rolling is less pronounced
than that of neutrophils. In the second step, an integrin-activating stimulus is mediated by
chemokines that are either localized on the endothelial surface or secreted locally. The thick
glycocalyx covering of the HEVs may function to retain these soluble chemoattractant factors
on the HEVs. If, as some have proposed, HEVs secrete lymphocyte-specific
chemoattractants, it would explain why neutrophils do not extravasate into lymph nodes at
the HEVs even though they express L-selectin. Chemokine binding to G-protein–coupled
receptors on the lymphocyte leads to activation of integrin molecules on the membrane, as
occurs in neutrophil extravasation. Once activated, the integrin molecules interact with Igsuperfamily adhesion molecules (e.g., ICAM-1), so the lymphocyte adheres firmly to the
endothelium. The molecular mechanisms involved in the final step, transendothelial
migration, are poorly understood.
Cluster of differentiation
The cluster of differentiation (cluster of designation) (often abbreviated as CD) is a
protocol used for the identification and investigation of cell surface molecules present on
White blood cells. CD molecules can act in numerous ways, often acting as receptors or
ligands (the molecule that activates a receptor) important to the cell. A signal cascade is
usually initiated, altering the behavior of the cell (see cell signaling). Some CD proteins do
not play a role in cell signaling, but have other functions, such as cell adhesion. CD for
humans is numbered up to 350 most recently (as of 2009).
Nomenclature
The CD nomenclature was proposed and established in the 1st International Workshop and
Conference on Human Leukocyte Differentiation Antigens (HLDA), which was held in Paris
in 1982.This system was intended for the classification of the many monoclonal antibodies
(mAbs) generated by different laboratories around the world against epitopes on the surface
molecules of leukocytes (white blood cells). Since then, its use has expanded too many other
cell types, and more than 320 CD unique clusters and subclusters have been identified. The
proposed surface molecule is assigned a CD number once two specific monoclonal antibodies
(mAb) are shown to bind to the molecule. If the molecule has not been well-characterized, or
has only one mAb, it is usually given the provisional indicator "w" (as in "CDw186").
Cell markers
The CD system is commonly used as cell markers, allowing cells to be defined based on what
molecules are present on their surface. These markers are often used to associate cells with
certain immune functions. While using one CD molecule to define populations is uncommon
(though a few examples exist), combining markers has allowed for cell types with very
specific definitions within the immune system.
CD molecules are utilized in cell sorting using various methods including flow cytometry.
Cell populations are usually defined using a '+' or a '–' symbol to indicate whether a certain
cell fraction expresses or lacks a CD molecule. For example, a "CD34+, CD31–" cell is one
that expresses CD34, but not CD31. This CD combination typically corresponds to a stem
cell, opposed to a fully-differentiated endothelial cell.
Type of cell
CD markers
stem cells
CD34+,CD31-
all leukocyte groups
CD45+
Granulocyte
CD45+,CD15+
Monocyte
CD45+,CD14+
T lymphocyte
CD45+,CD3+
T helper cell
CD45+,CD3+,CD4+
Cytotoxic T cell
CD45+,CD3+,CD8+
B lymphocyte
CD45+,CD19+ or CD45+,CD20+
Thrombocyte
CD45+,CD61+
Natural killer cell
CD16+,CD56+,CD3-
Two commonly-used CD molecules are CD4 and CD8, which are, in general, used as
markers for helper and cytotoxic T cells, respectively. These molecules are defined in
combination with CD3+, as some other leukocytes also express these CD molecules (some
macrophages express low levels of CD4; dendritic cells express high levels of CD8). Human
immunodeficiency virus (HIV) binds CD4 and a chemokine receptor on the surface of a T
helper cell to gain entry. The number of CD4 and CD8 T cells in blood is often used to
monitor the progression of HIV infection.
Other uses
It is important to note that, while CD molecules are very useful in defining leukocytes, they
are not merely markers on the cell surface. While only a fraction of known CD molecules
have been thoroughly characterised, most of them have an important function. In the example
of CD4 & CD8, these molecules are critical in antigen recognition.
ANTIBODIES
Antibodies are the antigen binding proteins present on the B-cell membrane and secreted
by plasma cells. Membrane-bound antibody confers antigenic specificity on B cells; antigen
specific proliferation of B-cell clones is elicted by the interaction of membrane antibody with
antigen. Secreted antibodies circulate in the blood, where they serve as the effectors of
humoral immunity by searching out and neutralizing antigens or marking them for
elimination.All antibodies share structural features, bind to antigen, and participate in a
limited number of effector functions.
BASIC STRUCTURE OF ANTIBODIES:
Blood can be separated in a centrifuge into a fluid and a cellular fraction. The fluid
fraction is the plasma and the cellular fraction contains red blood cells, leukocytes, and
platelets. Plasma contains all of the soluble small molecules and macromolecules of blood,
including fibrin and other proteins required for the formation of blood clots. If the blood or
plasma is allowed to clot, the fluid phase that remains is called serum. It has been known
since the turn of the century that antibodies reside in the serum. The first evidence that
antibodies were contained in particular serum protein fractions came from a classic
experiment by A. Tiselius and E. A.Kabat, in 1939. They immunized rabbits with the protein
ovalbumin (the albumin of egg whites) and then divided the immunized rabbits’ serum into
two aliquots. Electrophoresis of one serum aliquot revealed four peaks corresponding to
albumin and the alpha (_), beta (_), and gamma (_) globulins. The other serum aliquot was
reacted with ovalbumin, and the precipitate that formed was removed; the remaining serum
proteins, which did not react with the antigen, were then electrophoresed. A comparison of
the electrophoretic profiles of these two serum aliquots revealed that there was a significant
drop in the _-globulin peak in the aliquot that had been reacted with antigen (Figure 4-1).
Thus, the _-globulin fraction was identified as containing serum antibodies, which were
called immunoglobulins, to distinguish them from any other proteins that might be contained
in the _-globulin fraction. The early experiments of Kabat and Tiselius resolved serum
proteins into three major nonalbumin peaks—_, _ and _.We now know that although
immunoglobulin G (IgG), the main class of antibody molecules, is indeed mostly found in the
_-globulin fraction, significant amounts of it and other important classes of antibody
molecules are found in the _ and the _ fractions of serum.
ANTIBODIES ARE HETERODIMERS:
Antibody molecules have a common structure of four peptide chains (Figure 4-2). This
structure consists of two identical light (L) chains, polypeptides of about 25,000 molecular
weight, and two identical heavy (H) chains, largerpolypeptides of molecular weight 50,000
or more. Like the antibody molecules they constitute, H and L chains are also called
immunoglobulins. Each light chain is bound to a heavy chain by a disulfide bond, and by
such noncovalent interactions as salt linkages, hydrogen bonds, and hydrophobic bonds, to
form a heterodimer (H-L). Similar noncovalent interactions and disulfide bridges link the two
identical heavy and light (H-L) chain combinations to each other to form the basic four-chain
(H-L)2 antibody structure, The first 110 or so amino acids of the amino-terminal region of a
light or heavy chain varies greatly among antibodies of different specificity. These segments
of highly variable sequence are called V regions:VL in light chains and VH in heavy. All of
the differences in specificity displayed by different antibodies can be traced to differences in
the amino acid sequences of V regions. In fact, most of the differences among antibodies fall
within areas of the V regions called complementarity- determining regions (CDRs), and it is
these CDRs, on both light and heavy chains, that constitute the antigenbinding site of the
antibody molecule. The regions of relatively constant sequence beyond the variable regions
have been dubbed C regions, CL on the light chain and CH on the heavy chain.Antibodies are
glycoproteins; with few exceptions, the sites of attachment for carbohydrates are restricted to
the constant region.We do not completely understand the role played by glycosylation of
antibodies, but it probably increases the solubility of the molecules. Inappropriate
glycosylation, or its absence, affects the rate at which antibodies are cleared from the serum,
and decreases the efficiency of interaction between antibody and the complement system and
between antibodies and Fc receptors. Our knowledge of basic antibody structure was derived
from a variety of experimental observations. In a key experiment, brief digestion of IgG with
the enzyme papain produced three fragments, two of which were identical fragments and a
third that was quite different (Figure 4-3). The two identical fragments (each with a MW of
45,000), had antigen-binding activity and were called Fab fragments (“fragment, antigen
binding”). The other fragment (MW of 50,000) had no antigenbinding activity at all. Because
it was found to crystallize during cold storage, it was called the Fc fragment (“fragment,
crystallizable”).Digestion with pepsin, a different proteolytic enzyme, also demonstrated that
the antigen-binding properties of an antibody can be separated from the rest of the molecule.
