INNATE IMMUNITY
The innate immune defenses constitute an old and well-established system that form early barriers to infectious disease agents and play a critical role in
preventing infection.
GENERAL FEATURES
o
o
o
o
o
o
The two principal types of reactions of the innate immune system are inflammation and antiviral defense.
The innate immune system responds in essentially the same way to repeat encounters with a microbe
The innate immune system recognizes structures that are shared by various classes of microbes and are not present on normal host cells.
There are two broad categories of innate mechanisms, cell based systems (external body surface barriers and internal phagocytic cells) and a
circulating protein system (e.g. complement, acute phase proteins, defensins).
Instead of antigen specific receptors, the phagocytes have invariant receptors that recognize bacterial components.
While distinguishable from the adaptive system, the innate system has important roles in the activation of B and T lymphocytes and in the effector
functions of antibodies.
COMPONENTS OF THE INNATE SYSTEM
The major areas at which natural defense factors are found are:
o
o
o
o
The skin
The respiratory tract
The digestive tract, and
The genitourinary tract
The skin
o
o
Deeper layers of skin contain living keratinocytes that may secrete cytokines such as interleukin-8 (IL-8) and tumor necrosis
factor (TNF-a).
IL-8 is a potent chemoattractant for neutrophils, resting macrophages can be induced by TNF-a to synthesize proinflammatory cytokines, IL-1
(interleukin 1) and PG-E2 (prostaglandin E2)
1
o
o
The skin also contains Langerhans cells and dendritic cells, which present processed antigen to T cells of the SALT (skin associated lymphoid tissue)
and the local lymph node.
The tight junctions between cells at all locations provide a barrier to penetration by pathogens.
The upper respiratory tract
o
o
Contains mucus secreting goblet cells and ciliated epithelium, that traps particles and sweeps them upwards for expulsion by sneezing or coughing.
The lower tract is protected by surfactants (collectins) secreted by Type II pneumocytes.
Surfactants perform the dual role of lubricating the alveoli and preventing their collapse during expiration and facilitating phagocytosis by binding to
pathogens.
Gastrointestinal tract
o
o
o
Main defense is the stomach acidity and the intestinal flora.
The gut, especially the large intestine, contains the highest concentration of resident bacteria in the body and these perform a number of important
functions including the production of bacteriocins, vitamin synthesis and participation in sterol metabolism.
An intact epithelium and the presence of antimicrobial peptide secreting Paneth cells keep the intestinal organisms in check.
The genitourinary tract
o protected by the flushing action of urine, as well as by low pH of urine and vaginal secretions and the presence of bactericidal enzymes such as
lysozyme.
CELLS OF THE INNATE IMMUNE SYSTEM
The other principal cells of innate immunity are phagocytes (macrophages, neutrophils), dendritic cells, epithelial cells, and natural killer (NK) cells.
Phagocytes
o Widely distributed throughout the body and they acquire special designations depending on their location.
o Functional similar however and the basic mechanisms of recognition, activation and effector function are the same.
Dendritic cells
o Antigen-presenting cells, derived from BM precursors
o They may be classified as myeloid dendritic cells (mDC) derived from monocytes, plasmacytoid dendritic cells (pDC) derived from lymphoid cells
and follicular dendritic cells (FDC) derived from mesenchymal stem cells.
2
Epithelial cells
o provide a barrier to infection
o modulate the recruitment and activation of phagocytes.
Natural killer (NK) cells
o are large granular lymphocytes.
o Sometimes called “null” cells because they do not express characteristic B or T cell markers.
o Target virus-infected or abnormal cells and destroy such cells in the absence of a specific trigger.
o NK cells express receptors for interleukin-2 (IL- 2R) and can proliferate in response to T cell mitogens and IL-2.
o NK cells use killing mechanisms induced via receptors such as FcgRIII (CD16) and KAR.
Other substances that are necessary for effective functioning of innate immunity include complement components, acute phase proteins such as C-reactive
protein and mannan binding lectin, kinins and various enzymes (phospholipase, ribonuclease, amidase, etc.)
CELLULAR RECEPTORS FOR MICROBES
o
o
o
The receptors used by the innate immune system to react against microbes and damaged cells are expressed on phagocytes, dendritic cells, and many
other cell types.
The pattern recognition receptors of the innate immune system are nonclonally distributed; that is, identical receptors are expressed on all the cells of
a particular type, such as macrophages.
Therefore, many cells of innate immunity may recognize and respond to the same microbe.
There are five major families of cellular receptors in innate immunity: TLRs, CLRs (C-type lectin receptors), NLRs (NOD-like receptors), RLRs
(RIG- like receptors), and CDSs (cytosolic DNA sensors)
Toll Like Receptors- TLR
o
Signals generated by engagement of TLRs activate transcription factors that stimulate expression of genes encoding cytokines, enzymes, and other
proteins involved in the antimicrobial functions of activated phagocytes and other cell
3
o
TLR signals for transcription factors from the (NF-κB) family, which promote expres- sion of various cytokines and endothelial adhe- sion
molecules, and interferon regulatory factors (IRFs), which stimulate production of the antiviral cytokines, type I interferons.
NOD- Like Receptors
o
The NOD-like receptors (NLRs) are a large family of cytosolic receptors that sense DAMPs and PAMPs in the cytoplasm.
o
o
o
o
o
o
o
o
All NLRs contain a central NOD (nucleotide oligomerization domain) but have different N-terminal domains.
Three important NLRs are NOD-1, NOD-2, and NLRP-3.
NOD-1 and NOD-2 are cytosolic proteins containing N-terminal CARD (caspase relat- ed) domains. They are specific for bacterial
peptidoglycans, which are common compo- nents of bacterial cell walls. They both activate the NF-κB transcription factor.
NLRP-3 (NOD-like receptor family, pyrin domain containing 3) is a cytosolic NLR that responds to many unrelated microbial structures or
pathologic changes in the cytosol and reacts by enhancing production mainly of the inflammatory cytokine IL-1β.
NLRP-3 recognizes microbial products; substances that indicate cell dam- age and death.
NLRP-3 then oligomerizes with an adaptor protein and an inactive (pro) form of the enzyme caspase-1, resulting in generation of the active form
of the enzyme
Active caspase-1 cleaves a precursor form of the cytokine interleukin-1β (IL-1β) to gener- ate biologically active IL-1β
This cytosolic complex of NLRP-3 (the sensor), an adaptor protein, and caspase-1 is known as the inflammasome.
RIG- Like Receptors
o
Recognizes RNA produced by viruses in the cytosol and activates signaling pathways that lead to the production of type I interferon (IFN).
Cytosolic DNA sensors (CDSs)
o
Include several structurally related proteins that recognize cytosolic viral DNA and induce type I IFN production
Lectin (carbohydrate-recognizing) receptors
o
o
Specific for fungal glycans (these receptors are called dectins) and for terminal mannose residues (called mannose receptors);
Involved in the phagocytosis of fungi and bacteria and in inflammatory responses to these pathogens.
4
ACUTE INFLAMMATION
Cardinal Signs of Inflammation
There are 5 cardinal signs of inflammation:
o Inflammation kick starts with vasodilation of arterioles and capillaries near the site of tissue insult à“RUBOR” or redness.
o Vasodilation results in increased blood flow à “CALOR” or heat.
o Leakage of plasma fluids from the vessels and accumulation in the extra-vascular spaceà “TUMOR” or swelling.
o Chemical mediators of inflammation like histamine and prostaglandinsmayà“DOLOR” or pain.
o Swelling and pain may cause loss of function, of the tissue involvedà “FUNCTIOLAESA” or loss of function.
