Transcript III. Immunology and Complement

Unit 3 Immunology and Complement
Part 2
Terry Kotrla, MS, MT(ASCP)BB
Overview of Immunity
Immunoglobulins
 Humans produce specific proteins or immunoglobulins
which can be differentiated on the basis of:
 Size
 Biologic function
 Biochemical properties
 Serological activity
Basic Structure of Immunoglobulins
 An antibody digested by papain yields three fragments
 Two Fab which consist of antigen binding site, can sensitize.
 One Fc, whic is the region that determines biological properties of the Ig.
Basic Structure of Immunoglobulins
 An antibody digested by pepsin yields two fragments:
 One Fab2 which consist of 2 antigen binding sites joined together, able to
agglutinate.
 One Fc,the region that determines biological properties of the Ig.
Immunoglobulin Classes
IgM Class
IgM
 Largest of all the antibody molecules, consists of five of the basic units (pentamer)
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mu heavy chains joined together by a structure known as J-chain.
Accounts for about 5-10% of the immunoglobulin pool.
Restricted almost entirely to the intravascular space due to its large size.
Fixes complement, much more efficient than IgG in the activation of complement
and agglutination.
First antibody to be produced and is of greatest importance in the first few
days of a primary immune response to an infecting organism.
Does not cross the placenta.
Many blood group antibodies that are capable of agglutinating antigen positive RBCs
suspended in saline in tests performed at 22 C are IgM causing visible agglutination,
ie, ABO antibodies.
IgM antibodies are potent agglutinators that activate complement very efficiently.
IgG
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Most abundant of the immunoglobulins in the plasma
One basic structural unit, i.e.Y-shaped molecule having 2 light chains and 2 Gamma heavy chains.
Produced in response to a wide variety of antigens, including bacteria, viruses and RBC and WBC alloantigens.
Coats organisms to enhance phagocytosis by neutrophils and macrophages.
Through its ability to cross the placenta, maternal IgG provides the major line of defense against
infection for the first few weeks of a baby's life.
It is the predominant antibody produced in the secondary response.
The serologic behavior and characteristics of IgG antibodies make them one of the most clinically
significant in blood banking.
Most blood group antigens capable of eliciting an immune response result in the production of IgG
antibodies.
These antibodies are detected by serologic test procedures based on their behavior characteristics, such as
reactivity at 37 C, complement activation, indirect agglutination and hemolysis.
 Much of routine blood banking involves serologic test procedures designed to detect and identify IgG antibodies.
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Four subclasses which differ in their heavy chain composition and in some of their characteristics such as
biologic activities. IgG1, IgG2, IgG3 and IgG4.
IgA
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Found in saliva, tears, colostrum breast milk and in nasal, bronchial and intestinal
secretions.
IgA present in large quantities in colostrum and breast milk, is transferred across the gut mucosa in the
neonate and plays an important role in protecting the neonate from infection.
Produced in high concentrations by lymphoid tissues lining the gastrointestinal, respiratory and
genitourinary tracts.
Plays an important role in protection against respiratory, urinary tract and bowel infections and preventing
absorption of potential antigens in the food we eat.
Represents 10 to 15% of the total circulatory immunoglobulin pool.
In plasma IgA may exist as a single basic structural unit or as two or three basic units joined together.
The IgA present in secretions exists as two basic units (a dimer) attached to another molecule know as
secretory component.
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This substance is produced by the cells lining the mucous membranes.
It is thought to protect the IgA in secretions from destruction by digestive enzymes.
IgA does not cross the placenta and does not bind complement.
 For blood banking, an IgA deficient individual may produce anti-IgA which can cause severe, life-threatening
anaphylactic reactions during transfusion. Once identified these individuals must be transfused with blood
and components which lack IgA.
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IgA Structure
 The dimeric IgA molecule.
 1 H-chain,
 2 L-chain,
 3 J-chain,
 4 secretory component
IgE
 Trace plasma protein (only about 0.004%) in the plasma of non-parasitized
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individuals.
Major importance mediating some types of allergic reactions and is generally responsible for an
individual's immunity to invading parasites.
Fc region binds strongly to a receptor on mast cells and basophils and, when antigen
is bound it causes the basophil (or mast cell) to release histamines and heparin from
these cells, resulting in allergic symptoms.
Clinical effects of IgE mediated reactions include increased vascular permeability,
skin rashes, respiratory tract constriction (wheezing), and increased secretions from
epithelium (watery eyes, runny nose).
Not much else is known about its biologic role.
IgE does not fix complement and does not cross the placenta.
No blood group antibodies have been reported to belong to this class.
IgE
IgD
 Accounts for less than 1% of the total immunoglobulin pool.
 This is primarily a cell membrane immunoglobulin found on
the surface of B lymphocytes.
 IgD does not fix complement and does not cross the
placenta.
 Little is known about the function of this class of antibody.
 No blood group antibodies have been reported to belong to
this class.
Clinical Significance of Blood Group
Antibodies
 A blood group antibody is considered clinically significant if it has
been associated with the following:
 Has caused hemolytic transfusion reactions (destruction of
transfused red cells) or
 Implicated in Hemolytic disease of the fetus and newborn
(HDFN) (destruction of fetal cells)
Blood Group Antigens
 At least 30 blood groups with over 600 antigens.