Pepsin digestion generated a single 100,000- MW fragment composed of two Fab-like
fragments designated the F(ab_)2 fragment, which binds antigen. The Fc fragment was not
recovered from pepsin digestion because it had been digested into multiple fragments. A key
observation in deducing the multichain structure of IgG was made when the molecule was
subjected to mercaptoethanol reduction and alkylation, a chemical treatment that irreversibly
cleaves disulfide bonds. If the sample is chromate graphed on a column that separates
molecules by size following cleavage of disulfide bonds, it is clear that the intact 150,000MW IgG molecule is, in fact, composed of subunits. Each IgG molecule contains two
50,000-MW polypeptide chains, designated as heavy (H) chains, and two 25,000-MW chains,
designated as light (L) chains (see Figure 4-3). Immunoglobulin Fine Structure The structure
of the immunoglobulin molecule is determined by the primary, secondary, tertiary, and
quaternary organization of the protein. The primary structure, the amino acid sequence,
accounts for the variable and constant regions of the heavy and light chains. The secondary
structure is formed by folding of the extended polypeptide chain, back and forth upon itself
into an antiparallel _ pleated sheet (Figure 4-4). The chains are then folded into a tertiary
structure of compact globular domains, which are connected to neighboring domains by
continuations of the polypeptide chain that lie outside the _ pleated sheets. Finally, the
globular domains of adjacent heavy and light polypeptide chains interact in the quaternary
structure (Figure 4-5), forming functional domains that enable the molecule to specifically
bind antigen and, at the same time, perform a number of biological effector functions.
Antibody Classes and Biological Activities:
The various immunoglobulin isotypes and classes have been mentioned briefly already. Each
class is distinguished by unique amino acid sequences in the heavy-chain constant region that
confer class-specific structural and functional properties. In this section, the structure and
effector functions of each class are described in more detail. The molecular properties and
biological activities of the immunoglobulin classes are summarized in Table 4-2 (page 90).
The structures of the five major classes are diagramed in Figure 4-13 (page 91).
Immunoglobulin G (IgG):
IgG, the most abundant class in serum, constitutes about 80% of the total serum
immunoglobulin. The IgG molecule consists of two _ heavy chains and two light chains (see
Figure 4-13a). There are four human IgG subclasses, distinguished by differences in _-chain
sequence and numbered according to their decreasing average serum concentrations: IgG1,
IgG2, IgG3, and IgG4 (see Table 4-2). The amino acid sequences that distinguish the four
IgG subclasses are encoded by different germ-line CH genes, whose DNA sequences are
90%–95% homologous. The structural characteristics that distinguish these subclasses from
one another are the size of the hinge region and the number and position of the interchain
disulfide bonds between the heavy chains (Figure 4-14, page 92). The subtle amino acid
differences between subclasses of IgG affect the biological activity of the molecule: _ IgG1,
IgG3, and IgG4 readily cross the placenta and play an important role in protecting the
developing fetus. _ IgG3 is the most effective complement activator, followed by IgG1; IgG2
is less efficient, and IgG4 is not able to activate complement at all. _ IgG1 and IgG3 bind
with high affinity to Fc receptors on phagocytic cells and thus mediate opsonization. IgG4
has an intermediate affinity for Fc receptors, and IgG2 has an extremely low affinity.
Immunoglobulin M (IgM):
IgM accounts for 5%–10% of the total serum immunoglobulin, with an average serum
concentration of 1.5 mg/ml. Monomeric IgM, with a molecular weight of 180,000, is
expressed as membrane-bound antibody on B cells. IgM is secreted by plasma cells as a
pentamer in which five monomer units are held together by disulfide bonds that link their
carboxyl- terminal heavy chain domains (C_4/C_4) and their C_3/C_3 domains (see Figure
4-13e). The five monomer subunits are arranged with their Fc regions in the center of the
pentamer and the ten antigen-binding sites on the periphery of the molecule. Each pentamer
contains an additional Fc-linked polypeptide called the J (joining) chain, which is disulfidebonded to the carboxyl-terminal cysteine residue of two of the ten _ chains. The J chain
appears to be required for polymerization of the monomers to form pentameric IgM; it is
added just before secretion of the pentamer. IgM is the first immunoglobulin class produced
in a primary response to an antigen, and it is also the first immunoglobulin to be synthesized
by the neonate. Because of its pentameric structure with 10 antigen-binding sites, serum IgM
has a higher valency than the other isotypes. An IgM molecule can bind 10 small hapten
molecules; however, because of steric hindrance, only 5 or fewer molecules of larger antigens
can be bound simultaneously.Because of its high valency, pentameric IgM is more efficient
than other isotypes in binding antigens with many repeating epitopes such as viral particles
and red blood cells (RBCs). For example, when RBCs are incubated with specific antibody,
they clump together into large aggregates in a process called agglutination. It takes 100 to
1000 times more molecules of IgG than of IgM to achieve the same level of agglutination. A
similar phenomenon occurs with viral particles: less IgM than IgG is required to neutralize
viral infectivity. IgM is also more efficient than IgG at activating complement. Complement
activation requires two Fc regions in close proximity, and the pentameric structure of a single
molecule of IgM fulfills this requirement. Because of its large size, IgM does not diffuse well
and therefore is found in very low concentrations in the intercellular tissue fluids. The
presence of the J chain allows IgM to bind to receptors on secretory cells,which transport it
across epithelial linings to enter the external secretions that bathe mucosal surfaces. Although
IgA is the major isotype found in these secretions, IgM plays an important accessory role as a
secretory immunoglobulin.
Immunoglobulin A (IgA):
Although IgA constitutes only 10%–15% of the total immunoglobulin in serum, it is the
predominant immunoglobulin class in external secretions such as breast milk, saliva, tears,
and mucus of the bronchial, genitourinary, and digestive tracts. In serum, IgA exists primarily
as a monomer, but polymeric forms (dimers, trimers, and some tetramers) are sometimes
seen, all containing a J-chain polypeptide (see Figure 4-13d). The IgA of external secretions,
called secretory IgA, consists of a dimer or tetramer, a J-chain polypeptide, and a
polypeptide chain called secretory component (Figure 4-15a, page 93). As is explained
below, secretory component is derived from the receptor that is responsible for transporting
polymeric IgA across cell membranes. The J-chain polypeptide in IgA is identical to that
found in pentameric IgM and serves a similar function in facilitating the polymerization of
both serum IgA and secretory IgA. The secretory component is a 70,000-MW polypeptide
produced by epithelial cells of mucous membranes. It consists of five immunoglobulin-like
domains that bind to the Fc region domains of the IgA dimer. This interaction is stabilized by
a disulfide bond between the fifth domain of the secretory component and one of the chains
of the dimeric IgA. The daily production of secretory IgA is greater than that of any other
immunoglobulin class. IgA-secreting plasma cells are concentrated along mucous membrane
surfaces. Binding of secretory IgA to bacterial and viral surface antigens prevents attachment
of the pathogens to the mucosal cells, thus inhibiting viral infection and bacterial
colonization. Complexes of secretory IgA and antigen are easily entrapped in mucus and then
eliminated by the ciliated epithelial cells of the respiratory tract or by peristalsis of the gut.
Secretory IgA has been shown to provide an important line of defense
against bacteria such as Salmonella, Vibrio cholerae, and Neisseria gonorrhoeae and viruses
such as polio, influenza, and reovirus. Breast milk contains secretory IgA and many other
molecules that help protect the newborn against infection during the first month of life (Table
4-3). Because the immune system of infants is not fully functional, breast-feeding plays an
important role in maintaining the health of newborns.