Process of Acute Inflammation
A. Recognition / Cognitive function
o
o
o
o
An inflammatory response begins with an insult which could be exogenous or endogenous.
Any form of insult causes cell damage and damaged cells release molecules called “Damage Associated Molecular Patterns (DAMPs)”.
Similarly microbes have specific molecules called “Pathogen Associated Molecular Patterns (PAMPs)”
PAMP and DAMP are recognized by receptors on leukocytes like macrophages or dendritic cells called Pattern Recognition Receptors
(PRRs).
•
•
•
There are three types of PRRs:
Type 1 PRR: Secreted molecules that circulate in blood and lymph. Secreted PRRs include the mannan-binding lectin, a surfactant proteinlike collectin, that binds microbial carbohydrates to initiate the lectin pathway of complement. activation. Mannan binding protein is
synthesized in the liver and is secreted into the serum in an acute phase response. C-reactive protein is also a secreted PRR.
Type 2 PRR: e.g. scavenger receptors, mediate the uptake and delivery of pathogens into lysosomes where they are destroyed by
phagocytosis. Pathogen derived proteins are also processed and are presented in association with MHC molecules.
Phagocytosis PRRs appear to possess a high affinity for carbohydrates with large numbers of mannose residues.
Type 3 PRR:. Cell membrane signaling PRRs are called Toll-like receptors (TLRs). TLRs are found on and in macrophages, dendritic cells
and epithelial cells. TLRs function as PRRs that bind to microbial products, leading to the activation of a transcription factor NF-kB (nuclear
factor) signaling pathway. The initiation of this pathway turns on genes for such cytokines as TNF-a and IL-1. Mammals have many TLRs
(TLR-2, TLR-3, TLR-4, TLR-5, TLR-9), and it appears that each recognizes a different microbial product (PAMP).
5
o
o
o
o
o
o
o
o
§
For example:
- TLR-2 binds to peptidoglycan of Gram-positive bacteria, like Staphylococci and Streptococci
- TLR-3 binds to dsRNA
- TLR-4 binds to LPS (endotoxin) of Gram-negative bacteria
- TLR-5 binds to flagellin of motile bacteria.
•
•
•
•
The expression of pattern recognition receptors is not clonal.
All such receptors displayed by cells of a given type have identical specificities.
This explains why all phagocytes in any one individual are reactive to the same pathogens.
In addition, because the receptors are pre-existent, the response by phagocytes is immediate, an important feature of an innate
immune response.
On recognition leukocytes release pro-inflammatory cytokines
This result in the release of inflammatory mediators like histamine, prostaglandins, leukotrienes, and more pro-inflammatory cytokines.
Acute inflammation begins with vasodilation and increase permeability of arterioles and capillaries.
This is followed by a retraction of the endothelial cells in the vessels creating small gaps.
Plasma proteins leak out of the blood vessel through these gaps and accumulate in the extra-vascular space causing oedema.
This plasma rich fluid leaking out of the vessel and accumulating in the extravascular space is called an exudate.
Leakage of plasma proteins disturbs the axial flow of blood, increasing blood viscosity thereby slowing down or causing stasis of blood flow.
This results in red blood cells getting concentrated at the centre with leukocytes being pushed to the periphery near the vessel wall.
B. Recruitment of Neutrophils
Margination
o Leukocyte recruitment to the site of infection involves a series of steps.
o Once the blood flow slows down, leukocytes are pushed to the periphery and move along in close association to the endothelium.
Rolling
o Pro-inflammatory cytokines, IL-1 and TNF-α, and PG-E2 are released by macrophages and mast cells
o In response to TNF and IL-1, endothelial cells express an adhesion molecule of the selectin family called E-selectin.
o Circulating neutrophils and monocytes express surface carbohydrates that bind specifically to the selectins.
o The neutrophils become tethered to the endothelium, flowing blood disrupts this binding, the bonds reform downstream,
6
o
this repetitive process results in the rolling of the leukocytes along the endothelial surface
Adhesion
o Rolling and slowing down, helps leukocytes firmly adhere to the endothelium.
o Expression of integrins (ICAM-1) on the endothelium are induced by pro-inflammatory cytokines like IL-1 and TNF-α.
o Neutrophils express CD18. In the absence of CD18, neutrophils are unable to leave the blood vessels and this may result in a form of leucocyte
adhesion deficiency (LAD).
o (ICAM-1) on the endothelia cell begin to bind strongly to the neutrophil
o The firm binding of integrins to their ligands arrests the rolling leukocytes on the endothelium.
o The cytoskeleton of the leukocytes is reorganized, and the cells spread out on the endothelial surface.
Diapedesis
o The next step involves the transmigration of adherent leukocytes through the endothelial gaps to the extravascular space.
o Once in the extravascular space, leukocytes migrate towards the site of infection alonga concentration gradient, a process called chemotaxis.
o Chemotaxis is induced by a variety of chemokines produced by mast cells, complement proteinsand the microbes themselves.
o Examples of chemokines are IL-8, LTB4 and complement proteins like C5a.
o Neutrophils predominate in the site of infection for the first 6-24 hours followed by monocytes getting into action post 24-48 hours.
o After recruitment to the site of infection leukocytes recognize microbes or dead cells and kill them by a process called phagocytosis.
C. Activation and effector functions
Phagocytosis:
o
o
o
o
Phagocytosis is a process of ingestion of parti- cles larger than 0.5 μm in diameter.
It begins with membrane receptors binding to the microbe
Binding of the microbe to the cell is followed by extension of the phagocyte plasma membrane around the particle
The membrane then closes up and pinches off, and the microbe is internalized in a membrane-bound vesicle, called a phagosome.
o
o
The phagosomes fuse with lysosomes to form phagolysosomes.
Several enzymes in the phagolysosomes are activated.
7
o
o
o
o
o
o
o
o
Following activation, phagocytes begin to consume oxygen (the respiratory burst) the metabolic pathway shifts to the greater energy efficient hexose
monophosphate shunt.
Glucose-6-phosphate dehydrogenase is required for this shift to occur.
Associated with the HMP shunt is the enzyme nicotinamide-adenine phosphate (NADPH) oxidase.
The activity of this enzyme results in the conversion of oxygen to the super oxide radical and the formation of hydrogen peroxide and singlet oxygen.
Nitric oxide (NO) is also produced following the induction of NO synthetase.
Hydrogen peroxide, oxygen radicals and nitric oxide are potently microbicidal and are responsible for the effective killing of microbes by phagocytes.
In the presence of myeloperoxidase and halide ions, hydrogen peroxide is converted to hypohalide ions, and the reaction proceeds spontaneously to
produce water, halide ion and singlet oxygen.
Singlet oxygen is unstable and a return to the ground state emits light energy. This chemoluminescence is observed during phagocytosis.
The intracellular killing capacity of phagocytes may also be demonstrated by the reduction of nitroblue tetrazolium (NBT).
• A low NBT score indicates an inability to kill pathogens
• a high NBT score indicates adequate intracellular killing capacity.
