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Kurdistan Regional Government-Iraq
Ministry of Higher Education & Scientific Research
Salahaddin University/Collage of Science
Immunology Course Book
2014Syllabus and Class
Schedule
Asst. prof. Dr. Fikry Ali Qadir
E-mail: [email protected]
Academic year 2012/2013
Dr.Taban K.Rasheed
E-mail: [email protected]
Academic year 2012/2013
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Salahaddin University/ College of Science
Biology Department
Immunology (2 hours)
2014 Syllabus and Class Schedule
Lecturer : Dr.Taban K.Rasheed+ Dr. Fikry Ali Qadir
Office: Biology Department
Office hours: appointed by timetable schedule.
Class information
Biology Department
 8:30 am – 10:30 am
Monday
 Hall 8
Course Book: PowerPoint
Notes from the Faculty
I want to be supportive to everyone. This "Getting Started" page will help you understand how
College of Science/Biology Department environment works, what to do first, and who to contact
if you need help. I appreciate the participation and sharing from all students related to classroom
activities for the first time.
Whenever you have some questions or concerns about immunology and the course book, ask or
email me with any questions you may have about your concern. Sometimes a quick question at
time can save a lot of frustration later!
Our discussion goal in the class room is to be collaborative, not combative. This is important to
your success in the course and as a professional. Experience shows that even an innocent remark
in the class environment can be easily misconstrued. Please re-think your responses carefully
before you react with others in order not to be conceder as personal attacks. Be positive to others
and diplomatic with your words. I will try my best to do the same. Be careful when using
sarcasm and humor. Without face-to-face communications your joke may be viewed as criticism.
Remember you are not competing with each other for grades, but sharing information and
learning from one another.
The College of Science, Department of Biology, expects that all students exhibit professional
behavior.
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My Expectations of You
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Keep up with the assignments in the course
Participate in discussions.
Let me know when you experience problems so I have a chance to assist you
Maintain honesty and respect toward your classmates and instructor
What You Can Expect of Me
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Quick response to your inquiries
Concern for your success in this course.
A willingness to work with you within the rules of the course. However, I will not make
exceptions for one person that is not available to every other person in the course.
Respect for you and your ideas in return for the respect accorded to me and others.
Course Description
Explores innate and acquired immunity, Lymphoid organ, Antigen processing and presentation,
Immunoglobulin and Immune response, Complement and cytokines, and Hypersensitivity.
What remains however, is the research paper, written in the format of an article. You are learning
to write to many audiences; that is the essence of any Baccalaureate completion program. In this
course, your paper is written to the audience of your professional peers. It is not your opinion; it
is based on research journal articles.
Learning Objectives
After completion of this course, you will be able to:
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Define common terms used in immunology and the history of immunology.
Localization of the immune system in the body
Different structure and shape of immunoglobulin
Analyze serological test as a tool for diagnosis of different human disease.
Difference between active and passive immunity
Properties of the immunogen-Antigen presenting cell-Ag processing pathway
Structure of Ig-Type of Ig-Function of Ig
Mechanism of immune response-Primary and secondary immune response.
Source-Type-Function of cytokine
Anaphylactic hypersensitivity-Type 2 hypersensitivity-Immune complex
hypersensitivity-Delayed hypersensitivity
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Course Materials
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Ivan Roitt,I. Brostoff,J. and Male,D. (2002) Immunology (6th Ed.) Ediburgh, Mosby.
Parslow,T.G. , Stites,D.P. , Terr,A.I. , Imboden,J.B. (2001) Medical Immunology(10th
Ed.) NY, McGraw Hill
Brooks, G.F., Carroll, K.C., Butel, J.S. &Morse, S.A. (2007) Medical Microbiology (24th
Ed.) NY, McGraw Hill.
Learning Methods
Class time will consist of lectures, discussions in small and/or large groups, and other activities.
You should expect to spend approximately two hours outside of class preparing for the next class
session, with additional time set aside for completing assignments and preparing for exams.
Evaluation Methods
There will be a total of four (2) exams during the course of the class. Homework assignments
and a paper research completed with a partner will also be part of your grade. All assignments
are due at the beginning of the class period; no assignments will be accepted after this time.
Assessment
Course grades are based on the following:
Component
Exam1
Exam 2
Homework/assignments/
Discussion/attendance
Date
-/-/2014
-/-/2014
As assigned
Percent
40%
40%
20%
100%
Research Paper
The paper is a formal, research paper with a literature review. This is the only place in the
Baccalaureate curriculum where students write a formal paper, in the style of a publishable
article. Do not use the first person “I”; a formal paper is written in the objective third person.
Read the editorial page of a major newspaper, or the review the language in the articles that you
will use to see this formal style of writing. Imagine yourself writing an article to a group of
professional peers, your opinion will never be used.
Text books are unacceptable sources for the research paper. Part of the exercise is to familiarize
students with writing from professional research journals. Internet sources may be used in
provide data
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Exam policy
If an emergency occurs before an exam, the student must contact the professor within 24 hours
of the exam and must have the proper documentation for the absence. If you know that you will
be missing an exam ahead of time, you must speak with the professor at least one week before
the exam date. There will be no make-up exams for students who fail to follow this policy.
Missed exams will result in a “0”.
Classroom polices
Attendance: You are strongly encouraged to attend class on a regular basis as participation is
important to your understanding of the material. This is your opportunity to ask questions. You
are responsible for obtaining any information you miss due to absence, including class notes, and
homework assignments. Prolonged absence (greater than four classes throughout the semester)
will significantly impact your ability to succeed in the class. If you must miss a class, please let
the instructor know ahead of time whenever possible.
Lateness: Lateness to class is disruptive and disrespectful to both the professor and to your
fellow students. It is expected that you will arrive to class on time and ready for the topic of the
day. Please allow time for traffic or parking problems as these are frequent occurrences in
university. If you are late, please enter the room as quickly and as quietly as possible.
Electronic devices: All cell phones are to be turned off at the beginning of class and put away
during the entire class.
Talking: During class please refrain from side conversations. These can be disruptive to your
fellow students and your professor.
E-mail: The prefer method of communication outside of class is via e-mail. E-mails do not
express tone of voice or body language, so strive to use careful wording to convey your desired
message. Please take an extra minute when sending an e-mail to think about what you want to
say, spell-check your e-mail, and use appropriate and professional language. Your professor will
strive to do the same in all communications.
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Day
7/2
14/2
21/2
Date
Topic
Student Start With
Dr. Taban
February
Basic Immunology (Immunology-Hematopoiesis-Localization of
hematopoiesis).
February
Innate Immunity (Innate immunity-Factor influencing level of innate
immunity-Mechanism of innate immunity-Humoral factor-Cellular
factor-Mode of intracellular killing).
February
Acquired Immunity (Acquired Immunity-Active immunity-Passive
immunity-Difference between active and passive immunity).
28/2
February
7/3
March
28/3
4/4
March
April
Lymphoid Organ[Lymphoid Organ-A/Primary lymphoid tissue(Bone
marrow-Bursa of fabricius-Thymus)
B/Secondary lymphoid tissue (Lymphatic circulation-Lymph nodeSpleen) C/Tertiary lymphoid tissue (Mucosal associated lymphoid
tissue-Intraepithelial lymphocyte)].
First Examination
Antigen Processing and Presentation (Properties of the immunogenAntigen presenting cell-Ag processing pathway).
9/5
Student Start With
Dr. Fikry the rest
of the subject
April
Major Histocompatibility Complex and Alloreactivity and
transplantation rejection.
April
Immunoglobulin (Structure of Ig-Type of Ig-Function of Ig).
April
Immune response (Mechanism of immune response-Primary and
secondary immune response).
May
Complement System (Definition-Function-Path way of activation –
Regulation of complement activation).
May
Cytokines (Source-Type-Function of cytokine)
16/5
May
23/5
May
11/4
18/4
25/4
2/5
Hypersensitivity (Anaphylactic hypersensitivity-Type 2 hypersensiti
vity-Immune complex hypersensitivity-Delayed hypersensitivity).
Second Examination
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Example of Semester Examinations
Answer the following:
Q1: Define
1- T-dependent Antigen
2- C4b binding protein
3- Diageorge Syndrome
4- Secondary immune response
Q2: Fill in the blanks
1- Precursor T cells must migrate to thymus where they undergo differentiation into tow type of
T cells ____________ and __________.
2-Chemotactic factor for attracting phagocytic cells to site of inflammation includes
____________, ____________, and _________.
3- Fixation of first complement (C1) needed for immune complex and binding with Ig requires
___________ and ___________ ions.
4- _________________ blocks the association of factor-B complement with C3b in alternative
pathway.
5- NK cells are capable of killing ___________ and ___________ cells.
6- IgA has a ______________ which mad in ____________ cells as its passes into secretions.
7- Thymic nurse cells secreted __________, ____________, and ________ hormones to promote
maturation of T cell in thymus.
Q3: Explain with drawing the early events in Antibody production in lymph node.
Q4: Explain
A- The classical pathway for complement activation.
B- Detoxification reaction in PMN and Macrophage.
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Basic Principles of Immunology
Understanding of immunity date to 1798, when the English physician Edward
Jenner (1749-1823) published a report that people could be protected from deadly
small pox by sticking them with a needle dipped in the pus from a cowpox boil.
The great French biologist and chemist Louis Pasteur (1822-1895) theorized that
such immunization protects people against disease by exposing them to a version
of a microbe that is harmless but is enough like the disease-causing organism, or
pathogen, that the immune system learns to fight it. Modern vaccines against
diseases such as measles, polio, and chicken pox are based on this principle.
In the late nineteenth century, a scientific debate was waged between the German
physician Paul Ehrlich (1854-1915) and the Russian zoologist Elie Metchnikoff
(1845-1916). Ehrlich and his followers believed that proteins in the blood, called
antibodies, eliminated pathogens by sticking to them; this phenomenon became
known as humoral immunity. Metchnikoff and his students, on the other hand,
noted that certain white blood cells could engulf and digest foreign materials: this
cellular immunity, they claimed, was the real way the body fought infection.
Modern immunologists have shown that both the humoral and cellular responses
play a role in fighting disease. They have also identified many of the actors and
processes that form the immune response.
Fundamentals of Blood Cell Biology
The modern word “immunity” derives from the Latin immunis, meaning
exemption from military service, tax payments or other public services.
Immunology; is the study of the way in which the body defends itself from
infectious agents and other foreign substances in environment. Its include physical
barriers like skin, protective chemical substances in the blood and tissue fluids, and
the physiological reaction of tissue to injury or infection, but most dynamic and
effective defence strategies are carried out by cells that have evolved specialized
abilities to recognize and eliminate potentially infectious substances. Some of these
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cells circulate continually through the body in search of foreign invaders; others
are sentinels and lie in wait in solid tissue or at body surfaces.
All specialized defensive cells have two things in common: They all spend at least
part of their lives in blood stream, and they all derived from cells produced in the
bone marrow.
Hematopoiesis: The process by which blood cell generated, grow, divided, and
differentiated in the bone marrow.
Three general classes of cells are produced:
1- Red blood cells (erythrocytes): responsible for oxygen transport.
2- Platelets: responsible for the control of bleeding.
3- White blood cells (leukocytes): which involved in host defence.