Immunoglobulin E (IgE):
The potent biological activity of IgE allowed it to be identified in serum despite its extremely
low average serum concentration (0.3g/ml). IgE antibodies mediate the immediate
hypersensitivity reactions that are responsible for the symptoms of hay fever, asthma, hives,
and anaphylactic shock. The presence of a serum component responsible for allergic
reactions was first demonstrated in 1921 by K. Prausnitz and H. Kustner, who injected serum
from an allergic person intra-dermally into a nonallergic individual. When the appropriate
antigen was later injected at the same site, a wheal and flare reaction (analogous to hives)
developed there. This reaction, called the P-K reaction (named for its originators, Prausnitz
and Kustner), was the basis for the first biological assay for IgE activity. IgE binds to Fc
receptors on the membranes of blood basophils and tissue mast cells. Cross-linkage of
receptorbound IgE molecules by antigen (allergen) induces basophils and mast cells to
translocate their granules to the plasma membrane and release their contents to the
extracellular environment, a process known as degranulation. As a result, a variety of
pharmacologically active mediators are released and give rise to allergic manifestations
(Figure 4-16).
Immunoglobulin D (IgD):
IgD was first discovered when a patient developed a multiple myeloma whose myeloma
protein failed to react with antiisotype antisera against the then-known isotypes: IgA, IgM,
and IgG.When rabbits were immunized with this myeloma protein, the resulting antisera
were used to identify the same class of antibody at low levels in normal human serum. The
new class, called IgD, has a serum concentration of 30g/ml and constitutes about 0.2% of the
total immunoglobulin in serum. IgD, together with IgM, is the major membranebound
immunoglobulin expressed by mature B cells, and its role in the physiology of B cells is
under investigation. No biological effector function has been identified for IgD.
HEMATOPOIESIS
All blood cells arise from a type of cell called the hematopoietic stem cell
(HSC). Stem cells are cells that can differentiate into other cell types; they are selfrenewing—they maintain their population level by cell division. In humans, hematopoiesis,
the formation and development of red and white blood cells, begins in the embryonic yolk
sac during the first weeks of development.Here, yolk-sac stem cells differentiate into
primitive erythroid cells that contain embryonic hemoglobin. In the third month of gestation,
hematopoietic stem cells migrate from the yolk sac to the fetal liver and then to the spleen;
these two organs have major roles in hematopoiesis from the third to the seventh months of
gestation. After that, the differentiation of HSCs in the bone marrow becomes the major
factor in hematopoiesis, and by birth there is little or no hematopoiesis in the liver and spleen.
It is remarkable that every functionally specialized, mature blood cell is derived from the
same type of stem cell. Incontrast to a unipotent cell, which differentiates into a single cell
type, a hematopoietic stem cell is multipotent, or pluripotent, able to differentiate in various
ways and thereby generate erythrocytes, granulocytes, monocytes, mast cells, lymphocytes,
and megakaryocytes. hematopoietic stem cells are maintained at stable levels throughout
adult life; however, when there is an increased demand for hematopoiesis,
HSCs display an enormous proliferative capacityEarly in hematopoiesis, a
multipotent stem cell differentiates along one of two pathways, giving rise to either a
common lymphoid progenitor cell or a common myeloid progenitor cell (Figure 2-1). The
types and amounts of growth factors in the microenvironment of a particular stem cell or
progenitor cell control its differentiation. During the development of the lymphoid and
myeloid lineages, stem cells differentiate into progenitor cells, which have lost the capacity
for self-renewal and are committed to a particular cell lineage. Common lymphoid progenitor
cells give rise to B, T, and NK (natural killer) cells and some dendritic cells. Myeloid stem
cells generate progenitors of red blood cells (erythrocytes), many of the various white blood
cells (neutrophils, eosinophils, basophils, monocytes, mast cells, dendritic cells), and
platelets. Progenitor commitment depends on the acquisition of responsiveness to particular
growth factors and cytokines. When the appropriate factors and cytokines are present,
progenitor cells proliferate and differentiate into the corresponding cell type, either a mature
erythrocyte, a particular type of leukocyte, or a platelet-generating cell (the megakaryocyte).
Red and white blood cells pass into bone marrow channels, from which they enter the
circulation.
Development of B and T lymphocytes
Lymphocytes constitute 20%–40% of the body’s white blood cells and 99% of the cells
in the lymphThe lymphocytes can be broadly subdivided into three populations—B cells, T
cells, and natural killer cells—on the basis of function and cell-membrane components.
Natural killer cells (NK cells) are large, granular lymphocytes that do not express the set of
surface markers typical of B or T cells. Resting B and T lymphocytes are small, motile,
nonphagocytic cells,which cannot be distinguished morphologically. B and T lymphocytes
that have not interacted with antigen— referred to as naive, or unprimed—are resting cells in
the G0 phase of the cell cycle. Known as small lymphocytes, these cells are only about 6 _m
in diameter; their cytoplasm forms a barely discernible rim around the nucleus. Small
lymphocytes have densely packed chromatin, few mitochondria, and a poorly developed
endoplasmic reticulum and Golgi apparatus. The naive lymphocyte is generally thought to
have a short life span. Interaction of small lymphocytes with antigen, in the presence of
certain cytokines discussed later, induces these cells to enter the cell cycle by progressing
from G0 into G1 and subsequently into S, G2, and M (Figure 2-7a). As they progress through
the cell cycle, lymphocytes enlarge into 15 _m-diameter blast cells, called lymphoblasts;
these cells have a higher cytoplasm:nucleus ratio and more organellar complexity than small
lymphocytes (Figure 2-7b). Lymphoblasts proliferate and eventually differentiate into
effector cells or into memory cells. Effector cells function in various ways to eliminate
antigen. These cells have short lifespans, generally ranging from a few days to a few weeks.
Plasma cells—the antibody-secreting effector cells of the Bcell lineage—have a
characteristic cytoplasm that contains abundant endoplasmic reticulum (to support their high
rate of protein synthesis) arranged in concentric layers and also many Golgi vesicles (see
Figure 2-7). The effector cells of the T-cell lineage include the cytokine-secreting T helper
cell (TH cell) and the T cytotoxic lymphocyte (TC cell). Some of the progeny of B and T
lymphoblasts differentiate into memory cells. The persistence of this population of cells is
responsible for life-long immunity to many pathogens. Memory cells look like small
lymphocytes but can be distinguished from naive cells by the presence or absence of certain
cellmembrane molecules.
EXPRESSION OF IG GENES
As in the expression of other genes, post-transcriptional processing of
immunoglobulin primary transcripts is required to produce functional mRNAs (see Figures 54 and 5-5). The primary transcripts produced from rearranged heavy-chain and light-chain
genes contain intervening DNA sequences that include noncoding introns and J gene
segments not lost during V-(D)-J rearrangement. In addition, as noted earlier, the heavy-chain
C-gene segments are organized as a series of coding exons and noncoding introns. Each exon
of a CH gene segment corresponds to a constant-region domain or a hinge region of the
heavy-chain polypeptide. The primary transcript must be processed to remove the intervening
DNA sequences, and the remaining exons must be connected by a process called RNA
splicing. Processing of the primary transcript in the nucleus removes each of these
intervening sequences to yield the final mRNA product. The mRNA is then exported from
the nucleus to be translated by ribosomes into complete H or L chains.
DIFFERENTIAL RNA PROCESSING
Processing of an immunoglobulin heavy-chain primary transcript can yield several
different mRNAs,which explains how a single B cell can produce secreted or
membranebound forms of a particular immunoglobulin and simultaneously express IgM and
IgD.