This killing capacity is dependent on the activity of NADPH oxidase.
ANTIVIRAL DEFENSE
Defense against viruses is a special type of host response that involves interferons, NK cells, and other mechanisms.
NK cells kill infected and abnormal host cells via two main mechanisms.:
1. They possess killer activating receptors (KAR) that bind to PAMPs and initiate the release of perforin and granzyme.
•
•
•
A purely innate killing function.
This action may however be abrogated by the killer inhibition receptors (KIR) that bind MHC-1 on normal host cells.
KIR ensures that normal host cells are protected from NK action.
2. NK cells also possess FcgRIII (CD16) with specificity for Fc of IgG bound to microorganisms.
•
Engagement of CD16 results in the expression of FasL and binding to Fas leads to caspase activation.
8
•
•
This is an adaptive killing mechanism known as antibody mediated cellular cyttoxicity, ADCC.
It should be noted that CD16 is also present on other cells such as neutrophils and macrophages which kill using reactive oxygen species
Regulation
Several features of the innate immune system account for its inability to react against an individual’s own, or self, cells and molecules. First, the receptors of
innate immunity have evolved to be specific for microbial structures (and products of damaged cells) but not for sub- stances in healthy cell. Second, some
pattern rec- ognition receptors can recognize substances such as nucleic acids that are present in normal cells, but these receptors are located in cellular compartments (such as endosomes; see below) from where components of healthy cells are excluded. Third, normal mammalian cells express regulatory
molecules that prevent innate immune reactions. The adaptive immune system also discriminates between self and nonself; in the adaptive immune system,
lymphocytes capable of recognizing self antigens are produced, but they die or are inacti- vated on encounter with self antigens.
COMPLEMENT SYSTEM
The mammalian complement system is made up of several proteins that circulate in the blood. Many of these proteins are enzymes called zymogens,
which means that they normally exist in an inactive state and must undergo a biochemical change before they can be enzymatically active. When one
protein gets activated, it cleaves and activates the next protein in the sequence, which in turn cleaves the next.
The result is a molecular domino effect that leads to immune system engagement and pathogen killing.
Complement System Nomenclature
o
o
o
o
o
There are nine main complement proteins, which are named with the capital letter “C” and then a number.
When a complement protein is cleaved, the cleavage products are given a lowercase “a” or “b” after the number.
The “a” fragment is always the smaller fragment, an anaphylatoxin, while the “b” fragment is the larger binding portion, except in the case of C2.
For example, when C3 is cleaved, it is cleaved into C3a and C3b, with C3a being the smaller product.
To designate which complement proteins, make up a complex, the names of the proteins are strung along in the order in which they bind. For
example, the classical C3 convertase is called C4b2a, because is made up of C4b and C2a. When C3b binds to this complex to create a C5
convertase, the complex becomes C4b2a3b.
The Classical Pathway
o
The classical pathway begins with a protein complex called C1.
9
o
o
o
o
o
o
o
o
o
C1 is made up of three distinct subunits: C1q, C1r, and C1s.
C1r and C1s are proteases, which are enzymes that cleave proteins, but they are inactive during normal conditions.
During an infection, circulating C1q can bind directly to some bacterial surfaces, to antibodies bound to a pathogen, or to an acute-phase
protein called C-reactive protein, that also binds to bacterial surfaces.
C1q binding causes a conformational change resulting in autocatalytic activation of C1r.
Activated C1r then cleaves and activates C1s.
Once C1s is activated, it cleaves circulating complement proteins C4 and C2.
C4b covalently binds to the pathogen’s surface or to pathogen-bound antibodies, and C2a binds to C4b, forming the C4b2a complex.
This complex is known as the C3 convertase, and it cleaves circulating C3 into C3a and C3b.
Although the classical pathway of complement activation was the first to be discovered, it is likely the youngest evolutionarily.
Lectin Pathway
o Lectins are proteins that can recognize and bind to very specific carbohydrate groups.
o There are four lectins that activate complement after binding to microbial sugars. These include: mannose-binding lectin ( MBL), and ficolins 1, 2,
and 3.
o Mannose-binding lectin (MBL) is made in the liver and circulates in the bloodstream.
o It is a multimeric protein (MBL) with multiple binding sites that recognize the sugar groups fucose, mannose, and N-acetylglucosamine.
o These sugar groups are commonly exposed on microbial polysaccharides, but not on host cells.
o The ficolins are structurally similar to mannose-binding lectin, except that they bind acetylated sugars instead of mannose groups.
Now, here’s how lectin binding initiates complement.
o In the circulation, mannose-binding lectin or one of the ficolins complexes with the proteins MASP-1 and MASP-2, which are MBL-associated
serine proteases.
o These proteases are inactive until the lectin binds to the sugars on a pathogen’s surface.
o At this point, a conformational change activates MASP-2, allowing it to cleave and activate another molecule of MASP-2, which can then cleave
the complement proteins C4 and C2.
o C4b complexes with C2a to form the C3 convertase, and here the pathway converges with the classical pathway.
Alternative Pathway
o
o
o
o
The biggest difference between the alternative pathway and the other two is that it utilizes a distinct C3 convertase.
This convertase consists of C3b bound to a cleaved fragment of a plasma protein called Factor B.
When C3b is bound to the surface of a pathogen, circulating Factor B can bind. This allows another
plasma protein called Factor D to cleave Factor B, leaving behind the rather confusingly named C3bBb.
10
o
This is the alternative pathway C3 convertase.
Common Pathway- where the three pathways converge
The C3 convertases of the alternative and lectin pathway as well as the C3 convertase of the alternative pathway are bound to the pathogen surface
o When it is exposed to circulating C3, the convertase cleaves the C3 protein into 2 parts: C3a, the smaller part, and C3b, the larger.
o C3a is released into the circulation, where it recruits neutrophils and other phagocytes to the site of infection.
o C3b, on the other hand, can do one of two things:
(1) many molecules of C3b can bind to the surface of the pathogen, coating it in a process called opsonization. Opsonization marks the pathogen for
destruction by phagocytes like macrophages. Phagocytes have receptors that recognize C3b coating the surface of a pathogen, and will phagocytose
objects or organisms coated in C3b.
(2) C3b can bind to the existing C3 convertase (C2bC4aC3b), creating a C5 convertase.
o The C5 convertase is formed and can cleave C5.
o Like C3a, C5a is released into the circulation to recruit phagocytes.
o Meanwhile, C5b nucleates the membrane attack complex (MAC), a complex of complement proteins that form pores in pathogen cell
membranes, causing the pathogen to lyse.
Here’s how this works.
o C5b binds to circulating C6 to form C5b6, C5b6 then binds to C7.
o Binding causes a conformational change in C7, which exposes hydrophobic residues, allowing C5b67 to insert into the pathogen’s cell membrane.
o C8 then binds the complex, which also causes a conformational change allowing C8 to insert into the pathogen’s plasma membrane.
o Finally, 10- 16 molecules of the terminal complement protein C9 polymerize within the cell membrane to form a pore that allows water and
solutes to flow out of the pathogen, leading to death.
o This is possible, because while the outer surface of the pore is hydrophobic, allowing it to insert into the plasma membrane, the inside surface
is hydrophilic, allowing polar molecules like water and ions to travel freely through it.
o Although the membrane attack complex (MAC) is able to kill many kinds of bacteria and parasites, it is especially important for controlling
bacterial infection with Neisseria species, and may actually be dispensable for controlling other infections.