All three classes are hematopoitic stem cells (HSCs) which reside in the marrow
and have the unique ability to give rise to all of different mature blood cell types,
under the appropriate conditions. Both myeloid and lymphocytes are critical to
host defence.
Myeloid account 60%, lymphoid 15%, while erythroid 25% of marrow cells.
The myeloid progenitor (stem) cell in the bone marrow gives rise to erythrocytes,
platelets, neutrophils, monocytes/macrophages and dendritic cells whereas the
lymphoid progenitor (stem) cell gives rise to the NK, T cells and B cells. For T cell
development the precursor T cells must migrate to the thymus where they undergo
differentiation into two distinct types of T cells, the CD4+ T helper cell and the
CD8+ pre-cytotoxic T cell. Two types of T helper cells are produced in the thymus
the TH1 cells, which help the CD8+ pre-cytotoxic cells to differentiate into
cytotoxic T cells, and TH2 cells, which help B cells, differentiate into plasma cells,
which secrete antibodies.
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The HSCs are self-renewing cells, when they proliferate at least some of their
daughter cells remains as HSCs, so that the pool of stem cells dose note becomes
depleted.
Both self-replicating of HSCs and their ability to produce differentiated cells
depend on hormonal growth factor called cytokines (group of polypeptides,
secreted by both hematopoietic and non hematopoietic cell) many cytokines have
specific effect on growth, differentiation, survival, or function of blood cells.
Ontogeny of hematopoiesis
HSCs arise in the mesoderm of the yolk sac during the first week of embryonic
life. Within 2 months most HSCs migrate to fetal liver and its here bulk of
hematopoiesis occurs. Most embryonic and fetal hematopoisis is devoted to the
production of RBC, platelet production appears at 3 months of gestation and the
leukocytes do not appear until the fifth month. Later HSCs begin to colonize in
developing bone marrow cavities throughout the skeleton which contain a network
of epithelial cell which provide necessary environment for growth and
differentiation of HSC and their progeny. By birth, all of the bone marrow
occupied by developing hematopoietic cells, hematopoietic activity in the long
bones then declines with age, so that after puberty it’s largely confined to the axial
skeleton (The pelvis, sternum, ribs, vertebrae, and skull).
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If the bone marrow is injured by infection or malignancy or altered hematopoiesis
can resume in the liver and spleen of an adult to maintain the supply of blood cells.
Innate Immunity
Immunology: is the study of our protection from foreign macromolecules or
invading organisms and our responses to them. These invaders include viruses,
bacteria, protozoa or even larger parasites.
Our first lines of defence against foreign organisms are barrier tissues such as the
skin that stop the entry of organism into our bodies. If, however, these barrier
layers are penetrated, the body contains cells that respond rapidly to the presence
of the invader. These cells include macrophages and neutrophils that engulf foreign
organisms and kill them without the need for antibodies. Immediate challenge also
comes from soluble molecules that deprive the invading organism of essential
nutrients (such as iron) and from certain molecules that are found on the surfaces
of epithelia, in secretions (such as tears and saliva) and in the blood stream. This
form of immunity is the innate or non-specific immune system that is continually
ready to respond to invasion.
A second line of defence is the specific or adaptive immune system which may
take days to respond to a primary invasion. In the specific immune system, we see
the production of antibodies (soluble proteins that bind to foreign antigens) and
cell-mediated responses in which specific cells recognize foreign pathogens and
destroy them. In the case of viruses or tumors, this response is also vital to the
recognition and destruction of virally-infected or tumorigenic cells.
The response to a second round of infection is often more rapid than to the primary
infection because of the activation of memory B and T cells. We shall see how
cells of the immune system interact with one another by a variety of signal
molecules so that a coordinated response may be mounted. These signals may be
proteins such as lymphokines which are produced by cells of the lymphoid system,
cytokines and chemokines that are produced by other cells in an immune
response, and which stimulate cells of the immune system.
Although the innate and adaptive immune systems both function to protect against
invading organisms, they differ in a number of ways. The adaptive immune system
requires some time to react to an invading organism, whereas the innate immune
system includes defences that, for the most part, are constitutively present and
ready to be mobilized upon infection. Second, the adaptive immune system is
antigen specific and reacts only with the organism that induced the response. In
contrast, the innate system is not antigen specific and reacts equally well to a
variety of organisms. Finally, the adaptive immune system demonstrates
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immunological memory. It “remembers” that it has encountered an invading
organism and reacts more rapidly on subsequent exposure to the same organism. In
contrast, the innate immune system does not demonstrate immunological memory
Non-Specific Immune System
The elements of the non-specific (innate) immune system include anatomical
barriers, secretory molecules and cellular components. Among the mechanical
anatomical barriers are the skin and internal epithelial layers, the movement of the
intestines and the oscillation of broncho-pulmonary cilia. Associated with these
protective surfaces are chemical and biological agents.
A. Anatomical barriers to infections
1. Mechanical factors
The epithelial surfaces form a physical barrier that is very impermeable to most
infectious agents. Thus, the skin acts as our first line of defence against invading
organisms. The desquamation of skin epithelium also helps remove bacteria and
other infectious agents that have adhered to the epithelial surfaces.
Movement due to cilia or peristalsis helps to keep air passages and the
gastrointestinal tract free from microorganisms. The flushing action of tears and
saliva helps prevent infection of the eyes and mouth. The trapping affect of mucus
that lines the respiratory and gastrointestinal tract helps protect the lungs and
digestive systems from infection.
2. Chemical factors
Fatty acids in sweat inhibit the growth of bacteria. Lysozyme and phospholipase
found in tears, saliva and nasal secretions can breakdown the cell wall of bacteria
and destabilize bacterial membranes. The low pH of sweat and gastric secretions
prevents growth of bacteria. Defensins (low molecular weight proteins) found in
the lung and gastrointestinal tract have antimicrobial activity. Surfactants in the
lung act as opsonins (substances that promote phagocytosis of particles by
phagocytic cells).
3. Biologicalfactors
The normal flora of the skin and in the gastrointestinal tract can prevent the
colonization of pathogenic bacteria by secreting toxic substances or by competing
with pathogenic bacteria for nutrients or attachment to cell surfaces.
B. Humoral barriers to infection
The anatomical barriers are very effective in preventing colonization of tissues by
microorganisms. However, when there is damage to tissues the anatomical barriers
are breached and infection may occur. Once infectious agents have penetrated
tissues, another innate defence mechanism comes into play, namely acute
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inflammation. Humoral factors play an important role in inflammation, which is
characterized by oedema and the recruitment of phagocytic cells.
These humoral factors are found in serum or they are formed at the site of
infection.
1. Complement system – The complement system is the major humoral nonspecific defence mechanism. Once activated complement can lead to increased
vascular permeability, recruitment of phagocytic cells, and lysis and
opsonization of bacteria.
2. Coagulation system – Depending on the severity of the tissue injury, the
coagulation system may or may not be activated. Some products of the coagulation
system can contribute to the non-specific defences because of their ability to
increase vascular permeability and act as chemotacic agents for phagocytic cells.
In addition, some of the products of the coagulation system are directly
antimicrobial. For example, beta-lysin, a protein produced by platelets during
coagulation can lyse many Gram positive bacteria.
3. Lactoferrin and transferrin – By binding iron, an essential nutrient for bacteria,
and these proteins limit bacterial growth.
4. Interferon – Interferon are proteins that can limit virus replication in cells.
5. Lysozyme – Lysozyme breaks down the cell wall of bacteria.
6. Interleukin-1 – (IL-1) induces fever and the production of acute phase proteins,
some of which are antimicrobial because they can opsonize bacteria.
C. Cellular barriers to infection
Part of the inflammatory response is the recruitment of polymorphonuclear
eosinophiles and macrophages to sites of infection. These cells are the main line of
defence in the non-specific immune system.
1. Neutrophils – Polymorphonuclear cells (PMNs) are recruited to the site of
infection where they phagocytose invading organisms and kill them
intracellularly. In addition, PMNs contribute to collateral tissue damage that
occurs during inflammation.
2. Macrophages – Tissue macrophages and monocytes , which differentiate into
macrophages, also function in phagocytosis and intracellular killing of
microorganisms. In addition, macrophages are capable of extracellular killing of
infected or altered self target cells. Furthermore, macrophages contribute to tissue
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repair and act as antigen-presenting cells, which are required for the induction of
specific immune responses.
3. Natural killer (NK) and lymphokine activated killer (LAK) cells – NK and LAK
cells can non-specifically kill virus infected and tumor cells. These cells are not
part of the inflammatory response but they are important in nonspecific immunity
to viral infections and tumor surveillance.
4. Eosinophils – Eosinophils have proteins in granules that are effective in killing
certain parasites.
PHAGOCYTOSIS AND INTRACELLULAR KILLING
A. Phagocytic cells
1. Neutrophiles/Polymorphonuclear cells(Microphages)
PMNs are motile phagocytic cells that have lobed nuclei. They can be identified by
their characteristic nucleus or by an antigen present on the cell surface called
CD66. They contain two kinds of granules the contents of which are involved in
the antimicrobial properties of these cells. The primary or azurophilic granules,
which are abundant in young newly formed PMNs, contain cationic proteins and
defences that can kill bacteria, proteolytic enzymes like elastase, and lysozyme to
break down bacterial cell walls, and characteristically, myeloperoxidase, which is
involved in the generation of bactericidal compounds. The second type of granule
found in more mature PMNs is the secondary or specific granule. These contain
lysozyme, NADPH oxidase components, which are involved in the generation of
toxic oxygen products, and characteristically lactoferrin, an iron chelating protein
and B12-binding protein.
2. Monocytes/Macrophages - Macrophages are phagocytic cells that have a
characteristic kidney-shaped nucleus. They can be identified morphologically or by
the presence of the CD14 cell surface marker. Unlike PMNs they do not contain
granules but they have numerous lysosomes which have contents similar to the
PNM granules.
B. Response of phagocytes to infection
Circulating PMNs and monocytes respond to danger (SOS) signals generated at the
site of an infection. SOS signals include N-formyl-methionine containing peptides
released by bacteria, clotting system peptides, complement products and cytokines
released from tissue macrophages that have encountered bacteria in tissue. Some of
the SOS signals stimulate endothelial cells near the site of the infection to express
cell adhesion molecules such as ICAM-1 and selectins which bind to components
on the surface of phagocytic cells and cause the phagocytes to adhere to the
endothelium.
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Vasodilators produced at the site of infection cause the junctions between
endothelial cells to loosen and the phagocytes then cross the endothelial barrier by
“squeezing” between the endothelial cells in a process called diapedesis. Once in
the tissue spaces some of the SOS signals attract phagocytes to the infection site by
chemotaxis (movement toward an increasing chemical gradient). The SOS signals
also activate the phagocytes, which results in increased phagocytosis and
intracellular killing of the invading organisms.
C. Initiation of Phagocytosis
Phagocytic cells have a variety of receptors on their cell membranes through which
infectious agents bind to the cells. These include:
1. Fc receptors – Bacteria with IgG antibody on their surface have the Fc region
exposed and this part of the Ig molecule can bind to the receptor on phagocytes.
Binding to the Fc receptor requires prior interaction of the antibody with an
antigen. Binding of IgG-coated bacteria to Fc receptors results in enhanced
phagocytosis and activation of the metabolic activity of phagocytes (respiratory
burst).
