HBB 2306 CELL AND MOLECULAR IMMUNOLOGY 1

Introduction to the mammalian defense system (The human system)

IMPORTANCE

1. Understand the components (tissues, cells and molecules)and their role in body identity and
protection/defense
2. Use of body defense components in immunodiagnostics (ELISA, FIA, Western blot, microscopy)
Immunogenetics/molecular (PCR and hybridization)
3. Use of body defense components in therapy (antibody and cell mediated CART- cell therapy in
cancer treatment, biotech medicine)
4. Use of body defense components in tissue matching prior to organ grafting/transplants
5. Vaccination (whole organism, antigen cloned vector, purified or cloned antigen, gene transcript)

INNATE DEFENSE SYSTEM

- Skin
- Eyelashes, eyelids and conjunctiva
- Buccal cavity and alimentary canal
- Respiratory system (tract)
- Urogenital tract
- Ear wax
- Endocytosis/phagocytosis (Phagocytes and epithelial/endothelial cells)
- Inflammation
- Complement
- Blood clotting system
- Surveillance cells (Natural killer cells, macrophages, dentritic cells)

ADAPTIVE DEFENSE SYSTEM

1. Organs (Bone marrow, Thymus, spleen and secondary lymph nodes)

2. White blood cells (T and B Lymphocytes, large granular lymphocytes, Natural killer cells,
macrophages and monocytes, polymorphonuclear cells like basophils, neutrophils and
eosinophils, dendritic cells).

3. Proteins (Interferons, MHC 1 and MHC II proteins, cytokines, growth factors/hormones and
antibodies)

The Innate defence

It is protective, not inducible but constitutive and non-specific. It is enabled by the following anatomic
and physiologic features.

The skin has the epidermis and dermis which form an impervious barrier. It consists of the sebaceous
glands which line the sweat pores and secrete sebum. Sebum is rich in lactic acid and fatty acids. The
oily property of sebum prevents skin from cracking .The sticky and acid environment is hostile to
microbial growth.

Eyelids, lashes and conjunctiva form a physical barrier to microbial entry into the eye internal. Tears
secreted by lachrymal glands from the eye contain lysozyme which breaks down bacterial cell wall. This
fluid also mixes with sticky sebum from Meibomian and Moll/Zeis glands that open into the eyelid
follicles.

Buccal cavity and alimentary canal has an inner epidermal lining acting as an anatomical barrier.
Salivary glands secrete saliva which has sticky mucopeptides and lysozyme. The stomach is also lined by
an epidermal layer covered with protective mucopeptides (mucus). Gastric glands parietal, chief and
foveolar cells produce acid and mucin rich in hydrolytic enzymes. It creates a hostile environment for
bacterial growth.

Respiratory tract has an epidermal lining covered with protective mucus (from mucous secreting
goblet cells). It is lined with cilia that participates in mucociliary action. The act of sneezing and
coughing is instinctive/physiologic and serves to eject solids and parasites which gain entry into the
respiratory tract.

Ear contains wax which is sticky and therefore traps dust and microbes which gain entry into the inner
ear.

Urogenital tract has an epidermal lining covered with protective mucus containing lysozyme. The
flushing of this tract with urine lowers the pH and therefore making it hostile to microbial growth.

Interferons (alpha, beta and gamma) are released by body cells after viral infection. They are anti-viral.
They prepare the non infected cells to guard against invading viruses.

Endocytosis is practiced by the epidermal, epithelial and endothelial cells which line tissues and organs
of the body. This process allows cells to ingest soluble materials proximal to cell surface. The cells form
endocytic vesicles and endosomes. The ingestion is facilitated by receptors on the cell surface. The
endosomes fuse with cell lysosomes rich in hydrolytic enzymes. The ingested material is digested in
formed secondary lysosomes to harmless simple molecules readily recycled or secreted from the tissue.

Pinocytosis is similar to endocytosis. However the process is not facilitated by receptors.

Phagocytosis resembles endocytosis but practiced by phagocytes (macrophages, monocytes, dendritic

cells and neutrophils). Phagocytes engulf solid materials, cells and immune complexes to form
phagosomes. These vesicles fuse with lysosomes to form phagolysosomes where digestion occurs.

Inflammatory response occurs after the body is injured. The body may receive chemical, burn, bite or
cut injuries rendering it vulnerable to infection by viruses, bacteria or other parasites.

The observable signs of inflammation include;

1. Swelling
2. Pain
3. Redness which is conspicuous in light colored areas
4. High temperature
5. Loss of function where injury is extensive.