EXPRESSION OF MEMBRANE OR SECRETED IMMUNOGLOBULIN
A particular immunoglobulin can exist in either membrane-bound or secreted form. The
two forms differ in the amino acid sequence of the heavy-chain carboxyl-terminal domains
(CH3/CH3 in IgA, IgD, and IgG and CH4/CH4 in IgE and IgM). The secreted form has a
hydrophilic sequence of about 20 amino acids in the carboxylterminal domain; this is
replaced in the membrane-bound form with a sequence of about 40 amino acids containing a
hydrophilic segment that extends outside the cell, a hydrophobic transmembrane segment,
and a short hydrophilic segment at the carboxyl terminusThe C_4 exon contains a nucleotide
sequence (called S) at its 3_ end that encodes the hydrophilic sequence in the CH4 domain of
secreted IgM. Two additional exons called M1 and M2 are located just 1.8 kb downstream
from the 3_ end of the C_4 exon. The M1 exon encodes the transmembrane segment, and M2
encodes the cytoplasmic segment of the CH4 domain in membrane-bound IgM. Later DNA
sequencing revealed that all the CH gene segments have two additional downstream M1 and
M2 exons that encode the transmembrane and cytoplasmic segments. The primary transcript
produced by transcription of a rearranged _ heavy-chain gene contains two polyadenylation
signal sequences, or poly-A sites, in the C_ segment. Site 1 is located at the 3_ end of the
C_4 exon, and site 2 is at the 3_ end of the M2 exon (Figure 5-16b). If cleavage of the
primary transcript and addition of the poly-A tail occurs at site 1, the M1 and M2 exons are
lost. Excision of the introns and splicing of the remaining exons then produces mRNA
encoding the secreted form of the heavy chain. If cleavage and polyadenylation of the
primary transcript occurs instead at site 2, then a different pattern of splicing results. In this
case, splicing removes the S sequence at the 3_ end of the C_4 exon, which encodes the
hydrophilic carboxyl-terminal end of the secreted form, and joins the remainder of the C_4
exon with the M1 and M2 exons, producing mRNA for the membrane form of the heavy
chain. Thus, differential processing of a common primary transcript determines whether the
secreted or membrane form of an immunoglobulin will be produced.
SIMULTANEOUS EXPRESSION OF IgM and IgD
Differential RNA processing also underlies the simultaneous expression of
membrane-bound IgM and IgD by mature B cells. If the heavy-chain transcript is cleaved and
polyadenylated at site 2 after the C_ exons, then the mRNA will encode the membrane form
of the _ heavy chain (Figure 5-17b); if polyadenylation is instead further downstream at site
4, after the C exons, then RNA splicing will remove the intervening C_ exons and produce
mRNA encoding the membrane form of the heavy chain (Figure 5-17c). Since the mature B
cell expresses both IgM and IgD on its membrane, both processing pathways must occur
simultaneously. Likewise, cleavage and polyadenylation of the primary heavy-chain
transcript at poly-A site 1 or 3 in plasma cells and subsequent splicing will yield the secreted
form of the _ or _ heavy chains, respectively.
SYNTHESIS, ASSEMBLY, AND SECRETION OF IMMUNOGLOBULINS
Immunoglobulin heavy- and light-chain mRNAs are translated on separate
polyribosomes of the rough endoplasmic reticulum (RER). Newly synthesized chains contain
an amino-terminal leader sequence, which serves to guide the chains into the lumen of the
RER, where the signal sequence is then cleaved. The assembly of light (L) and heavy (H)
chains into the disulfide-linked and glycosylated immunoglobulin molecule occurs as the
chains pass through the cisternae of the RER. The complete molecules are transported to the
Golgi apparatus and then into secretory vesicles, which fuse with the plasma membrane
(Figure 5-18). The order of chain assembly varies among the immunoglobulin classes. In the
case of IgM, the H and L chains assemble within the RER to form half-molecules, and then
two half-molecules assemble to form the complete molecule. In the case of IgG, two H chains
assemble, then an H2L intermediate is assembled, and finally the complete H2L2 molecule is
formed. Interchain disulfide bonds are formed, and the polypeptides are glycosylated as they
move through the Golgi apparatus. If the molecule contains the transmembrane sequence of
the membrane form, it becomes anchored in the membrane of a secretory vesicle and is
inserted into the plasma membrane as the vesicle fuses with the plasma membrane (see
Figure 5-18, insert). If the molecule contains the hydrophilic sequence of secreted
immunoglobulins, it is transported as a free molecule in a secretory vesicle and is released
from the cell when the vesicle fuses with the plasma membrane.
PLASMA CELLS AND MEMORY CELLS
Lymphocytes are the central cells of the immune system, responsible for adaptive immunity
and the immunologic attributes of diversity, specificity, memory, and self/nonself
recognition. The other types of white blood cells play important roles, engulfing and
destroying microorganisms, presenting antigens, and secreting cytokines. The lymphocytes
can be broadly subdivided into three populations—B cells, T cells, and natural killer cells—
on the basis of function and cell-membrane components. B and T lymphocytes that have not
interacted with antigen— referred to as naive, or unprimed—are resting cells. When a naive
B cell (one that has not previously encountered antigen) first encounters the antigen that
matches its membrane bound antibody it enlarge into 15 _m-diameter blast cells, called
lymphoblasts; these cells have a higher cytoplasm: nucleus ratio and more organellar
complexity than small lymphocytes the binding of the antigen to the antibody causes the cell
to divide rapidly; its progeny differentiate into memory B cells and effector B cells called
plasma cells. Memory B cells have a longer life span than naive cells, and they express the
same membrane-bound antibody as their parent B cell. Plasma cells produce the antibody in a
form that can be secreted and have little or no membrane-bound antibody. Although plasma
cells live for only a few days, they secrete enormous amounts of antibody during this time. It
has been estimated that a single plasma cell can secrete more than 2000 molecules of
antibody per second. Secreted antibodies are the major effector molecules of humoral
immunity.
Plasma cells—the antibody-secreting effector cells of the Bcell lineage—have a
characteristic cytoplasm that contains abundant endoplasmic reticulum (to support their high
rate of protein synthesis) arranged in concentric layers and also many Golgi vesicles (see
Figure 2-7). Some of the progeny of B and T lymphoblasts differentiate into memory cells.
The persistence of this population of cells is responsible for life-long immunity to many
pathogens. Memory cells look like small lymphocytes but can be distinguished from naive
cells by the presence or absence of certain cellmembrane molecules.
THE LECTIN PATHWAY
Lectins are proteins that recognize and bind to specific carbohydrate targets. (Because
the lectin that activates complement binds to mannose residues, some authors designate this the
MBLectin pathway or mannan-binding lectin pathway.) The lectin pathway, like the alternative
pathway, does not depend on antibody for its activation.However, the mechanism is more like that of
the classical pathway, because after initiation, it proceeds, through the action of C4 and C2, to
produce a C5 convertase (see Figure 13-2). The lectin pathway is activated by the binding of
mannose- binding lectin (MBL) to mannose residues on glycoproteins or carbohydrates on the
surface of microorganisms including certain Salmonella, Listeria, and Neisseria strains, as well as
Cryptococcus neoformans and Candida albicans. MBL is an acute phase protein produced in
inflammatory responses. Its function in the complement pathway is similar to that of C1q, which it
resembles in structure. After MBL binds to the surface of a cell or pathogen, MBL-associated serine
proteases,MASP-1 and MASP-2, bind to MBL. The active complex formed by this association
causes cleavage and activation of C4 and C2. The MASP-1 and -2 proteins have structural similarity
to C1r and C1s and mimic their activities. This means of activating the C2–C4 components to form a
C5 convertase without need for specific antibody binding represents an important innate defense
mechanism comparable to the alternative pathway, but utilizing the elements of the classical pathway
except for the C1 proteins.
REGULATION OF THE COMPLEMENT SYSTEM
The complement system are capable of attacking host cells as well as foreign cells and
microorganisms, elaborate regulatory mechanisms have evolved to restrict complement activity to
designated targets. A general mechanism of regulation in all complement pathways is the inclusion
of highly labile components that undergo spontaneous inactivation if they are not stabilized by
reaction with other components. In addition, a series of regulatory proteins can inactivate various
complement components . For example, the glycoprotein C1 inhibitor (C1Inh) can form a complex
with C1r2s2, causing it to dissociate from C1q and preventing further activation of C4 or C2. The
reaction catalyzed by the C3 convertase enzymes of the classical, lectin, and alternative pathways is
the major amplification step in complement activation, generating hundreds of molecules of C3b.