2. Complement receptors – Phagocytic cells have a receptor for the 3rd component
of complement, C3b. Binding of C3b-coated bacteria to this receptor also results in
enhanced phagocytosis and stimulation of the respiratory burst.
3. Scavenger receptors – Scavenger receptors bind a wide variety of polyanions on
bacterial surfaces resulting in phagocytosis of bacteria.
4. Toll-like receptors – Phagocytes have a variety of Toll-like receptors (Pattern
Recognition Receptors or PRRs) which recognize broad molecular patterns called
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PAMPs (pathogen associated molecular patterns) on infectious agents. Binding of
infectious agents via Toll-like receptors results in phagocytosis and the release of
inflammatory cytokines (IL-1, TNF-alpha and IL-6) by the phagocytes.
D.Phagocytosis
After attachment of a bacterium, the phagocyte begins to extend pseudopods
around the bacterium. The pseudopods eventually surround the bacterium and
engulf it, and the bacterium is enclosed in a phagosome. During phagocytosis the
granules or lysosomes of the phagocyte fuse with the phagosome and empty their
contents. The result is a bacterium engulfed in a phagolysosome which contains the
contents of the granules or lysosomes.
E. Respiratory burst and intracellular killing
During phagocytosis there is an increase in glucose and oxygen consumption
which is referred to as the respiratory burst. The consequence of the respiratory
burst is that a number of oxygen-containing compounds are produced which kill
the bacteria being phagocytosed. This is referred to as oxygen-dependent
intracellular killing. In addition, bacteria can be killed by pre-formed substances
released from granules or lysosomes when they fuse with the phagosome. This is
referred to as oxygen-independent intracellular killing.
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1. Oxygen-dependent myeloperoxidase-independent intracellular killing.
During phagocytosis glucose is metabolized via the pentose monophosphate shunt
and NADPH is formed. Cytochrome B which was part of the specific granule
combines with the plasma membrane NADPH oxidase and activates it. The
activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the
production of superoxide anion. Some of the superoxide anion is converted to
H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion
can react with H2O2 resulting in the formation of hydroxyl radical and more
singlet oxygen. The result of all of these reactions is the production of the toxic
oxygen compounds superoxide anion (O2-), H2O2, singlet oxygen (1O2) and
hydroxyl radical (OH•).
2. Oxygen-dependent myeloperoxidase-dependent intracellular killing.
As the azurophilic granules fuse with the phagosome, myeloperoxidase is released
into the phagolysosome. Myeloperoxidase utilizes H2O2 and halide ions (usually
Cl-) to produce hypochlorite, a highly toxic substance. Some of the hypochlorite
can spontaneously break down to yield singlet oxygen. The result of these
reactions is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2).
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3. Detoxification reactions.
PMNs and macrophages have means to protect themselves from the toxic oxygen
intermediates. These reactions involve the dismutation of superoxide anion to
hydrogen peroxide by superoxide dismutase and the conversion of hydrogen
peroxide to water by catalase.
4. Oxygen-independent intracellular killing.
In addition to the oxygen-dependent mechanisms of killing there are also oxygen–
independent killing mechanisms in phagocytes: cationic proteins (cathepsin)
released into the phagolysosome can damage bacterial membranes; lysozyme
breaks down bacterial cell walls; lactoferrin chelates iron, which deprives bacteria
of this required nutrient; hydrolytic enzymes break down bacterial proteins. Thus,
even patients who have defects in the oxygen-dependent killing pathways are able
to kill bacteria. However, since the oxygen-dependent mechanisms are much more
efficient in killing, patients with defects in these pathways are more susceptible
and get more serious infections.
Oxygen-independent mechanisms of intracellular killing
Effecter Molecule and its Function:
1- Cationic proteins (including cathepsin): Damage to microbial membranes
2- Lysozyme: Splits mucopeptide in bacterial cell wall
3- Lactoferrin: Deprives proliferating bacteria of iron
4- Proteolytic and hydrolytic enzymes: Digestion of killed organisms
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NITRIC OXIDE-DEPENDENT KILLING
Binding of bacteria to macrophages, particularly binding via Toll-like receptors,
results in the production of TNF-alpha, which acts in an autocrine manner to
induce the expression of the inducible nitric oxide synthetase gene (i-nos )
resulting in the production of nitric oxide (NO). If the cell is also exposed to
interferon gamma (IFN-gamma) additional nitric oxide will be produced. Nitric
oxide released by the cell is toxic and can kill microorganism in the vicinity of the
macrophage.
NON-SPECIFIC KILLER CELLS
Several different cells including NK and LAK cells, K cells, activated
macrophages and eosinophils are capable of killing foreign and altered self target
cells in a non-specific manner. These cells play an important role in the innate
immune system.
A. NK and LAK cells
NK cells can be identified by the presence of CD56 and CD16 and a lack of CD3
cell surface markers. NK cells are capable of killing virus-infected and malignant
target cells but they are relatively inefficient in doing so. However, upon exposure
to IL-2 and IFN-gamma, NK cells become lymphokine-activated killer (LAK)
cells, which are capable of killing malignant cells.
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B. K cells
Killer (K) cells are not a morphologically distinct type of cell. Rather a K cell is
any cell that mediates antibody-dependent cellular cytotoxicity (ADCC). In ADCC
antibody acts as a link to bring the K cell and the target cell together to allow
killing to occur. K cells have on their surface an Fc receptor for antibody and thus
they can recognize, bind and kill target cells coated with antibody. Killer cells
which have Fc receptors include NK, LAK, and macrophages which have an Fc
receptor for IgG antibodies and eosinophils which have an Fc receptor for IgE
antibodies.
Specific Immune System
Adaptive immunity is often sub-divided into two major types depending on how
the immunity was introduced. Naturally acquired immunity occurs through
contact with a disease causing agent, when the contact was not deliberate, whereas
artificially acquired immunity develops only through deliberate actions such as
vaccination. Both naturally and artificially acquired immunity can be further
subdivided depending on whether immunity is induced in the host or passively
transferred from an immune host.
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Passive immunity is acquired through transfer of antibodies or activated T-cells
from an immune host, and is short lived, usually lasts only a few months, whereas
active immunity is induced in the host itself by antigen, and lasts much longer,
sometimes life-long. The diagram below summarizes these divisions of immunity.
A further subdivision of adaptive immunity is characterized by the cells involved;
humoral immunity is the aspect of immunity that is mediated by secreted
antibodies,
Whereas the protection provided by cell mediated immunity involves Tlymphocytes alone humoral immunity is active when the organism generates its
own antibodies and passive when antibodies are transferred between individuals.
Similarly, cell mediated immunity is active when the organisms’ own T-cells are
stimulated and passive when T cells come from another organism.
Passive immunity is the transfer of active immunity, in the form of readymade
antibodies, from one individual to another. Passive immunity can occur naturally,
when maternal antibodies are transferred to the fetus through the placenta, and can
also be induced artificially, when high levels of human (or horse) antibodies
specific for a pathogen or toxin are transferred to non- immune individuals. Passive
immunization is used when there is a high risk of infection and insufficient time
for the body to develop its own immune response, or to reduce the symptoms of
ongoing or immunosuppressive diseases. Passive immunity provides immediate
protection, but the body does not develop memory, therefore the patient is at risk
of being infected by the same pathogen later.
Naturally acquired passive immunity
Maternal passive immunity is a type of naturally acquired passive immunity, and
refers to antibody-mediated immunity conveyed to a fetus by its mother during
pregnancy. Maternal antibodies (MatAb) are passed through the placenta to the
fetus by an FcRn receptor on placental cells.
This occurs around the third month of gestation. IgG is the only antibody isotype
that can pass through the placenta. Passive immunity is also provided through the
22
transfer of IgA antibodies found in breast milk that are transferred to the gut of the
infant, protecting against bacterial infections, until the newborn can synthesize its
own antibodies.
Artificially acquired passive immunity
Artificially acquired passive immunity is a short-term immunization induced by
the transfer of antibodies, which can be administered in several forms; as human or
animal blood plasma, as pooled human immunoglobulin for intravenous (IVIG) or
intramuscular (IG) use, and in the form of monoclonal antibodies (MAb). Passive
transfer is used prophylactically in the case of immunodeficiency diseases, such as
hypogammaglobulinemia. It is also used in the treatment of several types of acute
infection, and to treat poisoning. Immunity derived from passive immunization
lasts for only a short period of time, and there is also a potential risk for
hypersensitivity reactions, and serum sickness, especially from gamma globulin of
non-human origin.
The artificial induction of passive immunity has been used for over a century to
treat infectious disease, and prior to the advent of antibiotics, was often the only
specific treatment for certain infections. Immunoglobulin therapy continued to be a
first line therapy in the treatment of severe respiratory diseases until the 1930’s,
even after sulfonamide antibiotics were introduced.
Passive transfer of cell-mediated immunity
Passive or "adoptive transfer" of cell-mediated immunity, is conferred by the
transfer of "sensitized" or activated T-cells from one individual into another. It is
rarely used in humans because it requires histocompatible (matched) donors, which
are often difficult to find. In unmatched donors this type of transfer carries severe
risks of graft versus host disease. It has, however, been used to treat certain
diseases including some types of cancer and immunodeficiency. This type of
transfer differs from a bone marrow transplant, in which (undifferentiated)
hematopoietic stem cells are transferred.
Active immunity
The time course of an immune response. Due to the formation of immunological
memory, reinfection at later time points leads to a rapid increase in antibody
production and effectors T cell activity. These later infections can be mild or even
in apparent.
When Bcells and T cells are activated by a pathogen, memory B-cells and T- cells
develop. Throughout the lifetime of an animal these memory cells will
“remember” each specific pathogen encountered, and are able to mount a strong
response if the pathogen is detected again. This type of immunity is both active and
adaptive because the body's immune system prepares itself for future challenges.
Active immunity often involves both the cell-mediated and humoral aspects of
immunity as well as input from the innate immune system. The innate system is
23
present from birth and protects an individual from pathogens regardless of
experiences, whereas adaptive immunity arises only after an infection or
immunization and hence is "acquired" during life.
Naturally acquired active immunity
Naturally acquired active immunity occurs when a person is exposed to a live
pathogen, and develops a primary immune response, which leads to immunological
memory. This type of immunity is “natural” because it is not induced by deliberate
exposure. Many disorders of immune system function can affect the formation of
active immunity such as immunodeficiency (both acquired and congenital forms)
and immunosuppression.
Artificially acquired active immunity
Artificially acquired active immunity can be induced by a vaccine, a substance that
contains antigen. A vaccine stimulates a primary response against the antigen
without causing symptoms of the disease. The term vaccination was coined by
Edward Jenner and adapted by Louis Pasteur for his pioneering work in
vaccination. The method Pasteur used entailed treating the infectious agents for
those diseases so they lost the ability to cause serious disease. Pasteur adopted the
name vaccine as a generic term in honor of Jenner's discovery, which Pasteur's
work built upon.
24
Lymphoid Organ
Lymphocytes are found circulating in the blood. A large proportion of them are
found either in discrete cluster or organised in specific tissue. The component of
this lymphoid system may be categorised as primary, Secondary or tertiary
lymphoid tissue.
• Primary lymphoid tissue: Bone marrow, Bursa of fabricius, Thymus.
• Secondary lymphoid tissue: Lymph node, Spleen.
• Tertiary lymphoid tissue: Mucosa-associated lymphoid tissue, Intraepithelial lymphocytes.