The injury induces mast cells to secrete histamine which has the following influence;

- Vasodilation of blood vessels

- Increased permeability of blood vessels which carry blood to the injured site. This allows blood
rich in natural antibodies, serum proteins like clotting factors, complement proteins, acute
phase proteins and kinnins to reach the injured site. Bradykinin is a product of high mol wt
kinnin hydrolysis by kallikren (released from blood clotting). It induces sensation of pain.
- Vasoconstriction of vessels which carry blood away from the injured site.
 - Influx of effector cells (leukocytes, basophils and platelets) and natural antibodies. Effector cells
release more physioactive agents like leukotriene, prostaglandins, interleukin (IL-1, 6), growth
factors (GM-CSF and TNF alpha) and serotonin enhancing inflammation.

This response creates an enabling environment for the body to fight/protect against disease and
initiates healing.

Show the scheme of blood clotting pathway (activated when blood vessels are injured)

It is comprised of clotting factors synthesized as inactive protein proteases (zymogens) by the liver. They
are activated after tissue injury. The role of clotting is preventing blood loss from leaking vessels and
entry of infectious agents/toxins in the systemic system (septicemia).

There are two pathways of activation

The Intrinsic path

This path is activated when injury is internal to the blood vessels (endothelial wall is disrupted):
Exposed Collagen activates platelets which become sticky and form a plug at site of injury. Factor 12
from liver is activated by plasma pre- kallikrein initiating the intrinsic activation path (Factor 11, 9, 10, 5,
Prothrombin to thrombin and fibrinogen to insoluble fibrin). Fibrin forms an insoluble network with
platelets closing the injured site. Activated factor 12 (F12a) activates Pre-kallikrein to kallikrein. Kallikrein
in turn breaks down HMWK to small peptides like bradykinin. Calcium ions are critical to the activation
process. (Factor 8 is a cofactor and enables hydrolysis of factor 10 by activated factor 9 (F9a). Factor 5
(labile factor) is also a cofactor which facilitates hydrolysis of prothrombin (factor 2) by factor 10a.)

The Extrinsic path

This path is activated by injury external to blood vessels. Tissue injury exposes sub-endothelial layer of
vessels releasing tissue factor. This factor activates factor VII. Factor VII activates factor 10. In presence
of factor 5, Prothrombin is hydrolysed to thrombin by factor 10. Soluble fibrinogen is hydrolysed to
insoluble fibrin that forms an insoluble plug (fibrin clot) with activated platelets.

The acute phase proteins comprise of C-reactive proteins for example mannan binding lectins (MBLs)
which bind carbohydrate structures on surface of microbes namely N-acetylglucosamine, mannosamine
thus generating MBL-microbial complex. This complex activates lectin path of complement.

Complement is a major defense system of the body. It is innate in character. It comprises about 20
major proteins of serum synthesized by the liver. It forms about 10 % of total serum proteins. The
proteins are synthesized as pro-enzymes but activated through hydrolysis in a cascade mechanism.

Role of complement

- Forms a microbial /cell attack complex

- Activates leukocytes
- Attracts leukocytes to site of infection
- Triggers mediator release from basophils, platelets and mast cells.
- Enhances vessel dilation, constriction and permeability.
- Removes immune complexes
- Opsonizes microbes for destruction

Three arms of complement activation;

- Lectin activation pathway

 - Alternate activation pathway
- Classical activation pathway

Show the scheme of complement activation system

Regulation of complement

Serpin: Inhibits complement factor C1. It inhibits activity of serine proteases C1r and C1s in classical
pathway. It also inhibits binding of MBLs to MASPs in lectin path.

The activities of C3 convertases are controlled by Decay Acceleration Factors (DAF) and Membrane
Cofactor Proteins (MCPs) located on the host surface membranes. They protect host cell damage by
complement. These inhibitors also destroy C5 convertases when free in plasma (not bound to cells).

Factor H and Factor I in plasma collaborate to destroy C3b and its convertase free in plasma. C3b is
converted to C3bH and iC3b which are degraded by serum proteases.

The C4b binding protein regulates activated factor C4b

The transient trimolecular complex has cell binding site therefore readily binds cells. When in free form
the site is vulnerable to hydrolysis. Fluid phase regulators like vitronectin and clusterin bind the complex
and prime it for destruction by proteases.

The binding of factor C8 to the transient complex in free/fluid form prevents it from forming MAC.

ADAPTIVE DEFENSE

This is a form of immune response which displays specificity, diversity, memory and self/non self
recognition. It is a form of defense which leads to acquired immunity against disease.