Regulation of the Complement System
Although C3a and C5a are sometimes forgotten because they don’t contribute directly to pathogen clearance, they still have potent biological effects.
o They can induce contraction of the smooth muscle cells lining blood vessels, increase vascular permeability, and signal endothelial cells to
increase expression of adhesion molecules necessary for immune cells to leave the bloodstream and enter an infected tissue.
11
o
o
o
o
o
o
o
o
o
o
o
o
While these effects are essential for recruiting phagocytes to the site of infection, when levels are too high, C3a and C5a can lead to anaphylactic
shock.
This is why they are often referred to as anaphylatoxins.
Because of how quickly the complement cascade can proceed and be amplified, and because of how potent the effects are, it is extremely
important to be able to regulate this response to keep it from getting out of control or from attacking host cells. There are several ways the
complement system can be regulated.
One major way to regulate the complement system is that all the enzymes involved in the complement system, except Factor D, are zymogens,
meaning they must be activated before they can perform their enzymatic functions.
Another way to maintain control of the complement system is that all of the enzymatic activity occurs at the pathogen’s surface, which prevents
active enzymes from circulating around in the bloodstream.
After cleavage, both C4b and C3b have a highly reactive exposed thioester that allows them to covalently attach to any adjacent carbohydrate or
protein, which is usually the pathogen surface or an antibody that has already attached to the pathogen’s surface. If they don’t bond quickly, this
reactive group is inactivated by hydrolysis, ensuring that host proteins aren’t indiscriminately tagged by C3b.
There are also specific proteins that regulate complement activation. The C1 inhibitor is a serine protease inhibitor that binds active C1r:C1s,
dissociates it from Cq, and prevents it from cleaving C2 and C4.
Decay-accelerating factor can compete with Factor B for C3b binding, preventing alternative complement activation. It can even displace bound
Bb, inactivating the alternative C3 convertase.
Decay-accelerating factor, as well as C4-binding protein and Complement receptor 1, can also bind to C4b in the classical C3 convertase and
displace C2a.
Factor H is another plasma protein that can outcompete Factor B. It preferentially binds to C3b on host cells, preventing complement activation on
host cells while not interfering with binding to pathogens. It can also displace C3b from the C5 convertase.
Plasma proteins like vitronectin can bind to any C5b67 complexes in the circulation, preventing them from inserting into host cell membranes and
forming the membrane attack complex.
Finally, protectin is a membrane-bound protein that prevents C9 from binding to the C5b678 complex, acting as yet another way to
prevent complement activation from harming host cells.
CYTOKINES
-
Cytokines are are the molecules through which leukocytes communicate with one another and with other cells of the body.
Cytokines are either proinflammatory (involved in inflammatory reactions), or anti-inflammatory (prevent or decrease inflammation).
12
-
-
Cytokines normally act over a short period of time and at low concentrations- an effective means of avoiding chronic inflammation and unnecessary
tissue damage. (Inflammation
is generally sustained if there is enough antigen present to maintain cytokine stimulation).
Cytokines are generally operate in an autocrine or paracrine fashion often both, but can sometimes act in an endocrine fashion
Different cytokines are often expressed together and complement each other’s work, and the effects of cytokines generally result in cascades that
radiate into network responses.
Cytokines are synthesized de novo. Cytokines are not stored in cells; they are synthesized upon stimulation.
Features of Cytokines
Different cell types can synthesize the same cytokine, different cytokines can be expressed by discreet cell types, just as a particular cell type
can synthesize different cytokines.
- Pleiotropism- Most cytokines exhibit several different functions
- Some cytokines may be limited to a single function
- Redundancy: cytokines possessing the same biological activity
Partial redundancy many cytokines may overlap in their biological function
- Cytokines may act
(1) additively, i.e. the combined effect of two or more cytokines is
equal to the sum of the effects of the cytokines taken separately
(2) synergistically, i.e. the combined effect of two or more cytokines is greater than the sum of the effects of the cytokines taken
separately or
(3) antagonistically, i.e. the effects of a cytokine inhibits the effects of the
other.
- Some cytokines are specialized in attracting cells: these cytokines are defined as chemokines.
- Finally, some cytokines also can be membrane-bound (mb), e.g. FasL (CD95L or CD178),
CD40L (CD154), mbIL-1b, mbIL-15, mbM-CSF, mbFlt3 ligand, mbTNF-α, mbLTα, membrane-bound fractalkine, mbTGF-β, and mbIFN-γ etc.
Cytokine signaling
1. Cytokine receptors
- Nine (9) cytokine receptors:
13
1. Type I cytokine receptor – used by the vast majority of cytokines (interleukins and Colony-Stimulating Factors or CSFs)/ mainly signals through a
JAK/STAT (JAnus Kinase/Signal Transducer and Activator of Transcription protein) pathway.
2. Type II cytokine receptor: main receptor by interferons, INF, and IL 10
3. TNF receptor family- TNF (CD40L & CD95L)
4. IL-1 receptor family- IL- 1β and IL-18
5. seven transmembrane G-coupled receptors- chemokines
6. The common g-chain family- IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21
Signaling pathways
JAK/STAT signaling pathway
The pathway used by most cytokines.
Steps of this pathway are as follows:
1. Cytokine engagement of cytokine receptor chains;
2. Dimerization of receptor chains bringing receptor-associated JAK molecules together;
3. Activation of JAKs by cross-phosphorylation (sometimes called trans-phosphorylation);
4. JAK-mediated phosphorylation of receptor ITAMs (Inducible Tyrosine-based Activation Motif);
5. Recruitment and docking of STATs;
6. JAK-mediated phosphorylation and dimerization of STATs;
7. STAT dimer nuclear translocation;
8. STAT dimer binding to promoter palindromic GAS-binding sites (Gamma interferon Activation Site);
9. Gene transcription.
NF-kB signaling pathway
This particular signaling pathway is almost omnipresent in immune reactions.
-
NF-kB pathway is used for cytokine signaling
Used by TLRs to signal cells as to characteristics of the ‘invading’ pathogen,
Used for T and B lymphocyte activation
14
-
triggers apoptosis through TNFRs paired with so-called death domains
This pathway is used by TNFRs, the IL-1R, TLRs, as well as antigen receptors
The following describes the events involved in the canonical NF-kB signaling pathway:
1. TNFR: TNFR trimerization → TRADD → TRAF2 → (RIP1)/TAK1/TAB1-2 → IKKa/IKKb/IKKg (NEMO) → IkBa phosphorylation and
proteasomal degradation → release and translocation of NF-kB transcription factor to the nucleus where it binds NF-kB-responsive elements, i.e.
cytokine gene as cytokine receptor gene promoters among others.
2. Some TLRs (3, 4): TLR → Myd88 adaptor protein (TLR4) → RIP1 → TAK1/TAB1-2/TRAF6 → IKKa/IKKb/IKKg (NEMO) → IkBa
phosphorylation and proteasomal degradation → release and translocation of NF-kB transcription factor to the nucleus where it binds NF-kBresponsive elements, i.e. cytokine gene as cytokine receptor gene promoters among others.