1- Primary lymphoid tissue: Major sites of lymphopoiesis (lymphocyte
differentiated from lymphoid stem cell, proliferat and mature to functional effector
cells) and include:
Bone marrow: all lymphocytes develop initially from haematopoietic stem cells in
the bone marrow. Immature B cells remain in the bone marrow and develop in the
mature cells.This processes are influenced by many factor including surface
ligands and cytokines (particularly IL-7).
Bursa of fabricius: The bursa is an epithelial and lymphoid organ that is found
only in birds. It function include development and differentiation of B-lymphocyte
. Its composed of numerous lobes each has a cortical and medullary area. The
cortex contain mostly large undifferentiated-lymphoid cells While the medulla
contains small, mature cells.
25
Thymus: thymus in mammals is a bilobed organ, located in the thoracic cavity, the
2 lobes divided by trabeculae in to lobules, each of which has an outer cortex and
an inner medulla.
Epithelial cell in the cortex “thymic nurse cells” made up of sequamous cells that
make and secret factors that attract T-cell precursors from the blood and promote
subsequent maturation within thymus. These factor include:
1- Chemokine: called thymus-expressed cytokine (TECK )
2- Hormones: thymulin, thymosin, and thymopoietine
Nave cells enter the thymus from subcapsular sinus. They migrate through the
cortex and medulla and undergo differentiation, express CD3 and either CD4 or
CD8 and leave thymus as mature T cells.
Thymus is relatively large and highly active at birth (22gm) reach its peak weight
at puberty (35gm) there after it begins to involutes as the lymphoid components
recede and replaced by fatty C.T. Little more than 6gm of thymic tissue persist in
adulthood.
26
If thymus surgically removed in neonates, they became severely immunodeficient
and fail to thrive and babies without functional thymus lead to disease called
Diageorge Syndrome.
Adult have developed enough mature T cell that removal of thymus or reduction of
its function has milder effect.
27
2- Secondary lymphoid tissue : are sites of accumulation and presentation of Ag
to both virgin and memory lymphocyte populations.
Lymphatic circulation: water and low molecular weight solutes leachout from
blood vessel walls into the lower pressure intersitial space.
Most of this fluid returns to blood stream through the walls of nearby venules, but
a substantial amount dose not. Instead, this portion flows through the
tissues,carring Ag and collected in a branching network of lymphatic vessels. Once
the fluid enters these vessels it is know as lymph.
After passing through secondary lymphoid organ, lymph empties in to larger
lymphatic vessels.Thoracic duct and right lymphatic duct which drain their
contents in to right and left subclavian veins in the thorax.
Lymph: interstitial fluid surrounding cells in tissue or organ low in protein content
than blood plasma.
Lymphatic vessels: (lymphocytes + dendritic)cell + lymph
Blood vessels: (lymphocytes + RBC + monocyte + neutrophil + Basophil
+eosinphil + Mф) + plasma
28
Lymph node: the lymph nodes form part of a network which filters antigens from
the interstitial tissue fluid and lymph during its passage from the periphery to the
thoracic duct and the major collecting duct.
Human lymph nodes are 2-10 mm in diameter and round or kidney shaped with
blood vessels enter and leave the node from hilus. Its surrounded by a collagenous
capsule. Radical trabeculae with reticular fibers support cellular components.
The lymph node consist of a B-cell area (cortex), a T-cell area (paracortex) and a
central medulla containing T- cells, B-cells, abundant plasma cells and
macrophages.lymph node cortex contain cellular aggregation called lymphoid
follicles which composed of memory B-lymphocytes, smaller number of T-cell and
follicular dendritic cell.
29
Lymphoid follicles re of 2 types:
• Primary follicles: contain mature resting B-cells.
• Secondary follicles: with germinal center contain various stages of activation
and blast transformation with numerous macrophage and occasional plasma
cells may be seen.
The paracortex contains specialized capillary vessels( high endothelial venulesHEV) that allow traffic of lymphocytes out of the circulation in to lymph node
(lymphocyte traffic). The lymph that flows into a node may carry with it
microorganism or other foreign matter from tissue. When such substance enters
lymphocytes and macrophage in the node respond by activation as a result, some
of the resident lymphocytes begins to proliferate, inflammatory mediators are
released locally, blood flow to the node
Increases and node may become noticeably enlarged when infection develop. The
swelling decrease when the infection ends.
Spleen: the spleen filters blood much as the lymph nodes filter lymph. Its located
just below the diaphragm on the left side of the abdomen, the spleen weight 150gm
in adult and enclused in a thin connective tissue capsule. Most of the spleen consist
of red pulp and whit pulp.
30
Function of red pulp:
Blood enters the spleen via the splenic artery, which divides into many arteries
called central arteries. These arteries become thiner arterioles, which eventually
enter the red pulp. The red pulp contains thin-walled blood vessels called venous
sinusoid and in between these sinusoids are called splenic cords.
Blood cells are emptied out of the arteries directly into the splenic cords. To reenter the blood circulation, blood cells must transverse the splenic cords and enter
the venous sinusoids. Venous sinusoids lined with macrophage and the splenic
cords are full of macrophage. These macrophage recognize and phgocytose old or
damaged red cells and pletelets preventing their re-entry in to the blood. Beside old
red cells membranes are less elastic and unable to squeeze through the wall of
endothelium of venous sinusoids to re-enter the blood stream.
31
Each arteriole is encased in lymphoid tissue that consist mainly of mature T- cells
and is called the periarteriolar lymphoid sheath(PALS). Adjacent to PALS is the Bcell area containing primary follicles and secondary follicle with germinal centres.
Between the whight pulp and the red pulp is the area called the marginal zone,
which contains B-cells and macrophage.
3- Tertiary lymphoid tissue: tissue in the body possess poorly organised
collections of lymphoid cells. Such collections include mucosa-associated
lymphoid tissue and intraepthelial lymphocytes (IEL).
Mucosa-associated lymphoid tissue: diffuse lymphoid tissue found in sub
mucosal regions and consititute the largest lymphoid organ, containing roughly
half the lymphoid cell in the body including: Gut associated lymphoid
tissue(GALT), Bronchus associated lymphoid tissue(BALT). GALT consist of
peyers patches which present beneath the mucosal epilethlium of the small
intestine. Its function is secrete antibodies across the mucosalsurface as a defense
against external pathogens. BALT consist of large collection of
lymphocytes(majority B cells) found along the main bronchi in the lungs.
At other sites, cells are organized in stable anatomic structures e.g. Tonsils (are
nodular aggregates of macrophage and lymphoid cells located immediately beneath
the stratified sequamous epithelium.
Intraepithelial lymphocytes: Large number of lymphocytes are intrinsically
associated with the epithelial surfaces of the body particularly reproductive tract,
the lung and skin. These collection of lymphoid cells play a key role in the
development of both local and systemic specific immune responses to antigens
present at the body surface.
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Antigens
A. Immunogen
A substance that induces a specific immune response.
B. Antigen (Ag)
A substance that reacts with the products of a specific immune response.
C. Hapten
A substance that is non-immunogenic but which can react with the products of a
specific immune response. Haptens are small molecules which could never induce
an immune response when administered by themselves but which can when
coupled to a carrier molecule. Free haptens, however, can react with products of
the immune response after such products have been elicited. Haptens have the
property of antigenicity but not immunogenicity.
D. Epitope or Antigenic Determinant
That portion of an antigen that combines with the products of a specific immune
response.
E. Antibody (Ab)
A specific protein which is produced in response to an immunogen and which
reacts with an antigen.
FACTORS INFLUENCING IMMUNOGENICITY
A. Contribution of the Immunogen
1. Foreignness
The immune system normally discriminates between self and non-self such that
only foreign molecules are immunogenic.
2. Size
There is not absolute size above which a substance will be immunogenic.
However, in general, the larger the molecule the more immunogenic it is likely to
be.
3. Chemical Composition
In general, the more complex the substance is chemically the more immunogenic it
will be. The antigenic determinants are created by the primary sequence of residues
in the polymer and/or by the secondary, tertiary or quaternary structure of the
molecule.
4. Physical form
In general particulate antigens are more immunogenic than soluble ones and
denatured antigens more immunogenic than the native form.
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5. Degradability
Antigens that are easily phagocytosed are generally more immunogenic. This is
because for most antigens (T-dependant antigens, see below) the development of
an immune response requires that the antigen be phagocytosed, processed and
presented to helper T cells by an antigen presenting cell (APC).
B. Contribution of the Biological System
1. Genetic Factors
Some substances are immunogenic in one species but not in another. Similarly,
some substances are immunogenic in one individual but not in others (i.e.
responders and non-responders). The species or individuals may lack or have
altered genes that code for the receptors for antigen on B cells and T cells or they
may not have the appropriate genes needed for the APC to present antigen to the
helper T cells.
2. Age
Age can also influence immunogenicity. Usually the very young and the very old
have a diminished ability to mount and immune response in response to an
immunogen.
C. Method of Administration
1. Dose
The dose of administration of an immunogen can influence its immunogenicity.
There is a dose of antigen above or below which the immune response will not be
optimal.
2. Route
Generally the subcutaneous route is better than the intravenous or intragastric
routes. The route of antigen administration can also alter the nature of the response
3. Adjuvants
Substances that can enhance the immune response to an immunogen are called
adjuvants. The use of adjuvants, however, is often hampered by undesirable side
effects such as fever and inflammation.
CHEMICAL NATURE OF IMMUNOGENS
A. Proteins
The vast majority of immunogens are proteins. These may be pure proteins or they
may be glycoproteins or lipoproteins. In general, proteins are usually very good
immunogens.
B. Polysaccharides
Pure polysaccharides and lipopolysaccharides are good immunogens.
C. Nucleic Acids
34
Nucleic acids are usually poorly immunogenic. However, they may become
immunogenic when single stranded or when complexed with proteins.
D. Lipids
In general lipids are non-immunogenic, although they may be haptens.
TYPES OF ANTIGENS
A. T-independent Antigens
T-independent antigens are antigens which can directly stimulate the B cells to
produce antibody without the requirement for T cell help In general,
polysaccharides are T-independent antigens. The responses to these antigens differ
from the responses to other antigens.
Properties of T-independent antigens
1. Polymeric structure
These antigens are characterized by the same antigenic determinant repeated many
times as illustrated in Figure .
2. Polyclonal activation of B cells
Many of these antigens can activate B cell clones specific for other antigens
(polyclonal activation). T-independent antigens can be subdivided into Type 1 and
Type 2 based on their ability to polyclonally activate B cells. Type 1 Tindependent antigens are polyclonal activators while Type 2 are not.
3. Resistance to degradation
T-independent antigens are generally more resistant to degradation and thus they
persist for longer periods of time and continue to stimulate the immune system.
B. T-dependent Antigens
T-dependent antigens are those that do not directly stimulate the production of
antibody without the help of T cells. Proteins are T-dependent antigens.
Structurally these antigens are characterized by a few copies of many different
antigenic determinants as illustrated in the Figure
35
SUPERANTIGENS
When the immune system encounters a conventional T-dependent antigen, only a
small fraction (1 in 104 -105) of the T cell population is able to recognize the
antigen and become activated (monoclonal/oligoclonal response). However, there
are some antigens which polyclonally activate a large fraction of the T cells (up to
25%). These antigens are called superantigens .