It is facilitated by the following organs;

1. Bone marrow is the site of hematopoiesis (stem cells grow and differentiate to become
functional white and red blood cells.
2. Thymus is the site for T lymphocyte maturation
3. Spleen is the site where blood is filtered, damaged blood cells are removed by phagocytes and
lymphocytes interact with antigens (secondary lymphoid organ)
4. Lymph nodes are secondary lymphoid organs where white blood cells (macrophages, dendritic
cells and helper T cells) interact with antigen initiating an immune response.

Major players of adaptive defense are lymphocytes. The B-lymphocytes originate and mature in the
bone marrow. The T-lymphocytes originate in the bone marrow but mature in the thymus.

There are two forms of immune response;

1. Humoral immune response; This response targets extracellular antigens. The major player is the
B cell and antibodies. Antibodies tag antigens for destruction by immune effector cells. They also
neutralize toxins. The B cell is activated to form a plasma cell or memory B cell.
2. The cell mediated response; This response targets intracellular antigens. It is mediated by T
cells. The T helper cells activate macrophages, monocytes and neutrophils to engulf foreign or
infected cells while the cytotoxic T cells destroy foreign and infected cells.

The structure and role of immune cells

The B lymphocyte
It is derived from the bone marrow stem cells. It matures in the bone marrow and leave expressing
unique antigen binding receptors (antibodies). B cells exit the bone marrow as naïve cells but when they
encounter an antigen in spleen or other secondary lymphoid organs, they are activated and divide
rapidly. They may eventually differentiate to form plasma cells. Plasma cells secrete antibodies into the
surrounding fluids (blood and lymph). They are capable of secreting 2000 antibody molecules per
second and have a short life span. The activated B cells may also form memory cells providing immunity
to the body. The memory cells express surface antibody (BCR) receptors for a unique antigen.

The structure of B-cells

The T lymphocytes

These cells are developed in the bone marrow from stem cells. They are subsequently released as
immature thymocytes which migrate to the thymus. In the thymus, the cells develop to form T cell
receptors of the type alpha and beta (heterodimers) or the rare type gamma and delta. The subunits are
linked by disulfide linkage. In addition they express CD3 receptor which facilitates signaling in the T cell
and therefore activation of the cells.

There are two types of T lymphocytes;

The helper T cell which expresses a unique co-receptor called CD4 receptor. This receptor assists the cell
to target antigen presenting cells which express antigen complexed to major histocompatibility complex
(MHC) protein on their surface. The CD4 cell once activated releases cytokines and growth factors. The
cell is capable of mediating humoral or cell mediated immunity. It is referred to as MHC II restricted
lymphocyte. Through production of cytokines this cell influences the following actions;

1. Hematopoiesis
2. Cytotoxic T cell activity
3. Attracting immune cells (chemotactic factors)
4. Suppressing immune response
5. Growth of other T cells
6. B cell function
7. Natural killer cell activity and macrophages

There are two subtypes of Helper T cell; the type one which secrete cytokines for cell mediated
response and type two which secrete cytokines for humoral response.

The cytotoxic T cell expresses the CD8 co-receptor alongside the TCR. This receptor allows this cell to
bind foreign antigens bound to MHC class 1 protein on the target cell surface. Cells targeted by this
lymphocyte include grafted, cancerous and intracellular infected. The CTL destroys cells by direct
cytotoxicity. The cell comprises internal vesicles rich in perforin (amphipathic), TNFα and granzymes A-G.

When bound to target cell it secretes the vesicular contents on surface of target poking holes and
inducing apoptosis. The cell also releases interferon gamma which reduces spread of viral infection. It
also secretes interleukin 2 which enhances and sustains its own activation (autoactivation) during an
immune response.

Show the structure of T-cells

Natural Killer cells

They constitute about 15% of blood lymphocytes and are capable of releasing interferon gamma,
interleukin-1 and GM-CSF cytokines. They express CD 16 and CD56 receptors on their surface. They play an
important role in immune surveillance. They are classified as large granular lymphocytes but lack CD 3
receptors on their surface. They attack their cell targets through antibodies. They have a receptor for the
constant antibody fragment (FcγRIII). The binding of the NKCs to target cells through targeting antibody
antigen complexes activates the immune cell. The NKCs are also capable of binding their target cells
through glycoprotein receptors, FAS receptors and phosphocholine receptors on target surface.

The NKCs eliminate their target cells by releasing cytotoxic factors from their cytoplasmic granules. They
release cytolysin/perforin onto the surface of target cells. In the presence of Mg2+ ions, the agent
polymerises with the cell membrane forming transmembrane pore (amphipathic). Other contents of the
NKCs granules include granzyme B (serine proteases) and tumor necrosis factor. Both flow into the
target cell internal inducing apoptosis.