The C3b generated by these enzymes has the potential to bind to nearby cells, mediating damage to
the healthy cells by causing their opsonization by phagocytic cells bearing C3b receptors or by
induction of the membraneattack complex. Damage to normal host cells is prevented because C3b
undergoes spontaneous hydrolysis by the time it has diffused 40 nm away from the C4_b2_a or
C3_bB_bconvertase enzymes, so that it can no longer bind to its target site. The potential
destruction of healthy host cells by C3b is further limited by a family of related proteins that
regulate C3 convertase activity in the classical and alternative pathways. These regulatory proteins
all contain repeating amino acid sequences (or motifs) of about 60 residues, termed short consensus
repeats (SCRs). All these proteins are encoded at a single location on chromosome 1 in humans,
known as the regulators of complementactivation (RCA) gene cluster. In the classical and lectin
pathways, three structurally distinct RCA proteins act similarly to prevent assembly of C3
convertase). These regulatory proteins include soluble C4b-binding protein (C4bBP) and two
membranes- bound proteins, complement receptor type 1 (CR1) and membrane cofactor protein
(MCP). Each of these regulatory proteins binds to C4b and prevents its association with C2a. Once
C4bBP, CR1, or MCP is bound to C4b, another regulatory protein, factor I, cleaves the C4b in to
bound C4d and soluble C4c). A similar regulatory sequence operates to prevent assembly of the C3
convertase C3_bB_b in the alternative pathway. In this case CR1,MCP, or a regulatory component
called factor H binds to C3b and prevents its association with factor B (Figure 13-9a(4)). A serum
protein called S protein can bind to C5b67, inducing a hydrophilic transition and thereby preventing
insertion of C5b67 into the membrane of nearby cells
CYTOKINES
Properties of Cytokines:
Cytokines are low-molecularweight regulatory proteins or glycoproteins secreted by white blood cells and
various other cells in the body in response to a number of stimuli.Cytokines bind to specific receptors on the
membrane of target cells, triggering signal-transduction pathways that ultimately alter gene expression in the
target cells (Figure 12-1a).The activity of cytokines was first recognized in the mid- 1960s. The susceptibility of
the target cell to a particular cytokine is determined by the presence of specific membrane receptors. In general,
the cytokines and their receptors exhibit very high affinity for each other. A particular cytokine may bind to
receptors on the membrane of the same cell that secreted it, exerting autocrine action; it may bind to receptors
on a target cell in close proximity to the producer cell, exerting paracrine action; in a few cases, it may bind to
target cells in distant parts of the body, exerting endocrine action. For example, cytokines produced by
activated TH cells can influence the activity of B cells, T C cells, natural killer cells, macrophages, granulocytes,
and hematopoietic stem cells, thereby activating an entire network of interacting cells. Cytokines exhibit the
attributes of pleiotropy, redundancy, synergy, antagonism, and cascade induction, which permit them to regulate
cellular activity in a coordinated, interactive way. Many cytokines are referred to as interleukins, a name
indicating that they are secreted by some leukocytes and act upon other leukocytes. Interleukins 1–25 have been
identified.
Cytokines Belong to Four Structural Families:
Once the genes encoding various cytokines had been cloned, sufficient quantities of purified preparations
became available for detailed studies on their structure and function. Cytokines generally have a molecular mass
of less than 30 kDa. Structural studies have shown that the cytokines share a similar polypeptide fold, with four
_-helical regions (A–D) in which the first and second helices and the third and fourth helices run roughly
parallel to one another and are connected by loops.
Cytokines Have Numerous Biological Functions:
Although a variety of cells can secrete cytokines, the two principal producers are the TH cell and the
macrophage. Cytokines released from these two cell types activate an entire network of interacting cells (Figure
12-5). Among the numerous physiologic responses that require cytokine involvement are development of
cellular and humoral immune responses, induction of the inflammatory response, regulation of hematopoiesis,
control of cellular proliferation and differentiation, and the healing of wounds. Although the immune response
to a specific antigen may include the production of cytokines, it is important to remember that cytokines act in
an antigen-nonspecific manner.
TRANSPLANTATION
Transplantation as the term used in immunology, refers to the act of transferring cells,
tissues, or organs from one site to another. Many diseases can be cured by implantation of a
healthy organ, tissue, or cells (a graft) from one individual (the donor) to another in need of
the transplant. The seriousness of the donor organ shortage is reflected in the fact that, as of
November 2000, an estimated 73,000 patients in the United States were on the waiting list for
an organ transplantation. The immune system has evolved elaborate and effective
mechanisms to protect the organism from attack by foreign agents, and these same
mechanisms cause rejection of grafts from anyone who is not genetically identical to the
recipient. Alexis Carrel reported the first systematic study of transplantation in 1908; he
interchanged both kidneys in a series of nine cats. Some of those receiving kidneys from
other cats maintained urinary output for up to 25 days. Although all the cats eventually died,
the experiment established that a transplanted organ could carry out its normal function in the
recipient. The first human kidney transplant, attempted in 1935 by a Russian surgeon, failed
because there was a mismatch of blood types between donor and recipient. This
incompatibility caused almost immediate rejection of the kidney, and the patient died without
establishing renal function. The rapid immune response experienced here, termed hyperacute
rejection, is mediated by antibodies. The first successful human kidney transplant, which was
between identical twins, was accomplished in Boston in 1954. Today, kidney, pancreas,
heart, lung, liver, bone-marrow, and cornea transplantations are performed among non
identical individuals with ever increasing frequency and success. A variety of
immunosuppressive agents can aid in the survival of the transplants, including drugs and
specific antibodies developed to reduce the immunologic attack on grafts, but the majority of
these agents have an overall immunosuppressive effect. New methods of inducing specific
tolerance to the graft without suppressing other immune responses are being developed and
promise longer survival of transplants without compromise of host immunity.
IMMUNOLOGIC BASIS OF GRAFT REJECTION
The degree of immune response to a graft varies with the type of graft. The
following terms are used to denote different types of transplants:
Autograft is self-tissue transferred from one body site to another in the same individual.
Transferring healthy skin to a burned area in burn patients and use of healthy blood vessels to
replace blocked coronary arteries are examples of frequently used autografts.
Isograft is tissue transferred between genetically identical individuals. In inbred strains of
mice, an isograft can be performed from one mouse to another syngeneic mouse. In humans,
an isograft can be performed between genetically identical (monozygotic) twins.
Allograft is tissue transferred between genetically different members of the same species. In
mice, an allograft is performed by transferring tissue or an organ from one strain to another.
In humans, organ grafts from one individual to another are allografts unless the donor and
recipient are identical twins.
Xenograft is tissue transferred between different species (e.g., the graft of a baboon heart
into a human). Because of significant shortages in donated organs, raising animals for the
specific purpose of serving as organ donors for humans is under serious consideration.
Autografts and isografts are usually accepted, owing to the genetic
identity between graft and host. Obviously, xenografts exhibit the greatest genetic
dissimilarity and therefore show a vigorous graft rejection.
TRANSPLANTATION ANTIGEN:
Tissues that are antigenically similar are said to be histocompatible; such tissues do not
induce an immunologic response that leads to tissue rejection. Tissues that display significant
antigenic differences are histoincompatible and induce an immune response that leads to
tissue rejection. The MHC in mice is called as the H-2 complex and in humans it is called as
HLA complex.
HLA Typing:
Since differences in blood group and major histocompatibility antigens are
responsible for the most intense graft-rejection reactions, various tissue-typing procedures to
identify these antigens have been developed to screen potential donor and recipient cells.
Initially, donor and recipient are screened for ABO blood-group compatibility. The bloodgroup antigens are expressed on RBCs, epithelial cells, and endothelial cells. Antibodies
produced in the recipient to any of these antigens that are present on transplanted tissue will
induce antibodymediated complement lysis of the incompatible donor cells. HLA typing of
potential donors and a recipient can be accomplished with a microcytotoxicity test (Figure
21-4a, b). In this test, white blood cells from the potential donors and recipient are distributed
into a series of wells on a microtiter plate, and then antibodies specific for various class I and
class II MHC alleles are added to different wells. After incubation, complement is added to
the wells, and cytotoxicity is assessed by the uptake or exclusion of various dyes (e.g., trypan
blue or eosin Y) by the cells. If the white blood cells express the MHC allele for which a
particular monoclonal antibody is specific, then the cells will be lysed upon addition of
complement, and these dead cells will take up a dye such as trypan blue. HLA typing based
on antibody-mediated microcytotoxicity can thus indicate the presence or absence of various
MHC alleles. Even when a fully HLA-compatible donor is not available, transplantation may
be successful. In this situation, a one-way mixed-lymphocyte reaction (MLR) can be used to
quantify the degree of class II MHC compatibility between potential donors and a recipient
(Figure 21-4c). Lymphocytes from a potential donor that have been x-irradiated or treated
with mitomycin C serve as the stimulator cells, and lymphocytes from the recipient serve as
responder cells. Proliferation of the recipient T cells, which indicates T-cell activation, is
measured by the uptake of [3H]thymidine into cell DNA. The greater the class II MHC
differences between the donor and recipient cells, the more [3H]thymidine uptake will be
observed in an MLR assay. Intense proliferation of the recipient lymphocytes indicates a poor
prognosis for graft survival. The advantage of the MLR over microcytotoxicity typing is that
it gives a better indication of the degree of TH-cell activation generated in response to the
class II MHC antigens of the potential graft. The disadvantage of the MLR is that it takes
several days to run the assay. If the potential donor is a cadaver, for example, it is not
possible to wait for the results of the MLR, because the organ must be used soon after
removalfrom the cadaver. In that case, the microcytotoxicity test, which can be performed
within a few hours, must be relied on. HLA matching is most important for kidney and bonemarrow transplants; liver and heart transplants may survive with greater mismatching.