Examples of superantigens include: Staphylococcal enterotoxins (food poisoning),
Staphylococcal toxic shock toxin (toxic shock syndrome), Staphylococcal
exfoliating toxins (scalded skin syndrome) and Streptococcal pyrogenic exotoxins
(shock). Although the bacterial superantigens are the best studied there are
superantigens associated with viruses and other microorganisms as well.
The diseases associated with exposure to superantigens are, in part, due to hyper
activation of the immune system and subsequent release of biologically active
cytokines by activated T cells.
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Antigen Processing and Presentation
Antigens are captured and taken from external environment in to a cell through
engulfment by:
A- phagocytosis: for micriorganism, part of microorganism and large partical of
protein.
B- endocytosis: small particles or individual proteins captured by receptors
C- pinocytosis: for free, soluble proteins.
Antigen-presenting cells(APC): refers to cell that express class II MHC molecules
so can present Ag to helper T-cells. Three major class of cells function as APCs
(Dendritic cells, Macrophage, and B-cells).
Mature dendritic cell most potent APCs because of their efficiency in capturing,
transporting, presentation of Ag and attracting and activation of specific T-cells.
Single dendritic cell can activate up to 3000 T-cells.
The antigen-processing pathways.
A- Endocytic pathway:
protein captured through any of engulfment pathway and taken into endosomal
vesicles and gradually broken down by exposure to an acidic ph and cellular
proteolytic enzymes. Protein is destroyed to short peptides which cleaveage by
proteinase then the peptides transported to the cell surface for presentation to T
cells. This pathway delivers peptides to MHC classII molecules which are
expressed by macrophage and other APCs that present Ag to CD4 Th lymphocytes.
B- Cytosolic pathway:
This way include pathogen that live inside infected host cells (e.g. Viruses,
intracellular bacteria (shigella), richettsia, chlamydia, listeria and intracellular
parasites(Toxoplasma).
37
Intracellular pathogen processed through a sequence of event: cytosolic antigenes
cleavage within proteasome enzyme to short peptides then pumped to lumen of
rough-endoplasmic reticulum (RER) through a channal called transporter of
antigenic peptides then associated with class I MHC proteins and are delivered to
the cell surface for presentation to CD8 T-lymphocytes.
This pathway expressed by every nucleated human cell ensuring that any cell that
becomes infected can present Ag to cytotoxic T-cells which lead to killing of
infected cell and help to limit spread of the pathogen while endocytic pathway
expressed only by APCs.
The Ag-processing occure in all normal cells even in the absence of infection e.g.
Unstable cellular protein from the cytosol cleaved in to peptide and then processed
by cytosolic pathway thus human cell normally presents a great peptides as MHC
Class I complex on its surface.
38
Immunoglobulins classes
Antibodies (also known as immunoglobulins,Ig)
Immunoglobulins are glycoprotein molecules that are produced by plasma cells in
response to an immunogen and which function as antibodies. They are typically
made of basic structural units—each with two large heavy chains and two small
light chains to form, for example, monomers with one unit, dimers with two units
or pentamers with five units. Antibodies are produced by a kind of white blood cell
called a plasma cell. There are different types of antibody which are grouped into
different isotypes based on which heavy chain they possess. Five different
antibody isotypes are known in mammals, which perform different roles, and help
direct the appropriate immune response for each different type of foreign object
they encounter.
GENERAL FUNCTIONS OF IMMUNOGLOBULINS
A. Antigen binding
Immunoglobulins bind specifically to one or a few closely related antigens. Each
immunoglobulin actually binds to a specific antigenic determinant. Antigen
binding by antibodies is the primary function of antibodies and can result in
protection of the host.
B. Effector Functions
39
The immunoglobulins mediate a variety of effector functions. Usually the ability to
carry out a particular effector function requires that the antibody bind to its
antigen. Such effector functions include:
• 1. Fixation of complement - This results in lysis of cells and release of
biologically active molecules.
• 2. Binding to various cell types - Phagocytic cells, lymphocytes, platelets,
mast cells, and basophils have receptors that bind immunoglobulins. This
binding can activate the cells to perform some function. Some
immunoglobulins also bind to receptors on placental trophoblasts, which
results in transfer of the immunoglobulin across the placenta. As a result, the
transferred maternal antibodies provide immunity to the fetus and newborn.
BASIC STRUCTURE OF IMMUNOGLOBULINS
The basic structure of the immunoglobulins is are built from the same basic units.
A. Heavy and Light Chains
All immunoglobulins have a four chain structure as their basic unit. They are
composed of two identical light chains (23kD) and two identical heavy chains (5070kD)
B. Disulfide bonds
1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy
chains are held together by inter-chain disulfide bonds and by non-covalent
interactions.
2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also
intra-chain disulfide bonds.
C. Variable (V) and Constant (C) Regions
When the amino acid sequences of many different heavy chains and light chains
were compared, it became clear that both the heavy and light chain could be
divided into two regions based on variability in
the amino acid sequences. These are the:
1. Light Chain - VL (110 amino acids) and CL (110 amino acids)
2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)
D. Hinge Region
This is the region at which the arms of the antibody molecule forms a Y. It is
called the hinge region because there is some flexibility in the molecule at this
point.
E. Domains
Three dimensional images of the immunoglobulin molecule show that it is not
straight but, it is folded into globular regions each of which contains an intra-chain
disulfide bond These regions are called domains.
1. Light Chain Domains - VL and CL
40
2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)
F. Oligosaccharides
Carbohydrates are attached to the CH2 domain in most immunoglobulins.
However, in some cases carbohydrates may also be attached at other locations.
Immunoglobulin classes
The immunoglobulins can be divided into five different classes, based on
differences in the amino acid sequences in the constant region of the heavy chains.
All immunoglobulins within a given class will have very similar heavy chain
constant regions. These differences can be detected by sequence studies or more
commonly by serological means (i.e. by the use of antibodies directed to these
differences).
1.
2.
3.
4.
5.
IgG - Gamma heavy chains
IgM - Mu heavy chains
IgA - Alpha heavy chains
IgD - Delta heavy chains
IgE - Epsilon heavy chains
41
STRUCTURE AND SOME PROPERTIES OF IG CLASSES AND
SUBCLASSES
A. IgG
1. Structure
The structures of the IgG subclasses are presented in figure. All IgG's are
monomers (7S immunoglobulin). The subclasses differ in the number of disulfide
bonds and length of the hinge region.
2. Properties
IgG is the most versatile immunoglobulin because it is capable of carrying out all
of the functions of immunoglobulin molecules.
a) IgG is the major Ig in serum - 75% of serum Ig is IgG
b) IgG is the major Ig in extra vascular spaces
c) Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer
is mediated by a receptor on placental cells for the Fc region of IgG. Not all
subclasses cross equally well; IgG2 does not cross well.
d) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix
complement
e) Binding to cells - Macrophages, monocytes, PMNs and some lymphocytes have
Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2
and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors
on PMNs, monocytes and macrophages is that the cell can now internalize the
antigen better.
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B. IgM
1. Structure
IgM normally exists as a pentamer but it can also exist as a monomer. In the
pentameric form all heavy chains are identical and all light chains are identical.
IgM has an extra domain on the mu chain (CH4) and it has another protein called
the J chain. This chain functions in polymerization of the molecule into a
pentamer.
2. Properties
a) IgM is the third most common serum Ig.
b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin
B cells when it is stimulated by antigen.
c) As a consequence of its pentameric structure, IgM is a good complement fixing
Ig. Thus, IgM antibodies are very efficient in leading to the lysis of
microorganisms.
d) As a consequence of its structure, IgM is also a good agglutinating Ig . Thus,
IgM antibodies are very good in clumping microorganisms for eventual
elimination from the body.
e) IgM binds to some cells via Fc receptors.
f) B cell surface Ig
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C. IgA
1. Structure
Serum IgA is a monomer but IgA found in secretions is a dimer as. When IgA exits
as a dimer, a J chain is associated with it.
When IgA is found in secretions is also has another protein associated with it
called the secretory piece ; Unlike the remainder of the IgA which is made in the
plasma cell, the secretory piece is made in epithelial cells and is added to the IgA
as it passes into the secretions. The secretory piece helps IgA to be transported
across mucosa and also protects it from degradation in the secretions.
44
Properties
a) IgA is the 2nd most common serum Ig.
b) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since
it is found in secretions secretory IgA is important in local (mucosal) immunity.
c) Normally IgA does not fix complement, unless aggregated.
d) IgA can binding to some cells - PMN's and some lymphocytes.
Figure 13 IgD Structure
D. IgD
1. Structure
2. IgD exists only as a monomer.
Properties
a) IgD is found in low levels in serum; its role in serum uncertain.
b) IgD is primarily found on B cell surfaces where it functions as a receptor for
antigen. IgD on the surface of B cells has extra amino acids at C-terminal end for
anchoring to the membrane. It also associates with the Ig-alpha and Ig-beta chains.
c) IgD does not bind complement.
E. IgE
Structure
IgE exists as a monomer and has an extra domain in the constant region.
45
Properties
a) IgE is the least common serum Ig since it binds very tightly to Fc receptors on
basophils and mast cells even before interacting with antigen.
b) Involved in allergic reactions - As a consequence of its binding to basophils an
mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE
on the cells results in the release of various pharmacological mediators that result
in allergic symptoms.
c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise
in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic
infections. Eosinophils have Fc receptors for IgE and binding of eosinophils to
IgE-coated helminths results in killing of the parasite.
d) IgE does not fix complement.
46
Immune response
Is a complex and regulated sequence of events involving several cell types. Its
triggered when an Ag enter the body and encounters a specialized class of APCs.
These cells capture the Ag and processed in a form that can be recognized by
helper T lymphocyte, the helper T cells activated and promote the activation of Bcells or cytotoxic T cells. The activated lymphocytes proliferate and carry out their
specific function(inactivate or eliminate the Ag).
At each stage in this process, the lymphocytes and APCs communicate with one
another through direct or by secreting regulatory cytokines.
47
When a person exposed to Ag B-cell response and concentration of serum Ab
against that Ag rise this is divided into several phase.
1- Lag phase: time between intial exposure to immunogen and appear of Ab in the
circulating which average 1 week in human.
2- Exponential phase: is rapid increase in quality of circulating Ab
3- Steady-State: Ab level remains constant because secretion and degradation is in
equal rates.
4- Decling-phase: Ab level decline(no new plasma cell produced and existing
plasma cell are dying).
This kind of immune response is weak, short-lived and called primary immune
response. subsequent encounters with same immunogen lead to response similar to
primary but lag period is short and Ab level rise more rapidly (because of presence
of primary memory T and B cells) to much higher level and the steady-state level
remaining in the same for much longer periods.
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49
Complement System
Complement is a group of serum proteins that are in an inactive state but when
appropriately stimulated, form an enzyme cascade designed to stimulate
inflammation and kill infectious agents. Complement comprises over 20 different
serum proteins that are produced by a variety of cells including, hepatocytes,
macrophages and gut epithelial cells.
Proteins of the Complement system
• Classical pathway
Activation Proteins: C1qrs, C2, C3, C4 Control Proteins: C1-INH, C4-BP
• Lectin Pathway
Mannan binding protein (MBP), mannan-asociated serine protease (MASP,
MASP2)
• Alternative Pathway
C3, Factors B & D*, Properdin (P) ,Factors I* & H, decay accelerating factor
(DAF), Complement receptor 1(CR1).