Show the structure of Natural Killer cell

THE POLYMORPHONUCLEAR GRANULOCYTES (POLYMORPHS)

They are comprised of eosinophils, neutrophils and basophils. They contribute to the white blood cell
population and categorized as phagocytes. They have regular plasma membranes and lobed nuclei.

The Eosinophil

This cell has a smooth membrane and bilobed nucleus. It has several cytoplasmic granules which stain
red with acid dyes like Eosin. It contributes to 2 – 5% of leukocyte population in blood. The cell is
capable of phagocytosis. It kills phagocytosed microbes with its toxins in the granules. The toxins include
major basic protein (MBP), a potent toxin for helminthes worms. The toxin also induces histamine
release from mast cells and activates neutrophils and platelets. The other toxin from the granules is
eosinophil derived neurotoxin (EDN). The cell is capable of releasing granular contents on surface of
target once activated and bound to the target. The target for this cell include extracellular worms like
schistosomes, plasmodium, tapeworms and roundworms. The cell has receptors for antibody E (FcԐRI)
which facilitate its binding to the target (antibody mediated cell binding).

Show the structure of eosinophil

The Basophil

This cell accounts for only 0.2% of leukocytes in blood. It has a multilobed nucleus and granules which
stain blue with basic dyes like haematoxylin. These granules are rich in physioactive agent like
leukotrienne which when released during its activation (degranulation of the cell), induces allergic
reaction and enhances inflammation. It has receptors for antibody E which facilitate its binding to
target. It has the potential to phagocytose foreign material in the body while bound to antibody E.

(Show the structure of basophil)

The Neutrophil

The cell constitutes about 60 – 70% of blood leukocytes and its granules do not stain with basic or acid
dyes. This cell has a regular membrane, several granules in the cytoplasm and multilobed nucleus. The
cell comprises two types of granules;

The azurophil granules which comprise lysozyme, myeloperoxidase, elastase, cathepsins, defensins like
seprocidins and cathericidins (antibiotic proteins) and bacterial permeability increasing factors. The
granules fuse with phagosomes enclosing phagocytosed microbes killing/digesting them.The cell also
has secondary granules which contain lysozyme, cytochrome b558 –NADPH oxidase, phosphatase,
lactoferrin and vitamin B12 binding proteins. The contents in these granules are secreted on the surface
of target through exocytosis. This strategy is used by these cells to kill extracellular parasites.
The cell has Fc receptors for binding antibody, Cr3/Cr4 for binding complement and receptors for
soluble opsonins like ficollins, collectins and pentraxins used to tag foreign cells for destruction.

(Show the structure of neutrophil)

The Mononuclear phagocytes

The monocyte which originates from the bone marrow has an irregular, undulating membrane and a
horseshoe nucleus. It is comprised of azurophilic granules and lysosomes with hydrolytic (peroxidase
and acid hyrolases)and cytotoxic contents which kill phagocytosed microbes. It is referred as the
immature form of macrophages and resides in the blood. The monocytes subsequently mature to form
macrophages which migrate to tissues.

Show the structure of monocyte

The Macrophages

They are found in the tissues and have an irregular undulating membrane which facilitates engulfment
of microbes and other foreign materials. They have granules and developed lysosomes with hydrolytic
enzymes for killing phygocytosed microbes once phagosome fuses with lysosome.

Examples of tissue macrophages; Kupffer cells in liver, Langerhan cells in skin, Osteoclasts in bone,
sinusoids in spleen, mesangial cells in kidneys, Microglial cells in brain.

Macrophages behave as antigen presenting cells. They express peptides processed through exogenous
pathway with MHC class II molecules. They are capable of eliminating intracellular microbes like
mycobacteria, rickettsia, leishmania, viruses and cancer cells.

They recognize targets through toll like receptors, carbohydrate receptors and scavenger receptors.
They also target foreign cells tagged by antibody through antibody mediated cell killing.

Show the structure of macrophage

The Dentritic cell

These cells are also referred as the professional APCs. They are capable of engulfing foreign material and
presenting processed peptides on their surface with MHC class II protein. They interact with helper T
lymphocyte to initiate an immune response.

There are two types;

The Follicular dentritic cell is found mainly in the lymphoid tissue where it presents trapped antigen as
immune complex in form of iccosomes to B- lymphocyte. These cells protect the naïve B – lymphocyte
from death. They stabilize the lymphocyte under influence from cytokines. The B lymphocyte once
stabilized with a unique antigen is influenced to grow and differentiate to become plasma cell and/or
memory B lymphocyte.

The Interdigitating dendritic cell is located in the thymus where it presents antigen complexed to MHC
class II proteins. The complex interacts with maturing T lymphocytes (helper and cytotoxic) to facilitate
negative selection.

Show the structure of dendritic cells.