MHC identity of donor and host is not the sole factor determining tissue
acceptance. When tissue is transplanted between genetically different individuals, even if
their MHC antigens are identical, the transplanted tissue can be rejected because of
differences at various minor histocompatibility loci. The tissue rejection induced by minor
histocompatibility differences is usually less vigorous than that induced by major
histocompatibility differences. Still, reaction to these minor tissue differences often results in
graft rejection. For this reason, successful transplantation even between HLA-identical
individuals requires some degree of immune suppression.
MECHANISM OF GRAFT REJECTION:
The process of graft rejection can be divided into two stages:
(1) A sensitization phase, in which antigen-reactive lymphocytes of the recipient proliferate
in response to allo- antigens on the graft.
(2) An effector stage, in which immune destruction of the graft takes place.
SENSITIZATION STAGE:
During the sensitization phase, CD4+ and CD8+ T cells recognize alloantigens
expressed on cells of the foreign graft and proliferate in response. Both major and minor
histocompatibility alloantigens can be recognized. The response to major histocompatibility
antigens involves recognition of both the donor MHC molecule and an associated peptide
ligand in the cleft of the MHC molecule. The peptides present in the groove of allogeneic
class I MHC molecules are derived from proteins synthesized within the allogeneic cell. The
peptides present in the groove of allogeneic class II MHC molecules are generally proteins
taken up and processed through the endocytic pathway of the allogeneic antigen-presenting
cell. Depending on the tissue, different populations of cells within a graft may function as
APCs. Because dendritic cells are found in most tissues and because they constitutively
express high levels of class II MHC molecules, dendritic cells generally serve as the major
APC in grafts. APCs of host origin can also migrate into a graft and endocytose the foreign
alloantigens (both major and minor histocompatibility molecules) and present them as
processed peptides together with self-MHC molecules. In some organ and tissue grafts (e.g.,
grafts of kidney, thymus, and pancreatic islets), a population of donor APCs called passenger
leukocytes has been shown to migrate from the graft to the regional lymph nodes. These
passenger leukocytes are dendritic cells, which express high levels of class II MHC
molecules (together with normal levels of class I MHC molecules) and are widespread in
mammalian tissues, with the chief exception of the brain. Because passenger leukocytes
express the allogeneic MHC antigens of the donor graft, they are recognized as foreign and
therefore can stimulate immune activation of T lymphocytes in the lymph node. Recognition
of the alloantigens expressed on the cells of a graft induces vigorous T-cell proliferation in
the host. Both dendritic cells and vascular endothelial cells from an allogeneic graft induce
host T-cell proliferation. The major proliferating cell is the CD4+ T cell, which recognizes
class II alloantigens directly or alloantigen peptides presented by host antigen-presenting
cells. This amplified population of activated TH cells is thought to play a central role in
inducing the various effector mechanisms of allograft rejection.
EFFECTOR STAGE
A variety of effector mechanisms participate in allograft rejection (Figure 21-6). The most
common are cell-mediated reactions involving delayed-type hypersensitivity and
CTLmediated cytotoxicity; less common mechanisms are antibodyplus- complement lysis
and destruction by antibody-dependent cell-mediated cytotoxicity (ADCC).
Graft rejection shows Sensitization and Memory:
The rate of allograft rejection varies according to the tissue involved. In general, skin grafts
are rejected faster than other tissues such as kidney or heart. If an inbred mouse of strain A is
grafted with skin from strain B, primary graft rejection, known as first-set rejection, occurs
(Figure 21-1b). The skin first becomes revascularized between days 3 and 7; as the reaction
develops, the vascularized transplant becomes infiltrated with lymphocytes, monocytes,
neutrophils, and other inflammatory cells. There is decreased vascularization of the
transplanted tissue by 7–10 days, visible necrosis by 10 days, and complete rejection by 12–
14 days. Immunologic memory is demonstrated when a second strain-B graft is transferred to
a previously grafted strain-A mouse. In this case, a graft-rejection reaction develops more
quickly, with complete rejection occurring within 5–6 days; this secondary response is
designated second-set rejection.
TUMOR IMMUNOLOGY
From an immunologic perspective, cancer cells can be viewed as altered
self-cells that have escaped normal growth-regulating mechanisms. The immune responses
that develop to cancer cells, as well as the methods by which cancers manage to evade those
responses, are then described. In most organs and tissues of a mature animal, a balance is
usually maintained between cell renewal and cell death. The various types of mature cells in
the body have a given life span; as these cells die a new cells are generated by the
proliferation and differentiation of various types of stem cells. Under normal circumstances,
the production of new cells is regulated so that the number of any particular type of cell
remains constant. Occasionally, though, cells arise that no longer respond to normal growthcontrol mechanisms. These cells give rise to clones of cells that can expand to a considerable
size, producing a tumor, or neoplasm. A tumor that is not capable of indefinite growth and
does not invade the healthy surrounding tissue extensively is benign. A tumor that continues
to grow and becomes progressively invasive is malignant; the term cancer refers specifically
to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit
metastasis; in this process, small clusters of cancerous cells dislodge from a tumor, invade
the blood or lymphatic vessels, and are carried to other tissues, where they continue to
proliferate. In this way a primary tumor at one site can give rise to a secondary tumor at
another site (Figure 22-1).
Types of Tumor (or) Cancer:
Malignant tumors or cancers are classified according to the embryonic origin of
the tissue from which the tumor is derived. Most (>80%) are carcinomas, tumors that arise
from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs
and glands. The majority of cancers of the colon, breast, prostate, and lung are carcinomas.
The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone
marrow and account for about 9% of cancer incidence in the United States. Leukemias
proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Sarcomas,
which arise less frequently are derived from mesodermal connective tissues such as bone, fat,
and cartilage.
Causes of Tumor:
Transformation-Treatment of normal cultured cells with chemical carcinogens, irradiation,
and certain viruses can alter their morphology and growth properties. In some cases this
process, referred to as transformation, makes the cells able to produce tumors when they are
injected into animals. Such cells are said to have undergone malignant transformation.
Chemical Agents- Various chemical agents (e.g.,DNA-alkylating reagents) and physical
agents (e.g., ultraviolet light and ionizing radiation) that cause mutations have been shown to
induce transformation. Methylcholanthrene and ultraviolet light are two carcinogens that
have been used extensively to generate lines of tumor cells
Viruses- A number of DNA and RNA viruses have been shown to induce malignant
transformation. Two of the best-studied are SV40 and polyoma. Most RNA viruses replicate
in the cytosol and do not induce malignant transformation. The exceptions are retroviruses,
which transcribe their RNA into DNA by means of a reverse-transcriptase enzyme and then
integrate the transcript into the host’s DNA. This process is similar in the cytopathic
retroviruses such as HIV-1 and HIV-2 and in the transforming retroviruses, which induce
changes in the host cell that lead to malignant transformation. In some cases, retrovirusinduced transformation is related to the presence of oncogenes, or “cancer genes,” carried by
the retrovirus. One of the best-studied transforming retroviruses is the Rous sarcoma virus.
Oncogenes-They are tumor causing genes. Oncogenes and tumor suppressor genes have been
shown to play an important role in this process, by regulating either cellular proliferation or
cell death.
TUMOR ANTIGENS:
The subdiscipline of tumor immunology involves the study of antigens on tumor cells
and the immune response to these antigens. Two types of tumor antigens have been identified
on tumor cells: tumor-specific transplantation antigens (TSTAs) and tumor-associated
transplantation antigens. Tumor-specific antigens are unique to tumor cells and do not
occur on normal cells in the body. Tumor-associated antigens, which are not unique to tumor
cells, may be proteins that are expressed on normal cells during fetal development when the
immune system is immature and unable to respond but that normally are not expressed in the
adult.