• Lytic Pathway
C5, C6, C7, C8, C9, Protein S.
COMPLEMENT FUNCTIONS
Historically, the term complement (C) was used to refer to a heat-labile serum
component that was able to lyse bacteria (activity is destroyed (inactivated) by
heating serum at 56 degrees C for 30 minutes). However, complement is now
known to contribute to host defenses in other ways as well.
1- C have intrinsic ability to lysis the cell membranes of many different bac. Sp. ,
virus, and fungi.
2- Activation of immune system by attracting phagocytic cell to site of reaction by
chemotaxis.
3- Coating bacterial surface with the opson protein “opsonization” which allow
phagocytic cell to recognize bac. through receptor for C on their surface.
4- C can participate in regulation of antibody responses and it can aid in the
clearance of immune complexes and apoptotic cells.
5- Complement can also have detrimental effects for the host; it contributes to
inflammation and tissue damage and it can trigger anaphylaxis.
50
PATHWAYS OF COMPLEMENT ACTIVATION.
Complement activation can be divided into four pathways: the classical pathway,
the lectin pathway, the alternative pathway and the membrane attack (or lytic)
pathway. Both classical and alternative pathways lead to the activation of C5
convertase and result in the production of C5b which is essential for the activation
of the membrane attack pathway.
• CLASSICAL PATHWAY
C1 activation
C1, a multi-subunit protein containing three different proteins (C1q, C1r and C1s),
binds to the Fc region of IgG and IgM antibody molecules that have interacted
with antigen. C1 binding does not occur to antibodies that have not complexed
with antigen and binding requires calcium and magnesium ions. The binding of
C1 to antibody is via C1q and C1q must cross link at least two antibody molecules
before it is firmly fixed. The binding of C1q results in the activation of C1r which
in turn activates C1s. The result is the formation of an activated “C1qrs”, which is
an enzyme that cleaves C4 into two fragments C4a and C4b.
51
C4 and C2 activation (generation of C3 convertase)
The C4b fragment binds to the membrane and the C4a fragment is released into the
microenvironment. Activated “C1qrs” also cleaves C2 into C2a and C2b. C2a
binds to the membrane in association with C4b, and C2b is released into the
microenvironment. The resulting C4bC2a complex is a C3 convertase, which
cleaves C3 into C3a and C3b.
52
• C3
activation
(generation
of
C5
convertase)
C3b binds to the membrane in association with C4b and C2a, and C3a is
released into the microenvironment. The resulting C4bC2aC3b is a C5
convertase. The generation of C5 convertase is the end of the classical
pathway.
If the classical pathway were not regulated there would be continued production of
C2b, C3a, and C4a. Thus, there must be some way to regulate the activity of the
classical pathway.
• All C1-INH: dissociates C1r and C1s from C1q
• C3a: C3a inactivator (C3a-INA;Carboxypeptidase B); inactivates C3a.
• C3b: Factors H and I; Factor H facilitates the degradation of C3b by Factor
I
• C4a: C3-INA
• C4b C4 binding protein(C4-BP) and Factor I; C4-BP facilitates
degradation of C4b by Factor I; C4-BP also prevents association of C2a with
C4b thus blocking the formation of C3 convertase
• LECTIN PATHWAY
The lectin pathway (figure 3) is very similar to the classical pathway. It is
initiated by the binding of mannose-binding lectin (MBL) to bacterial surfaces
with mannose-containing polysaccharides (mannans). Binding of MBL to a
pathogen results in the association of two serine proteases, MASP-1 and
MASP-2 (MBL-associated serine proteases). MASP-1 and MASP-2 are similar
to C1r and C1s, respectively and MBL is similar to C1q. Formation of the
MBL/MASP-1/MASP-2 tri-molecular complex results in the activation of the
MASPs and subsequent cleavage of C4 into C4a and C4b. The C4b fragment
binds to the membrane and the C4a fragment is released into the
microenvironment. Activated MASPs also cleave C2 into C2a and C2b. C2a
binds to the membrane in association with C4b and C2b is released into the
microenvironment. The resulting C4bC2a complex is a C3 convertase, which
cleaves C3 into C3a and C3b. C3b binds to the membrane in association with
C4b and C2a and C3a is released into the microenvironment. The resulting
C4bC2aC3b is a C5 convertase. The generation of C5 convertase is the end of
the lectin pathway
53
ALTERNATIVE PATHWAY
The alternative pathway begins with the activation of C3 and requires Factors B
and D and Mg++ cation, all present in normal serum.
1. Amplification loop of C3b formation
In serum there is low level spontaneous hydrolysis of C3 to produce C3i. Factor B
binds to C3i and becomes susceptible to Factor D, which cleaves Factor B into Bb.
The C3iBb complex acts as a C3 convertase and cleaves C3 into C3a and C3b.
Once C3b is formed, Factor B will bind to it and becomes susceptible to cleavage
by Factor D. The resulting C3bBb complex is a C3 convertase that will continue to
generate more C3b, thus amplifying C3b production. If this process continues
unchecked, the result would be the consumption of all C3 in the serum. Thus, the
spontaneous production of C3b is tightly controlled.
54
2. Control of the amplification loop.
As spontaneously produced C3b binds to autologous host membranes, it interacts
with DAF (decay accelerating factor), which blocks the association of Factor B
with C3b thereby preventing the formation of additional C3 convertase. In
addition, DAF accelerates the dissociation of Bb from C3b in C3 convertase that
has already formed, thereby stopping the production of additional C3b. Some cells
possess complement receptor 1 (CR1). Binding of C3b to CR1 facilitates the
enzymatic degradation of C3b by Factor I. In addition, binding of C3 convertase
(C3bBb) to CR1 also dissociates Bb from the complex. Thus, in cells possessing
complement receptors, CR1 also plays a role in controlling the amplification loop.
Finally, Factor H can bind to C3b bound to a cell or in the in the fluid phase and
facilitate the enzymatic degradation of C3b by Factor I. Thus, the amplification
loop is controlled by either blocking the formation of C3 convertase, dissociating
C3 convertase, or by enzymatically digesting C3b.
The importance of controlling this amplification loop is illustrated in patients with
genetic deficiencies of Factor H or I. These patients have a C3 deficiency and
increased susceptibility to certain infections.
55
3. Stabilization of C convertase by activator (protector) surfaces
When bound to an appropriate activator of the alternative pathway, C3b will bind
Factor B, which is enzymatically cleaved by Factor D to produce C3 convertase
(C3bBb). However, C3b is resistant to degradation by Factor I and the C3
convertase is not rapidly degraded, since it is stabilized by the activator surface.
The complex is further stabilized by properdin binding to C3bBb. Activators of
the alternate pathway are components on the surface of pathogens and include:
LPS of Gram-negative bacteria and the cell walls of some bacteria and yeasts.
Thus, when C3b binds to an activator surface, the C3 convertase formed will be
stable and continue to generate additional C3a and C3b by cleavage of C3.
4. Generation of C5 convertase
Some of the C3b generated by the stabilized C3 convertase on the activator surface
associates with the C3bBb complex to form a C3bBbC3b complex. This is the C5
convertase of the alternative pathway. The generation of C5 convertase is the end
56
of the alternative pathway. The alternative pathway can be activated by many
Gram-negative (most significantly, Neisseria meningitidis and N. gonorrhoea),
some Gram-positive bacteria and certain viruses and parasites, and results in the
lysis of these organisms. Thus, the alternative pathway of C activation provides
another means of protection against certain pathogens before an antibody response
is mounted. A deficiency of C3 results in an increased susceptibility to these
organisms. The alternate pathway may be the more primitive pathway and the
classical and lectin pathways probably developed from it.
• MEMBRANE ATTACK (LYTIC) PATHWAY
C5 convertase from the classical (C4b2a3b), lectin (C4b2a3b) or alternative
(C3bBb3b) pathway cleaves C5 into C5a and C5b. C5a remains in the fluid phase
and the C5b rapidly associates with C6 and C7 and inserts into the membrane.
Subsequently C8 binds, followed by several molecules of C9. The C9 molecules
form a pore in the membrane through which the cellular contents leak and lysis
occurs. Lysis is not an enzymatic process; it is thought to be due to physical
damage to the membrane. The complex consisting of C5bC6C7C8C9 is referred to
as the membrane attack complex (MAC).C5a generated in the lytic pathway has
several potent biological activities. It is the most potent anaphylotoxin. In addition,
it is a chemotactic factor for neutrophils and stimulates the respiratory burst in
them and it stimulates inflammatory cytokine production by macrophages. Its
activities are controlled by inactivation by carboxypeptidase B (C3-INA).
Some of the C5b67 complex formed can dissociate from the membrane and enter
the fluid phase. If this were to occur it could then bind to other nearby cells and
lead to their lysis. The damage to bystander cells is prevented by Protein S
(vitronectin). Protein S binds to soluble C5b67 and prevents its binding to other
cells.
57
BIOLOGICALLY ACTIVE PRODUCTS OF COMPLEMENT ACTIVATION
Activation of complement results in the production of several biologically active
molecules which contribute to resistance, anaphylaxis and inflammation.
• Kinin production
C2b generated during the classical pathway of C activation is a prokinin which
becomes biologically active following enzymatic alteration by plasmin. Excess
C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH)
also known as serpin which displaces C1rs from the C1qrs complex (Figure 10). A
genetic deficiency of C1-INH results in an overproduction of C2b and is the cause
of hereditary angioneurotic edema. This condition can be treated with Danazol
which promotes C1-INH production or with ε-amino caproic acid which decreases
plasmin activity.
• Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which
cause basophil/mast cell degranulation and smooth muscle contraction.
Undesirable effects of these peptides are controlled by carboxypeptidase B (C3aINA).
• Chemotactic Factors
C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of
neutrophils, basophils and macrophages and causes induction of adhesion
molecules on vascular endothelial cells.
• Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on
phagocytic cells and promote phagocytosis.
• Other
Biologically
active
products
of
C
activation
Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells
by distinct receptors and modulate their functions.
58
Cytokines
Cytokines are low molecular weight (generally under 30 kDa) typically
functioning as intercellular (between cells) messengers, mediating their effect via
specific receptors on target cells. They may also occur in membrane-bound forms
which still bind to the receptor following contact with another cell. Cytokines,
despite being antigen nonspecific, regulate the intensity and duration of the
inflammatory/immune response by stimulating/inhibiting activation, proliferation
and/or differentiation and migration of multiple cell types and by regulating the
synthesis and secretion of immunoglobulins and other cytokines.
Most cytokines are single polypeptide chains, although these may be in aggregated
forms in biological fluids, for example, tumor necrosis factor (TNF)-alpha
circulates as a homotrimer. Exceptions to this rule are IL-12 and IL-23 which are
comprised of two different polypeptide chains (heterodimers)
Cytokines act through specific receptors, binding with high affinity and are
therefore extremely potent, often having effects at picomolar concentrations. As a
result the production of cytokines is tightly regulated.
Important general properties of cytokine and chemokine.
1. cytokines may act in an autocrine (binds to receptors on same cell as secreted
the cytokine), paracrine (binds to receptors on a nearby cell, a variation on
paracrine is "juxtacrine" meaning binds a neighbouring cell) or, in some cases,
endocrine fashion (binds to receptors on distant target cells).