3. IL-1R and other TLRs (1/2, 2/6, 5, 7, 8, 9, 10): TLR → Myd88 adaptor protein → IRAK → TAK1/TAB1-2/TRAF6 → IKKa/IKKb/IKKg (NEMO)
→ IkBa phosphorylation and proteasomal degradation → release and translocation of NF-kB transcription factor to the nucleus where it binds NFkB-responsive elements, i.e. cytokine gene as cytokine receptor gene promoters among others.
Important soluble mediators of inflammation
Mediator
IL-1b (cytokine):
Function
TNF-a (cytokine):
initiation of inflammatory response, induction of other proinflammatory cytokine synthesis and secretion (IL-1b, TNF-a,
IL-6, & CXCL8) as well as other inflammatory mediators (LTs, prostaglandins) and adhesion molecules (endothelial
cell activation & transendothelial migration, i.e. expression of selectins and integrins);
vasodilation, chemotaxis, endothelial activation, transendothelial
migration, and activation of neutrophils;
CXCL8
Chemotaxis
IL-12 (cytokine):
activation of Natural Killer cells (NK cells) for the synthesis and release
of InterFeroN-gamma (IFN-g);
IFN-g (cytokine
C5a
activation of macrophage microbicidal functions;
activation of mast cells (degranulation) and chemotaxis;
15
C3a
activation of mast cells (degranulation) and chemotaxis;
Histamine
Leukotrienes
vasodilation and constriction of smooth muscle cells;
pruritous (itching);
constriction of smooth muscle cells
Prostaglandins
pain, vascular smooth muscle regulation, chemotaxis;
Bradykinin (peptide):
vasodilation; burning pain and itching.
Important cellular mediators of inflammation
Mediator
Macrophages
Function
phagocytosis, synthesis and release of proinflammatory cytokines,
microbial killing;
phagocytosis, release of DAMPs, microbial killing
Neutrophils
Mast cells
Endothelial cells
release of soluble inflammatory mediators
infiltration of plasma and leukocytes.
Systematic Inflammation
Mediator
IL-1b (cytokine):
TNF-a (cytokine):
IL-6 (cytokine)
Function
fever
fever, vasodilation, increased muscle and fat catabolism (cachexia);
fever, synthesis of acute-phase reactants by the liver (e.g. C-reactive protein (CRP), mannose-binding lectin
(MBL or mannose-binding protein (MBP)), alpha-1 antitrypsin, fibrinogen, complement factors, serum amyloid
A (SAA_
16
ADAPTIVE IMMUNITY
There are two types of adaptive immune responses: antibody-mediated, and cell mediated immunity
Antibody- Mediate Immunity
Antigens
o Molecules recognized by the immune system
o Antigens possess the following characteristics to provoke an immune response:
Size:
§
§
The typical antigen is a molecule with a minimum molecular mass of about 10,000 Daltons.
Smaller molecules tend to be weakly antigenic or haptenic.
Complexity:
§
§
§
Protein molecules are the most antigenic on the basis of their complex nature.
Carbohydrates, lipids and nucleic acids, which tend to contain repeating subunits, are poorly antigenic.
Nucleic acids and phospholipids are highly conserved across biologic systems and tend to need special conditions to stimulate an
immune response.
Hormones, metabolic by-products, lipids, simple sugars, and other small molecules are antigenic, but typically need to be
conjugated to a larger macromolecule to stimulate an immune response.
Foreignness:
§ the immune system is programmed to react against “nonself”
§ molecules that are not recognized as foreign are not antigenic.
Solubility:
§
§
molecules that are soluble or easily solubilized are better antigens than those that are insoluble.
Insoluble or nondegradable materials tend to pass through the body virtually unchanged.
So, not all antigens are immunogens.
Obviously, since all immunogens are antigens, immunogens possess the properties of antigens. In addition to
17
the following, the following factors contribute to the immunogenicity of an antigen:
Immunogenicity
Not all antigens recognized by immune cells induce an immune response. Those that can are referred to as immunogens, and this property is referred to as
immunogenicity.
Dose: The amount of an antigen determines its immunogenicity. Very small quantities might be rapidly cleared and be unable to induce a
response while very large quantities might overwhelm the system and induce a state of tolerance, depending on the nature of the antigen
and on many other
factors.
Route: The route of administration determines availability to immune cells. Many pathogens enter the body by crossing a mucosal surface, such
as the genitourinary, respiratory or gastrointestinal surfaces. Some antigens can enter through breaks in the skin. Materials introduced via
the healthy
digestive tract are rapidly hydrolyzed and destroyed with a reduced chance of exposure to immune cells whereas parenterally introduced materials
generally remain intact and are able to induce a robust immune response. The route of administration also impacts primary type of response that
is induced.
Host factors: T cells only recognize peptide antigens. These peptides are loaded into Major Histocompatibility Complex (MHC) molecules
for display to T cells. While MHC molecules are encoded by a large family of alleles, a single individual will carry only a few alleles. The different
MHC alleles vary in their affinity for distinct peptides. Also, there are several alleles that encode the machinery that degrade the proteins
into peptides that will be loaded into MHC molecules. So, depending on the individual’s MHC and peptide processing alleles, different
individuals will likely display different peptides (epitopes) from the same antigen. This variability in which peptides are generated and presented can
influence the immunogenicity of an antigen.
Adjuvant effects: The presence of added chemical substances (e.g. Alum) may enhance the immune response to the antigen. Adjuvants act through
three basic mechanisms – by functioning prolonging the persistence of the antigen, by stimulating or modulating immune cells and by
enhancing macrophage
function.
18
Epitopes
The portions of an antigen that bind the recognition molecules of the immune system are referred to as epitopes (or antigenic determinants).
o
o
o
o
o
o
o
o
The antigen is a complex molecule and may possess several epitopes.
Each epitope is a cluster of ligands held together in a mosaic.
Each epitope may contain amino acid sequences that are located on different parts of the polypeptide chain.
Epitopes that comprise amino acids on the same chain are called linear or continuous epitopes.
Epitopes formed by three-dimensional conformations are nonlinear or conformational epitopes.
The number of epitopes on an antigen molecule is the valence.
The total valence is the number of all epitopes on the molecule.
However, because of the convoluted shape of the antigen, not all epitopes are readily accessible.
The effective valence of the antigen in terms of its reactivity is the number of accessible epitopes.
When an antigen is degraded, the conformational epitopes are the first to be lost as the molecule unfolds.
Haptens
o Small molecules that are non-immunogenic.
o Haptens can be recognized by antibodies but the binding is below the threshold required for immune response.
o Thus, haptens possess reactivity, but because of their small size, typically only possess one epitope.
o Haptens may become immunogenic when they become linked to larger molecules, referred to as carrier proteins, to bring the total mass above the
threshold.
o Hapten-carrier conjugates behave like true antigens.
The Antigen Elimination Curve
It is useful to determine how intravenously injected antigen is removed from the circulation and how this
relates to the formation of antibody.
Three phases of removal are easily detected:
1. The first is the phase of equilibration. This takes place 10-30 minutes after injection and represents the time required for equilibration of
the antigen with tissues and fluids. Antigen removed during this phase can be found in the liver, spleen and lung.
19
2. The second phase is that of a gradual catabolic degradation and antigen removal. This proceeds over the following 4-7 days.