Tumor specific antigen (e.g) Chemically or Physically induced Tumor antigens, viral
antigens.
Tumor associated antigen (e.g)
1)Oncofetal tumor antigen: Oncofetal tumor antigens, as the name implies, are found
not only on cancerous cells but also on normal fetal cells. These antigens appear early
in embryonic development, before the immune system acquires immunocompetence;
if these antigens appear later on cancer cells, they are recognized as nonself and
induce an immunologic response.
2)Oncogene proteins as tumor antigen: A number of tumors have been shown to
express tumorassociated antigens encoded by cellular oncogenes. These antigens are
also present in normal cells encoded by the corresponding proto-oncogene. In many
cases, there is no qualitative difference between the oncogene and proto-oncogene
products; instead, the increased levels of the oncogene product can be recognized by
the immune system.
IMMUNE RESPONSE TO TUMOR:
Humoral And Cell-Mediated Immune Response: In experimental animals, tumor antigens
can be shown to induce both humoral and cell-mediated immune responses that result in the
destruction of the tumor cells. In general, the cell-mediated response appears to play the
major role.
CTLS Tumor Specific: A number of tumors have been shown to induce tumor-specific
CTLs that recognize tumor antigens presented by class I MHC on the tumor cells. However
as discussed below, expression of class I MHC molecules are decreased in a number of
tumors, thereby limiting the role of specific CTLs in their destruction.
Role of NK cells: The recognition of tumor cells by NK cells is not MHC restricted. Thus,
the activity of these cells is not compromised by the decreased MHC expression exhibited by
some tumor cells. In some cases, Fc receptors on NK cells can bind to antibody-coated tumor
cells, leading to ADCC.
Macrophages: Numerous observations indicate that activated macrophages also play a
significant role in the immune response to tumors. For example, macrophages are often
observed to cluster around tumors, and their presence is often correlated with tumor
regression.
IMMUNE SURVEILLANCE MECHANISM:
The immune surveillance theory was first conceptualized in the early 1900s by
Paul Ehrlich.He suggested that cancer cells frequently arise in the body but are recognized as
foreign and eliminated by the immune system. Some 50 years later, Lewis Thomas suggested
that the cell-mediated branch of the immune system had evolved to patrol the body and
eliminate cancer cells.According to these concepts; tumors arise only if cancer cells are able
to escape immune surveillance, either by reducing their expression of tumor antigens or by
impairment in the immune response to these cells. Among the early observations that seemed
to support the immune surveillance theory was the increased incidence of cancer in
transplantation patients on immunosuppressive drugs. Other findings, however, were difficult
to reconcile with this theory. Nude mice, for example, lack a thymus and consequently lack
functional T cells. According to the immune surveillance theory, these mice should show an
increase in cancer, instead, nude mice are no more susceptible to cancer than other mice.
Furthermore, although individuals on immunosuppressive drugs do show an increased
incidence of cancers of the immune system, other common cancers (e.g., lung, breast, and
colon cancer) are not increased in these individuals, contrary to what the theory predicts. One
possible explanation for the selective increase in immune system cancers is that the
immunosuppressive agents themselves may exert a direct carcinogenic effect on immune
cells. Experimental data concerning the effect of tumor-cell dosage on the ability of the
immune system to respond also are incompatible with the immune surveillance theory. For
example, animals injected with very low or very high doses of tumor cells develop tumors,
whereas those injected with intermediate doses do not. The mechanism by which a low dose
of tumor cells “sneaks through” is difficult to reconcile with the immune surveillance theory.
Finally, this theory assumes that cancer cells and normal cells exhibit qualitative antigen
differences. In fact, as stated earlier, many types of tumors do not express tumor-specific
antigens, and any immune response that develops must be induced by quantitative differences
in antigen expression by normal cells and tumor cells. However, tumors induced by viruses
would be expected to express some antigens encoded by the viral genome. These antigens are
qualitatively different from those expressed by normal tissues and would be expected to
attract the attention of the immune system. In fact, there are many examples of specific
immune responses to virally induced tumors. Nevertheless, apart from tumors caused by
viruses, the basic concept of the immune surveillance theory—that malignant tumors arise
only if the immune system is somehow impaired or if the tumor cells lose their
immunogenicity, enabling them to escape immune surveillance—at this time remains
unproved. In spite of this, it is clear that an immune response can be generated to tumor cells,
and therapeutic approaches aimed at increasing that response may serve as a defense against
malignant cells.
IMMUNOLOGIC ENHANCEMENT OF TUMOR GROWTH:
Although the immune system clearly can respond to tumor cells, the fact that so many
individuals die each year from cancer suggests that the immune response to tumor cells is
often ineffective. Following the discovery that antibodies could be produced to tumorspecific antigens, attempts were made to protect animals against tumor growth by active
immunization with tumor antigens or by passive immunization with antitumor antibodies.
Much to the surprise of the researchers, these immunizations did not protect against tumor
growth; in many cases, they actually enhanced growth of the tumor.
Modulation of Tumor antigens: Certain tumor-specific antigens have been observed to
disappear from the surface of tumor cells in the presence of serum antibody and then to
reappear after the antibody is no longer present. This phenomenon, called antigenic
modulation, is readily observed when leukemic T cells are injected into mice previously
immunized with a leukemic T-cell antigen
Reduction in Class I MHC molecule: Since CD8+ CTLs recognize only antigen associated
with class I MHC molecules, any alteration in the expression of class I MHC molecules on
tumor cells may exert a profound effect on the CTL-mediated immune response. Malignant
transformation of cells is often associated with a reduction (or even a complete loss) of class I
MHC molecules, and a number of tumors have been shown to express decreased levels of
class I MHC molecules.
ENZYME-LINKED IMMUNOSORBENT ASSAY
Enzyme-linked immunosorbent assay, commonly known as ELISA (or EIA), is similar in
principle to RIA but depends on an enzyme rather than a radioactive label. An enzyme
conjugated with an antibody reacts with a colorless substrate to generate a colored reaction
product. Such a substrate is called a chromogenic substrate. A number of enzymes have
been employed for ELISA, including alkaline phosphatase, horseradish peroxidase, and
galactosidase. These assays approach the sensitivity of RIAs and have the advantage of being
safer and less costly.
There Are Numerous Variants of ELISA:
A number of variations of ELISA have been developed, allowing qualitative detection or
quantitative measurement of either antigen or antibody. Each type of ELISA can be used
qualitatively to detect the presence of antibody or antigen. Alternatively, a standard curve
based on known concentrations of antibody or antigen is prepared, from which the unknown
concentration of a sample can be determined.
INDIRECT ELISA:
Antibody can be detected or quantitatively determined with an indirect ELISA
(Figure 6-10a). Serum or some other sample containing primary antibody (Ab1) is added to
an antigen- coated microtiter well and allowed to react with the antigen attached to the well.
After any free Ab1 is washed away, the presence of antibody bound to the antigen is detected
by adding an enzyme-conjugated secondary anti-isotype antibody (Ab2), which binds to the
primary antibody. Any free Ab2 then is washed away, and a substrate for the enzyme is
added. The amount of colored reaction product that forms is measured by specialized
spectrophotometric plate readers, which can measure the absorbance of all of the wells of a
96-well plate in seconds. Indirect ELISA is the method of choice to detect the presence of
serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.
In this assay, recombinant envelope and core proteins of HIV are adsorbed as solid-phase
antigens to microtiter wells. Individuals infected with HIV will produce serum antibodies to
epitopes on these viral proteins. Generally, serum antibodies to HIV can be detected by
indirect ELISA within 6 weeks of infection.
SANDWICH ELISA:
Antigen can be detected or measured by a sandwich ELISA (Figure 6-10b). In this
technique, the antibody (rather than the antigen) is immobilized on a microtiter well. A
sample containing antigen is added and allowed to react with the immobilized antibody. After
the well is washed, a second enzyme- linked antibody specific for a different epitope on the
antigen is added and allowed to react with the bound antigen. After any free second antibody
is removed by washing, substrate is added, and the colored reaction product is measured.