2. Cytokines may have various attributes: a cytokine may have different biological
effects on different target cells (pleiotropy). Two or more cytokines may have the
same effect on a target cell (redundancy, eg., IL-2 and IL-15). Cytokines may also
synergize (an effect greater than the additive effect of each cytokine used alone)
59
with each other or may antagonize each other. Finally, cytokines often stimulate
other cytokines, as in a cascade, forming cytokine networks
3. the term cytokine encompasses several more specific terms: lymphokines
(secreted by lymphocytes), monokines (secreted by macrophages and monocytes),
chemokines (cytokines with chemoattractant properties), interferons, tumor
necrosis factors and interleukins (IL), so named because of their role in
communication between leukocytes. There are multiple members in each grouping,
for example there are presently 25 interleukins described and cloned and over 50
chemokines.
60
4. Cytokines are classified into four groups based on structure: hematopoietin
family (examples, IL-2, IL-4); interferon family (example, interferon-beta);
tumor necrosis factor family (example, TNFalpha) and chemokine family
(examples, IL-8, MCP-1).
Cytokines can be divided into functionally distinct groups based on the receptors
they bind.
– Growth Factors (e.g., CSF-1, SCF, RANKL, Flt3L)
– IL-1 Family (e.g., IL-1, IL-18 & natural products/PAMPs)
– TNF Family (e.g., TNF-α, CD40L, FasL, LT, TRAIL, BAFF)
– TGF-β Family (e.g., TGF-β )
– Type I & II Cytokines (4 Helix Bundle Cytokines; e.g., IL-2, IL-4, IL6, IL-7 IL-10, IL-12, IL-21, IL-22 IL-23, IL-27, G-CSF, GM-CSF, IFN-γ,
IFN-α
– Chemokines (e.g., CC and CXC families)
– Other (e.g., steroid hormones, prostaglandins and IL-17)
Chemokines
Chemokines are small polypeptides that selectively control the adhesion,
chemotaxis (movement) and activation of leukocytes. Some are constitutively
expressed and likely are involved in homeostatic or developmental roles. Others
are expressed only following stimulation of the cell.
There are four classes of chemokines based on the position of two of four
conserved cysteines (C)
1. CXC, also known as alpha chemokines, where "X" could be any amino acid.
Two further subclasses are distinguished: with an ELR motif are neutrophil
chemoattractants, without the ELR motif are mononuclear chemoattractants
2. CC, mononuclear cell chemoattractants, also called the beta chemokines
3. C, two members, also referred to as the gamma chemokines
4. CX3C, a single member, fractalkine, is a neutrophil chemoattractant and is
membrane-bound, also called the delta chemokines.
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Chemokine are secreted by many cell types
Functional Classification of Chemokines
• Homeostatic Chemokines - Development of immune tissues
– These chemokines direct the basal or homeostatic distribution of
leukocytes to immune tissues.
– Homeostatic chemokines include: “S1P”, CCL19, CCL21, CXCL12,
CXCL13
• Inflammatory Chemokines - Acute and chronic inflammation
– e.g., Danger signals, many chemokines are involved in directing
leukocyte traffic during infection & inflammation (chronic & acute).
– Inflammatory chemokines include: CCL2, CCL5, CCL11, CXCL8,
CXCL9, CXCL10.
• There are some “double dippers”.
Cytokine secretion by Th1 and Th2 cells:
1. T helper cells (CD4+ lymphocytes) can be divided into Th1 and Th2 subsets,
each with distinct cytokine secretion profiles. Th1 make IL-2 and IFN-gamma
(involved in cell-mediated immunity); Th2 make IL-4, IL-5, IL-10 (involved in B
cell activation and antibody responses).
62
2. the Th1 subset is involved in responses to intracellular pathogens including the
production of opsonizing antibodies. Th1 cytokines promote the differentiation of
CD8+ cells to become cytotoxic.
The Th2 subset mediates the responses to extracellular pathogens, including
eosinophil activation and promoting production of IgM and IgE and
noncomplement-activating IgG isotypes, much of which contributes to allergic
reactions.
3. cytokines produced by Th1 and Th2 cells exhibit cross-regulation. IFN-gamma
inhibits Th2 proliferation while IL-10 indirectly (by acting on antigen presenting
cells) downregulates IFNgamma and IL-2 production by Th1 cells (required for
Th1 proliferation). IL-4 directly antagonizes IFN-gamma activity.
Both subsets arise from a common precursor cell, Th0, which appears capable of
making cytokines from both subsets. The differentiation of this cell is determined
by the cytokine environment during antigen activation. IL-4 is essential for
development of Th2 while IFN-gamma, IL-12 and IL-18 are important for the
development of Th1. IL-12 and IL-18 may be derived from the antigenpresenting
cell. The influence of IL-4 is predominant. A cytokine that promotes differentiation
of one helper subset may also suppress the development of the alternate subset, an
effect known as cross-regulation. Cross-regulation explains the inverse
relationship between classical cell-mediated and antibody responses leading to
allergy.
Cytokine receptors:
Most cytokine receptors are grouped into 5 major families on the basis of
conserved structural features, often involving the position of cysteines. Recall there
were four classes of cytokines.
The receptor classes are:
(i) immunoglobulin superfamily receptors
(ii) Class I, or hematopoietin receptor family (includes most cytokine receptors
involved in immune function)
(iii) Class II, or interferon receptor family.
(iv) TNF receptor family.
(v) the chemokine receptor family.
Receptors may be composed of more than one polypeptide subunit; for example, a
heterodimer may contain a cytokine-specific polypeptide and a signal-transducing
peptide subunit. Subfamilies of hematopoietin class of receptors may even share
the signaling polypeptide.
The IL-2R, consisting of 3 subunits (alpha, beta and gamma), is among the best
characterized cytokine receptor belonging to the hematopoietin receptor family.
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IL-2R may be present in 3 forms; low affinity monomeric IL-2R (alpha chain
only); intermediate affinity dimeric IL-2R (beta and gamma chains); and a high
affinity trimeric IL-2R (alpha,beta,gamma chains). The affinity of the receptor for
IL-2 increases with increasing numbers of polypeptides but signal transduction
requires both the beta and gamma chains. Gamma chain expression is constitutive
by T cells. Expression of
alpha and beta chains by T cells is enhanced following antigenic stimulation,
ensuring these cells become capable of binding IL-2 with high affinity only if
activated. NK cells express the dimeric IL-2R constitutively and bind IL-2 with
intermediate affinity.
The idea of heterodimer receptors is taken to another level in some cases in which
multiple cytokine receptor types utilize the same polypeptide unit, often as the
intracellular signaling unit. This explains the redundancy and antagonism between
some cytokines. Continuing with the IL-2R example, the gamma chain of this
receptor is also used in the IL-4, -7, -9, and IL-15 receptors. Each of these
receptors has a unique low affinity alpha chain which is responsible for the
specificity of the receptor to the particular cytokine; however, the common gamma
chain implies that the transmembrane signal is similar for the different receptors.
The alpha chain associates with the cytokine then non-covalently with the signal
transducing chain. The dimeric receptor also exhibits increased affinity for the
cytokine. In fact, the alpha units (for GM-CSF in the textbook) may compete for
associating with beta signaling units in the plasma membrane.
The extreme in sharing is seen with the signaling chain of IL-6, pg130. This
polypeptide acts as the signal transducing chain of the IL-6, IL-11, ciliary
neurotrophic factor (CNTF), LIF and Oncostatin M receptors. It is no surprise that
these cytokines display overlapping activities.
An interesting twist in the utilization of cytokine receptors was the discovery in
1996 that certain chemokine receptors acted as co-receptors (with CD4) for HIV
infection. Approximately 90% of HIV strains are thought to infect using CCR5
(macrophage tropic), the virus can mutate to use CXCR4 (T
cell tropic) as patients develop AIDS. Neither G protein signaling nor
internalization of the receptor is necessary. The ligands for these receptors
(RANTES, MIP-1alpha, MIP-1beta) can acts as antagonists to HIV infection.
Soluble Cytokine Receptors
Receptors may occur in soluble forms which typically retain high affinity for the
cytokine and thus are capable of binding the cytokine in solution. One of two main
mechanisms results in solublization:
64
1. proteolytic cleavage of the extracellular domain, releasing the receptor from the
cell membrane. This is often a result of some specific activation event acting on the
cell; for example the M-CSF receptor is cleaved from the cell surface by a protease
induced by the activation of protein kinase C. Also, the TNF receptors, p55 and
p75 are solublized by this mechanism. In fact it appears that solublization of p75
can occur following binding of TNF to p55.
2. splicing out of the transmembrane encoding exon of the primary RNA transcript
resulting in a mRNA that encodes a protein that is secreted, and not anchored in
the plasma membrane. Examples of cytokine receptors that are solublized by this
mechanism includes IL-1, IL-4, IL-7, some of which appear to occur
constitutively, and perhaps not due to specific activation signals.
Mechanism/roles of soluble receptors include:
1. receptor down-regulation; the receptor can no longer serve as the signaling
molecule to the cell, limiting the response of the cell to the cytokine ligand.
2. the soluble receptor may become a binding protein that protects the ligand from
degradation or clearance in the extracellular space. The receptor now has no role in
signaling but facilitates the delivery of the ligand to additional membrane-bound
receptors.
3. the soluble receptor binds to the cytokine preventing it from binding further
membrane-bound receptors- becoming a direct antagonist. Examples include the
IL-1, IL-4 and TNF receptors.
4. Receptor families consisting of multichain receptors, such as the IL-6R family,
binding of the soluble alpha receptor chain to the ligand can confer sensitivity to
another cell which may have only the beta chain (gp130). This greatly expands the
number and types of cells sensitive to the soluble receptor/ligand complex.
Cytokine Receptor signaling
The receptor is responsible for transmitting a signal into the cell upon binding the
appropriate ligand.Many of the class I and class II cytokine receptors lack intrinsic
tyrosine kinase domains. Yet to a great extent this is achieved by phosphorylation
of proteins already present in the cytoplasm which results in a rapid pattern of
alterations in multiple proteins. The present unifying model to explain signaling
(using Class I and II receptors) is as follows:
! the receptor is composed of multiple chains: an alpha chain binds the cytokine
while a beta chain is necessary for signal transduction (but still may play a role in
binding the cytokine, already discussed)
! different inactive protein tyrosine kinases are associated with different subunits of
the receptor. The alpha chain is associated with the "Janus kinase" or JAK, even
in the absence of the cytokine ligand. However, in the absence of cytokine the JAK
lacks protein tyrosine kinase activity
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! cytokine binding induces the association of the two separate cytokine receptor
subunits and activation of the associated JAKs by each other
! activated JAKs create docking sites for the signal transducers and activators of
transcription or "STAT" transcription factors by phosphorylation of specific
tyrosine residues on the receptors. This 3115 cytokine lecture notes 5 docking is
between the "SH2" domain of the STAT and the phophorylated tyrosine on the
receptor. In turn, the JAK then phosphorylate the docked STAT
! after phosphorylation the STATs translocate from the receptor as dimers, to the
nucleus to initiate the transcription of specific genes. Which genes are transcribed
is determined by specific DNA sequences to which monomeric or dimeric STATs
bind in the promotor region of the gene.