3. The third stage is that of immune elimination. This is a phase of accelerated removal due to binding of newly formed antibody with
antigen. Antigen specific antibodies are not detectable at this point as they are bound to antigen. At the end of this phase, it is possible to detect free
irculating antibody.
Antibodies
o
o
o
Antigen recognition by B lymphocytes is facilitated by the B cell receptor (BCR).
Recognition of antigen through the BCR results in clonal expansion and differentiation of B cells into plasma cells.
Plasma cells are essentially factories for the production of antibodies, or immunoglobulins, which are the secreted form of the BCR.
Generation of Antibody Diversity and Specificity
o
Single B cells make antibodies of only one specificity and that specificity is determined by the structure of the antigen-binding site.
o
o
The structure of the antigen-binding site is determined by the amino acid sequence of said antigen binding site.
That amino acid sequence is generated by random gene rearrangement of immunoglobulin gene segments.
Within a particular B cell, the immunoglobulin germ line genes are rearranged to include a single set out of many possible Variable (V),
Diversity (D) and Joining (J) gene segments.
§ For the k light chain, for example, there are functional 30 V gene segments, 5 J gene segments and 1 C gene segment.
§ For the heavy chain, there are up to 65 functional V gene segments, 27 D gene segments, 6 J gene segments and 9 C gene segments.
The rearrangement of these genes results in billions of possible combinations and this process of rearrangement accounts for antibody specificity.
Rearrangement of the immunoglobulin genes occurs during the earliest stages of B cell development.
Additional diversity is generated by the process of somatic hypermutation.
This process results in the addition of random nucleotides into the antigen-binding region (V region) of the BCR and further modifies the
avidity and affinity of antibody molecules.
Somatic hypermutation occurs in during the late stage of B cell differentiation, after contact with antigen.
o
o
o
o
Antibody Structure
o
The molecule is comprised of 4 polypeptide chains, 2 identical heavy chains and 2 identical light chains.
The two heavy chains are linked together by disulfide bonds and each light chain is linked to a heavy chain by disulfide bonds.
20
o
o
o
o
o
o
o
o
The antigen binding region of heavy and light chains are located at the amino-terminal of the molecule and are referred to as the variable
or V domains (VH and VL, respectively).
The constant domains (CH and CL) make up the constant region and define the effector function of the molecule.
While the specificity of an antibody molecule is defined by the amino acid sequence of the V region, the sequence variability is not
evenly distributed across the V region.
There are three regions within both the VH and VL chains that have particular variability.
These hypervariable regions are separated by framework regions that provide the structural framework.
When the antibody molecule assembles into its three-dimensional structure, the hypervariable regions form loops at the surface of the molecule that
will interact with antigen.
These loops are often termed the complementarity-determining regions (CDRs).
The shape of the antibody molecule is variable, from a straight line to a ‘T’ although a Y shape is the most stable, due to steric and other factors.
Studies on Antibody Structure
Studies using proteolytic enzymes (proteases) have revealed a lot about the structure of the antibody molecule and the functions of its component
parts:
Papain cleaves the antibody molecule into 3 fragments:
o Two (2) identical Fab (Fragment antigen binding) fragments and one Fc (Fragment crystallizable) fragment.
o The Fab fragments contain the variable regions of both the heavy and light chains;
o They contain a complete light chain paired with the VH and CH1 domains of the heavy chain.
o Importantly, the Fab portion contains antigen-binding activity of the antibody molecule.
o The Fc fragment contains the CH2 and CH3 domains of the heavy chain and comprises the portion of the antibody molecule that determines the
effector function.
o The term Fc reflects the properties of the domain: constant sequence, crystallizable, complement binding, complement activating, the
domain with the carboxyl (COOH) terminal and cell binding via the FcR.
Pepsin cuts the antibody molecule on the carboxyl-terminal side of the disulfide bonds. This cleavage produces
o the F(ab’)2 fragment made up of the 2 antigen-binding arms of the antibody molecule linked together.
Studies using these fragments showed that the Fab fragment was unable to cross human placenta while the Fc fragment could
cross the placenta.
o This result provided conclusive evidence that the ability of IgG to cross the placenta was an active process involving binding of the Fcg
domain, rather than passive diffusion of the entire molecule.
21
Allotypes, Idiotypes and Isotypes
Allotypic variations
o
o
o
o
o
o
o
refer to genetic differences between individuals within a species involving different alleles at a given locus.
Allotypic variations have no known functional significance.
Allotypes occur mostly as variants of heavy chain constant regions.
Allotypes are inherited as dominant Mendelian traits but have not been found in all Ig classes.
Each of the four IgG subclasses has a set of allotypes (Gm allotypes, e.g. IgG3m).
IgA allotypes (Am allotypes) have also been described.
No allotypes have been found for the other Ig classes.
Idiotypic variation
o refers to the variations in the variable domain, particularly in the hypervariable region (CDR).
o These variations determine the antigen binding specificity of a given antibody.
o Private idiotypes are those that are specific for individual B-cell clones.
o Public idiotypes are shared between different B-cell clones.
Isotypic Variation
o Antibodies exist in different forms, or isotypes or classes.
o This isotypic variation is achieved through the linking of different constant region genes (located downstream of the V-genes on the heavy chain
gene locus) with the same variable region.
o The effector function of the antibody molecule is defined by its isotype.
o Antibody isotypes possess different C regions but have identical Fab regions.
o Therefore, isotypic variation results in antibodies with the same specificity but different biological functions.
Antibody Isotypes
o An antibody’s heavy chain determines it’s tissue distribution and effector function.
o There are five heavy chain classes, Immunoglobulin (Ig)M, IgD, IgG, IgA and IgE.
22
IgM
-
IgM is the first antibody secreted in an immune response. It is the “early” antibody.
The presence of IgM antibody indicates that the antigenic exposure was a recent event.
IgM is secreted as a pentamer with five identical units bound by a joining “J” chain.
The molecule is thus very large with 10 binding sites.
IgM is found mainly in the blood and can be found in the lymph.
IgM binds very strongly to pathogens with repetitive epitopes, such as polysaccharides.
IgM is also a powerful activator of the complement cascade due to its size and the ready formation of immune complexes.
IgG
-
the second antibody to be secreted, late antibody,
is secreted in very large amounts and it has a longer half-life than IgM.
IgG is therefore the most abundant antibody in blood and lymph.
IgG subclasses (IgG1, IgG2, IgG3 and IgG4) possess various biological activities and greatly enhance the functional capacity of IgG.
They can readily diffuse into extracellular space due to their great flexibility, IgG1 and IgG3 readily cross the human placenta, providing
protection for the developing fetus.
IgG antibodies can activate the classical complement pathway.
They also serve an important role in opsonization, or coating pathogens and marking them for phagocytosis by phagocytic cells expressing
Fcg receptors.
NK cells express an Fc receptor, CD16/FcgRIII, which is specific for IgG1 and IgG3. CD16 engagement results in antibody dependent cellmediated cytotoxicity, ADCC.
IgA
-
occurs as a monomer and as a dimer.
Trimeric and tetrameric forms of IgA have also been described.
Monomeric IgA is secreted into the bloodstream.
Dimeric IgA contains a J chain and a secretory component (SC).
It is transported across the epithelium and is found in the lumen of the gut as well as in secretions (saliva, tears,
milk) and on mucus membranes (respiratory and intestinal).
More IgA is produced in the gut than all other Ig from other sources combined.