COMPETITIVE ELISA
Another variation for measuring amounts of antigen is competitive ELISA (Figure 610c). In this technique, antibody is first incubated in solution with a sample containing
antigen. The antigen-antibody mixture is then added to an antigencoated microtiter well. The
more antigen present in the sample, the less free antibody will be available to bind to the
antigen-coated well. Addition of an enzyme-conjugated secondary antibody (Ab2) specific
for the isotype of the primary antibody can be used to determine the amount of primary
antibody bound to the well as in an indirect ELISA. In the competitive assay, however, the
higher the concentration of antigen in the original sample, the lower the absorbance.
IMMUNOFLUORESCENCE
In 1944, Albert Coons showed that antibodies could be labeled with molecules that
have the property of fluorescence. Fluorescent molecules absorb light of one wavelength
(excitation) and emit light of another wavelength (emission). If antibody molecules are
tagged with a fluorescent dye, or fluorochrome, immune complexes containing these
fluorescently labeled antibodies (FA) can be detected by colored light emission when excited
by light of the appropriate wavelength. Antibody molecules bound to antigens in cells or
tissue sections can similarly be visualized. The emitted light can be viewed with a
fluorescence microscope,which is equipped with a UV light source. In this technique, known
as Immunofluorescence or Fluorescent antibody technique, fluorescent compounds such
as fluorescein and rhodamine are in common use, but other highly fluorescent substances are
also routinely used, such as phycoerythrin, an intensely colored and highly fluorescent
pigment obtained from algae. These molecules can be conjugated to the Fc region of an
antibody molecule without affecting the specificity of the antibody. Each of the
fluorochromes below absorbs light at one wavelength and emits light at a longer wavelength:
Fluorescein- an organic dye that is the most widely used label for immunofluorescence
procedures, absorbs blue light (490 nm) and emits an intense yellow-green fluorescence (517
nm).
Rhodamine- another organic dye, absorbs in the yellow-green range (515 nm) and emits a
deep red fluorescence (546 nm). Because it emits fluorescence at a longer wavelength than
fluorescein, it can be used in two-color immunofluorescence assays. An antibody specific to
one determinant is labeled with fluorescein, and an antibody recognizing a different antigen
is labeled with rhodamine.
Phycoerythrin-is an efficient absorber of light (~30-fold greater than fluorescein) and a
brilliant emitter of red fluorescence, stimulating its wide use as a label for
immunofluorescence.
Fluorescent-antibody staining of cell membrane molecules or tissue sections can be direct or
indirect (Figure 6-14).
In direct staining, the specific antibody (the primary antibody) is directly conjugated with
fluorescein;
In indirect staining, the primary antibody is unlabeled and is detected with an additional
fluorochrome-labeled reagent. A number of reagents have been developed for indirect
staining.
APPLICATION:
1. Immunofluorescence has been applied to identify a number of subpopulations of
lymphocytes, notably the CD4_ and CD8_ T-cell subpopulations.
2. The technique is also suitable for identifying bacterial species.
3. For detecting Ag-Ab complexes in autoimmune disease.
4. For detecting complement components in tissues, and localizing hormones and other
cellular products stained in situ.
5. Major application of the fluorescent-antibody technique is the localization of antigens in
tissue sections or in subcellular compartments.
RADIO IMMUNO ASSAY
One of the most sensitive techniques for detecting antigen or antibody is
radioimmunoassay (RIA). The technique was first developed in 1960 by two
endocrinologists, S. A. Berson and Rosalyn Yalow, to determine levels of insulin–anti-insulin
complexes in diabetics. It’s used for measuring hormones, serum proteins, drugs, and
vitamins at concentrations of 0.001 micrograms per milliliter or less. In 1977, some years
after Berson’s death, the significance of the technique was acknowledged by the award of a
Nobel Prize to Yalow.
The principle of RIA involves competitive binding of radiolabeled antigen and
unlabeled antigen to a high-affinity antibody.
The labeled antigen is mixed with antibody at a concentration that saturates the antigenbinding sites of the antibody. Then test samples of unlabeled antigen of unknown
concentration are added in larger amounts. The antibody does not distinguish labeled from
unlabeled antigen, so the two kinds of antigen compete for available binding sites on the
antibody. As the concentration of unlabeled antigen increases, more labeled antigen will be
displaced from the binding sites. The decrease in the amount of radiolabeled antigen bound to
specific antibody in the presence of the test sample is measured in order to determine the
amount of antigen present in the test sample. The antigen is generally labeled with a gamma-
emitting isotope such as 125I, but beta-emitting isotopes such as tritium (3H) are also routinely
used as labels. The radiolabeled antigen is part of the assay mixture; the test sample may be a
complex mixture, such as serum or other body fluids, that contains the unlabeled antigen. The
first step in setting up an RIA is to determine the amount of antibody needed to bind 50%–
70% of a fixed quantity of radioactive antigen (Ag) in the assay mixture. This ratio of
antibody to Ag is chosen to ensure that the number of epitopes presented by the labeled
antigen always exceeds the total number of antibody binding sites. Consequently, unlabeled
antigen added to the sample mixture will compete with radiolabeled antigen for the limited
supply of antibody. Even a small amount of unlabeled antigen added to the assay mixture of
labeled antigen and antibody will cause a decrease in the amount of radioactive antigen
bound, and this decrease will be proportional to the amount of unlabeled antigen added. To
determine the amount of labeled antigen bound, the Ag-Ab complex is precipitated to
separate it from free antigen (antigen not bound to Ab), and the radioactivity in the
precipitate is measured. A standard curve can be generated using unlabeled antigen samples
of known concentration (in place of the test sample), and from this plot the amount of antigen
in the test mixture may be precisely determined. Several methods have been developed for
separating the bound antigen from the free antigen in RIA. One method involves precipitating
the Ag-Ab complex with a secondary anti-isotype antiserum. After removal of the complex
by either of these methods, the amount of free labeled antigen remaining in the supernatant
can be measured in a radiation counter; subtracting this value from the total amount of
labeled antigen added yields the amount of labeled antigen bound.Various solid-phase RIAs
have been developed that make it easier to separate the Ag-Ab complex from the unbound
antigen. In some cases, the antibody is covalently crosslinked to Sepharose beads. The
amount of radiolabeled antigen bound to the beads can be measured after the beads have been
centrifuged and washed.Alternatively, the antibody can be immobilized on polystyrene or
polyvinylchloride wells and the amount of free labeled antigen in the supernatant can be
determined in a radiation counter. In another approach, the antibody is immobilized on the
walls of microtiter wells and the amount of bound antigen determined. For example, a
microtiter RIA has been widely used to screen for the presence of the hepatitisB virus (Figure
6-9). RIA screening of donor blood has sharply reduced the incidence of hepatitis B
infections in recipients of blood transfusion.
IMMUNOLOGY-Important questions
1. Inflammatory response
2. Complement fixation test
3. Phagocytosis
4. Immunoflurescense
5. Primary and secondary immune response
6. Cascade regulation
7. Interferon & Interleukins
8. Precipitation test
9. Agglutination test
10. Write about Humoral and cell mediated immune response.
11. Write in detail about the different pathways of complement activation
12. Give the principle and procedures of immunoelectrophoresis
13. Describe the various antigen-antibody reactions and their characteristics
14. How B cells & T cells are get activated
15. Write about the structure and function of lymphoid organs with neat Diagram
17. Describe hypersensitive reactions and its types
18. Write in detail about the cells involved in the immune response
19. Explain the innate and Acquired immune response of our body
20. Define antibody? Explain the different types of immunoglobulins with neat labeled sketch
21. Write about monoclonal antibody and its application
22. Define Hematopoiesis? Development of B and t lymphocytes
23. Give detailed notes on immunoglobulin Gene expression
24. Write about primary and secondary Immunodeficiency disorderes.
25. Write about auto immune diseases
26. Write about transplantation and mechanism of graft rejection
27. Give detailed notes on Tumor immunology and different types of ELISA
28. Write about the principle and application of RIA
29. Give detailed notes on HLA typing
30. Write about Cytokine structure and its function
31. Explain Tolerance and Suppression in Immunoregulation
32. Describe Plasma and Memory cells
33. Explain antigen its types and application
34. Write in detail about cell mediated immune response
35. Write about the historical developments in Immunology
All the Best