There are multiple JAKs and STATs acting in different permutations:
Ultimately the specificity of a cytokine effect is due to three factors: 1) the
particular JAK/STAT pathway; 2) STAT specific sequences in the promotor
regions of genes; 3) only certain target genes can be activated in a particular cell
type. In any given cell type only a subset of the potential target genes of a
particular STAT may be permitted expression.
Th cell cytokine cross -regulation can be explained at the level of intracellular
signaling. The expression of the transcription factor T-Bet drives the cell to Th1
differentiation and suppresses Th2 differentiation. Expression of the transcription
factor GATA-3 promotes development of Th2 but inhibits development of Th1.
Heterodimerization of STATs can be accomplished by the simultaneous activation
of different cytokine receptors that result in phosphorylation of different STATS,
e.g. IL-6 results in STAT3 and interferongamma results in STAT1 phosphorylation
and when both cytokines bind receptors on the same cell, then STAT1/STAT3
heterodimers can result.
In marked contrast to the Type 1 and Type II receptors, the chemokine family of
receptors signals through an entirely different mechanism. Chemokine receptors
are coupled with heterotrimeric large G proteins. The signal transduction process
generates second messengers such as cAMP, IP3, iCa2+ and activated small G
proteins.
Yet even chemokine receptors are reportedly able to signal through JAK-STAT
phosphorylation events which seem contingent on dimerization of chemokine
receptors. The dimerization can include homodimers or heterodimers.
IL-1 and TNF signal through NF-kappaB activation.
IL-1 and TNF signal through another mechanism which also involves serial
serine/threonine (not tyrosine) phosphorylation of different proteins but ultimately
the activation of NFkappaB. Working backwards up the pathway, nuclear factor
kappa B (NFkappaB), a heterodimer, is sequestered in the cytoplasm by another
molecule, IkappaB (also a heterodimer). IkappaBalpha becomes phosphorylated by
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IKK (IkappaB kinase) and upon phosphorylation is released from NFkappaB. The
phosphorylated IkappaB becomes ubiquinated which targets it for destruction by
the proteosome.
Meanwhile the liberated NFkappaB is free to translocate to the nucleus where it
direct gene transcription similar to the STATs. The range of genes activated by
NFkappaB is wide and thus this transcription factor has proven important in efforts
to control the inflammatory response, and is considered by many as the "Master
regulator" of inflammation.
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HYPERSENSITIVITY REACTIONS
Hypersensitivity refers to undesirable (damaging, discomfort-producing and
sometimes fatal) reactions produced by the normal immune system.
Hypersensitivity reactions require a pre-sensitized (immune) state of the host.
Hypersensitivity reactions can be divided into four types: type I, type II, type III
and type IV, based on the mechanisms involved and time taken for the reaction.
Frequently, a particular clinical condition (disease) may involve more than one
type of reaction.
Type I Hypersensitivity
Type I hypersensitivity is also known as immediate or anaphylactic
hypersensitivity. The reaction may involve skin (urticaria and eczema), eyes
(conjunctivitis), nasopharynx (rhinorrhea, rhinitis), bronchopulmonary tissues
(asthma) and gastrointestinal tract (gastroenteritis). The reaction may cause a range
of symptoms from minor inconvenience to death. The reaction usually takes 15 30 minutes from the time of exposure to the antigen, although sometimes it may
have a delayed onset (10 - 12 hours). Immediate hypersensitivity is mediated by
IgE. The primary cellular component in this hypersensitivity is the mast cell or
basophil. The reaction is amplified and/or modified by platelets, neutrophils and
eosinophils. A biopsy of the reaction site demonstrates mainly mast cells and
eosinophils.
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The mechanism of reaction involves preferential production of IgE, in response to
certain antigens (allergens). IgE has very high affinity for its receptor on mast
cells and basophils. A subsequent exposure to the same allergen cross links the
cell-bound IgE and triggers the release of various pharmacologically active
substances (figure 1). Cross-linking of IgE Fc-receptor is important in mast cell
triggering. Mast cell degranulation is preceded by increased Ca++ influx, which is
a crucial process; ionophores which increase cytoplasmic Ca++ also promote
degranulation, whereas, agents which deplete cytoplasmic Ca ++ suppress
degranulation.
The agents released from mast cells and their effects are listed in Table 1. Mast
cells may be triggered by other stimuli such as exercise, emotional stress,
chemicals (e.g., photographic developing medium, calcium ionophores, codeine,
etc.), anaphylotoxins (e.g., C4a, C3a, C5a, etc.). These reactions, mediated by
agents without IgE-allergen interaction, are not hypersensitivity reactions
although they produce the same symptoms.
69
Table 1. Pharmacologic Mediators of Immediate Hypersensitivity
MEDIATOR
Preformed mediators in granules
histamine
bronchoconstriction, mucus secretion, vasodilatation, vascular
permeability
tryptase
proteolysis
kininogenase
kinins and vasodilatation, vascular permeability, edema
ECF-A
(tetrapeptides)
attract eosinophil and neutrophils
Newly formed mediators
leukotriene B4
basophil attractant
leukotriene C4,
D4
same as histamine but 1000x more potent
prostaglandins
D2
edema and pain
PAF
platelet aggregation and heparin release: microthrombi
70
The agents released from mast cells and their effects are listed in Table 1. Mast
cells may be triggered by other stimuli such as exercise, emotional stress,
chemicals (e.g., photographic developing medium, calcium ionophores, codeine,
etc.), anaphylotoxins (e.g., C4a, C3a, C5a, etc.). These reactions, mediated by
agents without IgE-allergen interaction, are not hypersensitivity reactions
although they produce the same symptoms.
Type II Hypersensitivity
Type II hypersensitivity is also known as cytotoxic hypersensitivity and may
affect a variety of organs and tissues. The antigens are normally endogenous,
although exogenous chemicals (haptens) which can attach to cell membranes can
also lead to type II hypersensitivity. Drug-induced hemolytic anemia,
granulocytopenia and thrombocytopenia are such examples. The reaction time is
minutes to hours. Type II hypersensitivity is primarily mediated by antibodies of
the IgM or IgG classes and complement. Phagocytes and K cells may also play a
role (ADCC).
The lesion contains antibody, complement and neutrophils. Diagnostic tests
include detection of circulating antibody against the tissues involved and the
presence of antibody and complement in the lesion (biopsy) by
immunofluorescence. The staining pattern is normally smooth and linear, such as
that seen in Goodpasture's nephritis (renal and lung basement membrane) and
pemphigus (skin intercellular protein, desmosome).
71
Type III Hypersensitivity
Type III hypersensitivity is also known as immune complex hypersensitivity. The
reaction may be general (e.g., serum sickness) or may involve individual organs
including skin (e.g., systemic lupus erythematosus, Arthus reaction), kidneys (e.g.,
lupus nephritis), lungs (e.g., aspergillosis), blood vessels (e.g., polyarteritis), joints
(e.g., rheumatoid arthritis) or other organs. This reaction may be the pathogenic
mechanism of diseases caused by many microorganisms.
The reaction may take 3 - 10 hours after exposure to the antigen (as in Arthus
reaction). It is mediated by soluble immune complexes. They are mostly of the
IgG class, although IgM may also be involved. The antigen may be exogenous
(chronic bacterial, viral or parasitic infections), or endogenous (non-organ specific
autoimmunity: e.g., systemic lupus erythematosus, SLE). The antigen is soluble
and not attached to the organ involved. Primary components are soluble immune
complexes and complement (C3a, 4a and 5a). The damage is caused by platelets
and neutrophils (Figure 4). The lesion contains primarily neutrophils and deposits
of immune complexes and complement. Macrophages infiltrating in later stages
may be involved in the healing process.
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The affinity of antibody and size of immune complexes are important in
production of disease and determining the tissue involved. Diagnosis involves
examination of tissue biopsies for deposits of Ig and complement by
immunofluorescence. The immunofluorescent staining in type III hypersensitivity
is granular (as opposed to linear in type II such as seen in Goodpasture's
syndrome). The presence of immune complexes in serum and depletion in the
level of complement are also diagnostic. Polyethylene glycol-mediated turbidity
(nephelometry), binding of C1q and Raji cell test are utilized to detect immune
complexes. Treatment includes anti-inflammatory agents.
Type IV Hypersensitivity
Type IV hypersensitivity is also known as cell mediated or delayed type
hypersensitivity. The classical example of this hypersensitivity is tuberculin
(Montoux) reaction which peaks 48 hours after the injection of antigen (PPD or
old tuberculin). The lesion is characterized by induration and erythema.
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Type IV hypersensitivity is involved in the pathogenesis of many autoimmune
and infectious diseases (tuberculosis, leprosy, blastomycosis, histoplasmosis,
toxoplasmosis, leishmaniasis, etc.) and granulomas due to infections and foreign
antigens. Another form of delayed hypersensitivity is contact dermatitis (poison
ivy (figure above), chemicals, heavy metals, etc.) in which the lesions are more
papular. Type IV hypersensitivity can be classified into three categories
depending on the time of onset and clinical and histological presentation. (Table
3).
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Table 3 - Delayed hypersensitivity reactions
Type
contact
tuberculin
granuloma
Reaction
time
Clinical
appearance
Histology
Antigen and site
48-72 hr
eczema
lymphocytes,
followed by
macrophages;
edema of
epidermis
epidermal (
organic
chemicals, poison
ivy, heavy metals,
etc.)
48-72 hr
local
induration
lymphocytes,
monocytes,
macrophages
intradermal
(tuberculin,
lepromin, etc.)
hardening
macrophages,
epitheloid and
giant cells,
fibrosis
persistent antigen
or foreign body
presence
(tuberculosis,
leprosy, etc.)
21-28
days
Mechanisms of damage in delayed hypersensitivity include T lymphocytes and
monocytes and/or macrophages. Cytotoxic T cells (Tc) cause direct damage
whereas helper T (TH1) cells secrete cytokines which activate cytotoxic T cells
and recruit and activate monocytes and macrophages, which cause the bulk of the
damage (figure 4). The delayed hypersensitivity lesions mainly contain
monocytes and a few T cells.
Major lymphokines involved in delayed hypersensitivity reaction include
monocyte chemotactic factor, interleukin-2, interferon-gamma, TNF alpha/beta,
etc.
Diagnostic tests in vivo include delayed cutaneous reaction (e.g. Montoux test
(figure 5)) and patch test (for contact dermatitis). In vitro tests for delayed
hypersensitivity include mitogenic response, lympho-cytotoxicity and IL-2
production.
Corticosteroids and other immunosuppressive agents are used in treatment
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Table 5 - Comparison of Different Types of hypersensitivity
characteristics
type-I
(anaphylactic)
type-II
(cytotoxic)
type-III
(immune
complex)
type-IV
(delayed
type)
antibody
IgE
IgG, IgM
IgG, IgM
None
antigen
exogenous
cell surface
soluble
tissues &
organs
response time
15-30 minutes
minutes-hours
3-8 hours
48-72 hours
appearance
weal & flare
lysis and
necrosis
erythema
and edema,
necrosis
erythema
and
induration
histology
basophils and
eosinophil
antibody and
complement
complement
and
neutrophils
monocytes
and
lymphocytes
transferred with
antibody
antibody
antibody
T-cells
erythroblastosis
examples
allergic
asthma, hay
fever
fetalis,
Goodpasture's
nephritis
SLE,
farmer's
lung
disease
tuberculin
test, poison
ivy,
granuloma
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