23
-
As IgA is predominately found along mucosal surfaces where complement and phagocytes are not present, the main function of IgA antibodies is
to neutralize pathogens.
-
-
Binds to high affinity receptors (FceR) on mast cells (which are found beneath the skin and mucosa, and along blood vessels in connective
tissue) and basophils.
As such, is found at very low levels in the blood and extracellular fluid.
Bound to these Fcε receptors, IgE is highly efficient at triggering mast cells to release their granule contents and trigger the elimination of certain
pathogens, namely helminthic parasites.
IgE also plays a major role in the pathogenesis of allergic reactions (Type I hypersensitivity).
-
The least understood of the immunoglobulins.
Secreted IgD has no known function.
IgD is however expressed on the surface of B cells, and, with IgM, acts as the B cell receptor (BCR) for antigen.
IgE
-
IgD
Fc Receptors
o
o
o
o
o
o
Fc receptors are specialized receptors expressed by immune cells.
They bind to the Fc portions of antibodies and trigger a variety of effector functions.
Different Fc receptors bind different Fc regions on the various antibody isotypes.
When antibodies bind to multimeric antigens or multimeric antigenic particles, they aggregate;
Fc receptors can bind the Fc portions of the antibodies in these aggregates and this triggers the cell to initiate effector functions like phagocytosis
or ADCC.
In the case of Fce receptor (which is expressed by mast cells, eosinophils and basophils), IgE tends to stay bound to the Fce receptor,
when the specific antigen comes along, those Fce receptor-bound IgEs will aggregate on the cell surface and trigger release of granules.
Helper T cells regulate isotype switching
The cytokines produced by an activated CD4 T cell can induce activated B cells to switch to a particular isotype.
The activated helper T cell (which was influenced by the cytokines produced by the APC) now influences the activated B cell – all in order to make the
antibody best suited for elimination of the pathogen!
24
Antigen-Antibody reactions
o
o
o
o
Antigen-antibody reactions are highly reversible because weak non-covalent forces hold the reactants together:
Ag + Ab
«
(AgAb)
The determinants of reaction equilibrium include the relative concentrations of the reactants, reaction, temperature and pH and the binding affinity of
the reactants.
In general, the biological outcomes of antigen-antibody reactions depend on the nature of the antigen.
Agglutination – whole cells or aggregated antigens
Precipitation – soluble antigens
Flocculation – particulate antigens
Neutralization – toxins or live organisms
ANTIGENS RECOGNIZED BY T LYMPHOCYTES
o
o
o
o
o
The majority of T lymphocytes recognize peptide antigens that are bound to and displayed by major histocompatibility complex (MHC)
molecules of antigen-presenting cells.
The MHC is a genetic locus whose principal protein products function as the peptide dis- play molecules of the immune system
The cells that capture microbial antigens and display them for recognition by T lymphocytes are called antigen-presenting cells (APCs).
Naive T lymphocytes must see protein antigens presented by dendritic cells to initiate clonal expansion and differentiation of the T cells into effector
and memory cells.
For this reason, dendritic cells are considered the most efficient and specialized APCs, and are therefore sometimes called professional APCs.
CAPTURE OF PROTEIN ANTIGENS BY ANTIGEN-PRESENTING CELLS
o
o
o
o
The epithelia and subepithelial tissues contain a network of dendritic cells; the same cells are present in the T cell–rich areas of peripheral lymphoid
organs and, in smaller numbers, in most other organs
Dendritic cells use various membrane receptors to bind microbes, such as lectin receptors for carbohydrate structures typical of
These microbes or their antigens enter the dendritic cells by phagocytosis or receptor-mediated endocytosis
At the same time, the innate immune response is also stimulated.
25
o
o
o
o
o
o
o
o
o
This occurs when the products of the microbe bind to TLR and other pattern recognition receptors. This leads to an inflammatory response with the
release of TNF and IL-1
TLR signaling and cytokine release causes an activation of the dendritic cells
Upon activation by these signals, classical dendritic cells lose their adhesiveness for epithelia and begin to express the chemokine receptor CCR7,
which is specific for chemoattracting cytokines (chemokines) produced by lymphatic endothelium and by stromal cells in the T cell zones of lymph
nodes.
These chemokines direct the dendritic cells to exit the epithelium and migrate through lymphatic vessels to the lymph nodes draining that epithelium
During the process of migration, the dendritic cells mature from cells designed to capture antigens into APCs capable of stimulating T lymphocytes.
This maturation is reflected by increased synthesis and stable expression of MHC molecules, which display antigens to T cells, and of costimulators.
The net result of this sequence of events is that the protein antigens of microbes that enter the body are transported to and concentrated in the regions
of lymph nodes where the antigens are most likely to encounter T lymphocytes.
Recall that naive T lymphocytes continuously recirculate through lymph nodes and also express CCR7, which promotes their entry into the T cell
zones of lymph nodes
Therefore, dendritic cells bearing captured antigen and naive T cells poised to recognize anti- gens come together in lymph nodes.
STRUCTURE AND FUNCTION OF MAJOR HISTOCOMPATIBILITY COMPLEX MOLECULES
o
o
o
o
o
MHC molecules are membrane proteins on APCs that display peptide antigens for recognition by T lymphocytes.
The collection of genes that make up the MHC locus is found in all mammals, and includes genes that encode MHC molecules and other proteins.
Human MHC proteins are called human leukocyte antigens (HLAs) because they were discovered as antigens of leukocytes that could be identified with
specific antibodies.
In all mammals, the MHC locus contains two sets of highly polymorphic genes, called the class I and class II MHC genes.
Class I and class II MHC molecules are membrane proteins that each contains a peptide- binding cleft at the amino-terminal end. Although the two
classes of molecules differ in subunit composition, they are very similar in overall structure
26
(HLAàMHC locusà Class 1, Class2)
Class I MHC Molecules
o
o
o
o
o
o
o
o
Class I MHC molecules consist of two polypeptide chains: A polymorphic a chain responsible for class I MHC specificity which is non-covalently
associated with the smaller constant (i. e. non-polymorphic) chain b2-microglobulin.
The α chain consists of three extracellular domains (a1, a2 and a3) followed by short transmembrane and cytoplasmic domains
The amino-terminal α1 and α2 domains of the α chain molecule form a peptide-binding cleft, or groove, that can accommodate peptides typically 8 to 9 amino acids
long.
The floor of the peptide-binding cleft is the region that binds peptides for display to T lymphocytes, and the walls of the cleft are the regions that make contact with
the T cell receptor (which also contacts part of the displayed peptide.
The polymorphic residues of class I molecules—that is, the amino acids that dif fer among different individuals’ MHC mole- cules—are located in the α1 and α2
domains of the α chain.
Some of these polymorphic residues contribute to variations in the floor of the peptide-binding cleft and thus in the abil- ity of different MHC molecules to bind
peptides.
Other polymorphic residues contribute to variations in the walls of the clefts and thus influence recognition by T cells.
The α3 domain is invariant and contains a site that binds the CD8 T cell coreceptor but not CD4.
27
o
Therefore, CD8+ T cells can only respond to peptides displayed by class I MHC molecules, the MHC molecules to which the CD8 coreceptor binds.
Class II MHC Molecules
Each class II MHC molecule consists of two transmembrane chains, called α and β. Each chain has two extracellular domains, fol- lowed by the transmembrane and
cytoplasmic regions.
o
o
o
The amino-terminal regions of both chains, called the α1 and β1 domains, contain poly- morphic residues and form a cleft that is large enough to accommodate
peptides of 10 to 30 residues.
The nonpolymorphic α2 and β2 domains con- tain the binding site for the CD4 T cell core- ceptor.
Because CD4 binds to class II MHC molecules but not to class I, CD4+ T cells can only respond to peptides presented by class II MHC molecules.
Properties of MHC Genes and Proteins
Several features of MHC genes and molecules are important for the normal function of these molecules
•
•
•
•
MHC genes are highly polymorphic- The polymorphism of MHC genes is so great that any two individuals in an outbred population are extremely unlikely to
have exactly the same MHC genes and molecules. Therefore, MHC polymorphism ensures that a population will be able to deal with the diversity of microbes and at
least some individ- uals will be able to mount effective immune responses to the peptide antigens of these microbes. Thus, everyone will not succumb to a newly
encountered or mutated microbe.
MHC genes are codominantly expressed, meaning that the alleles inherited from both parents are expressed equally. Co- dominant inheritance maximizes the
number of HLA genes, and therefore proteins, present in each individual and thus enables each individual to display a large number of peptides. Because every
individual expresses both sets of MHC alleles inherited from each parent, there is a 1 in 4 chance of siblings expressing all the same MHC molecules.
There are 3 sets of class I MHC genes called HLA-A, HLA-B and HLA-C, present on all somatic, nucleated cells (hence erythrocytes and gametes do not
normally express HLAs); similarly, there are 3 pairs of class II MHC gene clusters referred to as HLA-DR, HLA-DP and HLA-DQ, and these are usually
restricted to pAPCs. But because many individuals possess an extra b chain in their HLA-DR cluster, there actually exist 4 types of class II MHC
molecules. HLA-A, HLA-B and HLA-C all encode the a chain of class I MHC molecules, whereas HLA-DR, HLA-DP and HLA-DQ each encode both
the a and b chains of class II MHC molecules (except for HLA-DR which encodes an extra b chain).
The potential number of molecule expressed by each class is as follows:
o MHC-I: minimum of 3 and maximum of 6, average of 6
o MHC-II: minimum of 3 and maximum of 12
28
Processing of Internalized Antigens for Display by Class II MHC Molecules
A. Internalization and digestion of antigens.
o
o
o
o
o
o
Antigens destined for the class II MHC pathway are usually internalized from the extracellular environment.
Dendritic cells and macrophages may ingest extracellular microbes or microbial proteins by several mechanisms, including phagocytosis and receptor-mediated
endocytosis.
Microbes may bind to surface receptors specific for microbial products or to receptors that recognize antibodies or products of complement activation (opsonins)
attached to the microbes.
B lymphocytes efficiently internalize proteins that specifically bind to the cells’ antigen receptors
These APCs may also pinocytose proteins without any specific recognition event. After internalization into APCs by any of these pathways, the micro- bial proteins
enter acidic intracellular vesicles, called endosomes or phagosomes, which may fuse with lysosomes.
In these vesicles the proteins are broken down by proteolytic en- zymes, generating many peptides of varying lengths and sequences.
B. Binding of peptides to MHC molecules.
o
o
o
o
o
Peptides bind to newly synthesized MHC molecules in specialized vesicles. Class II MHC–expressing APCs constantly synthesize these MHC molecules in the
endoplasmic reticulum (ER).
Each newly synthesized class II molecule carries with it an attached protein called the invariant chain (Ii), which contains a sequence called the class II invariant
chain peptide (CLIP) that binds to the peptide- binding cleft of the class II molecule.
Thus, the cleft of the newly synthesized class II molecule is occupied and prevented from accepting peptides in the ER that are destined to bind to class I MHC
molecules
This class II molecule with its associated Ii is targeted to the late endosomal/lysosomal vesicles that contain peptides derived from ingested extracellular proteins. In
these vesicles, the invariant chain is degraded, leaving only CLIP in the peptide- binding cleft.
Late endosomes/lysosomes also contain a class II MHC–like protein called DM, whose function is to exchange CLIP in the class II MHC molecule with other
peptides that may be available in this compartment and can bind to the MHC molecule with higher affinity.
C. Transport of peptide-MHC complexes to the cell surface.
29
o
o
Peptide loading stabilizes class II MHC molecules, which are exported to the cell surface. Once the class II MHC molecule binds tightly to one of the
peptides generated from the ingested proteins, this peptide-MHC complex becomes stable and is delivered to the cell surface.
If the MHC mol- ecule does not find a peptide it can bind, the empty molecule is unstable and is eventually degraded by lysosomal proteases.
Processing of Cytosolic Antigens for Display by Class I MHC Molecules
Proteolysis of cytosolic proteins.
o
o
o
o
o
The peptides that bind to class I MHC molecules are derived from cytosolic proteins following digestion by the ubiquitin-proteasome
pathway. Antigenic proteins may be produced in the cytoplasm from viruses that are living inside infected cells, from some phagocytosed microbes
that may leak from or be transported out of phagosomes into the cytosol, and from mutated or altered host genes that encode cytosolic or nuclear
proteins, as in tumors.
All of these proteins, as well as the cell’s own misfolded cytosolic and nuclear proteins, are targeted for destruction by proteolysis by the ubiquitin-proteasome pathway.
These proteins are unfolded, covalently tagged with multiple copies of a peptide called ubiquitin, and threaded through a protein complex called the
proteasome that is composed of stacked rings of proteolytic enzymes.
The unfolded proteins are degraded by the proteasomes into peptides.
In cells that have been exposed to inflammatory cytokines (as in an infection), the enzymatic composition of the proteasomes changes. As a result,
these cells become very efficient at cleaving cytosolic and nuclear proteins into peptides with the size and sequence properties that enable the peptides to bind well to class I MHC molecules.
Binding of peptides to class I MHC molecules
o
o
o
o
o
To form peptide-MHC complexes, the peptides must be transported into the endoplasmic reticulum.
The peptides produced by proteasomal digestion are in the cytosol, while the MHC molecules are being synthesized in the ER, and the two need to come
together. This transport function is provided by a molecule, called the trans- porter associated with antigen process- ing (TAP), located in the ER
membrane.
TAP binds proteasome-generated peptides on the cytosolic side of the ER membrane, then actively pumps them into the interior of the ER.
Newly synthesized class I MHC mol- ecules, which do not contain bound peptides, associate with a bridging protein called tapa- sin that links them to TAP
molecules in the ER membrane.
Thus, as peptides enter the ER, they can easily be captured by the empty class I molecules. (Recall that in the ER, the class II MHC molecules are not able to
bind peptides because of the associated invariant chain.)
Transport of peptide-MHC complexes to the cell surface.
30
o If a class I molecule finds a peptide with the right fit, the complex is stabilized, released from association with TAP, and transported to the cell surface.
Cross Presentation
o
o
There are instances where both pathways intersect, a process called cross-presentation.
Cross-presentation occurs when exogenous Ag “leaks” into the cytosol from endosomes by a process called retrotranslocation; in such cases,
exogenous peptides leak into the endogenous pathway (Sec61 channels?) and can be displayed by MHC class I molecules
•
31
Question 5
32
33