 Individuals may produce antibodies to blood group antigens
they do not possess when exposed to blood through
transfusion or pregnancy.
 Second exposure may result in immune hemolysis of red
blood cells.
Transfusion Reaction
 Term used to describe an unfavorable response by a recipient
to the infusion of blood or blood products and include the
following:
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In-vivo hemolysis (either immediate or delayed)
Decreased survival of transfused cells
Anaphylaxis
Graft-versus-host disease
Post-transfusion purpura
Alloimmunization
Sepsis due to bacterial contaminated components,
Disease transmission.
 Will be discussed in detail later
Severity
 Depends on a number of factors, including the characteristics
of the antibody class involved.
 Antibodies to the ABO system antigens are predominantly IgM,
cause complement activation and intravascular hemolysis.
 Other RBC antigens induce formation of IgG class antibodies
which may cause accelerated RBC destruction extravascularly.
 Symptoms of response to incompatible ABO transfusion
may include fever, low back pain, nausea and vomiting,
circulatory shock, anemia, jaundice, and kidney failure
which may ultimately result in death.
 Primary immune response may be asymptomatic due to
slow destruction of RBCs.
 Secondary response symptomatic due to memory B cells
and rapid antibody production.
Antibody Mediated Hemolysis
 Hemolysis can be intravascular or extravascular.
 INTRAVASCULAR: Antibodies destroy the red cells IN THE
CIRCULATION. Due to of IgM and activation of complement with
destruction of RBCs, VERY BAD, will see RED serum/plasma.
 EXTRAVASCULAR: hemolysis is due to RBCs being coated with IgG and
destroyed OUTSIDE the circulation in the RES system. If it occurs slowly
may not be detectable.
Transfusion Reactions
 Screening donor blood for disease markers significantly
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decreased transfusion transmitted diseases.
Reactions to donor WBCs and platelets relatively common
but usually not severe.
ABO reactions severe and PREVENTABLE by following
protocols.
“Other” blood group antibodies may or may not be
detectable.
It is YOUR duty to provide serologically compatible blood
and blood components for transfusion.
Complement
 Spend quality time on your notes from Serology.
Complement
 Integral part of the immune system.
 Three pathways
 Classical
 Alternative or properdin
 Lectin
 Three primary functions:
 Lysis of antibody coated cells, such as bacteria and RBCs.
 Mediation of opsonization, preparation of foreign cells for
phagocytosis.
 Generation of peptide fragments that regulate features of the
inflammatory and immune response.
Importance in Blood Banking
 Two major areas:
 Some antigen-antibody complexes cause sufficient quantities of
complement to be bound to RBCs to complete activation cycle,
causing hemolysis.
 Antigen-antibody complexes initiate complement binding in
such a way that allows demonstration of the existence of such
complexes by the use of serologic techniques.
 Fresh serum necessary to detect complement mediated
in-vitro reactions.
The Classic Pathway
 Eleven components involved, numbered C1 to C9.
 Complement cascade requires presence of cations, both
calcium and magnesium.
 Activation of the classic pathway almost always initiated by
immunoglobulin.
 Requires only 1 molecule of IgM (has 5 Fc).
 Requires 2 molecules of IgG (has 1 Fc).
Two IgG, One IgM
The Classic Pathway
 Recognition Phase - Recognition unit: C1q,C1r,C1s.
 Activation Phase -Activation Unit: C4b,C2b,C3b,C5b
 Attack Phase – Attack Unit: C5b,C6,C7,C8 and C9
 Classic pathway: C1,C4,C2,C3,C5,C6,C7,C8,C9
 Must go to completion for hemolysis to occur. The
next two slides are to assist you in your studies.
Classical Pathway
Alternative (Properdin) Pathway
 Proteins in the alternative pathway perform activities similar to those in the
classic pathway but are usually non-antibody triggered.
 Any one of a variety of substances can initiate complement activation
including:
 bacterial polysaccharides and lipopolysaccharides,
 endotoxins,
 cobra venom,
 trypsin like enzymes,
 aggregates of IgA and IgG4 that do not activate C1.
 C1, C4 and C2 do not participate.
 Alternative pathway: C3,C5,C6,C7,C8,C9
Alternative Pathway
Lectin Pathway
 Activation begins when mannan-binding protein (MBP) binds to the mannose groups of
microbial carbohydrates.
 Two more lectin pathway proteins called MASP1 and MASP2 (equivalent to C1r and C1s of
the classical pathway) now bind to the MBP.
 This forms an enzyme similar to C1 of the classical complement pathway that is able to cleave
C4 and C2 to form C4bC2a, the C3 convertase capable of enzymatically splitting hundreds of
molecules of C3 into C3a and C3b.
 The beneficial results are the same as in the classical complement pathway above:
 trigger inflammation (C5a>C3a>c4a);
 chemotactically attract phagocytes to the infection site (C5a);
 promote the attachment of antigens to phagocytes via enhanced attachment or opsonization
(C3b>C4b);
 serves as a second signal for the activation of naive B-lymphocytes (C3d);
 cause lysis of gram-negative bacteria and human cells displaying foreign epitopes (MAC);
 and remove harmful immune complexes from the body (C3b>C4b).
Lectin Pathway - FYI
 Overview of the lectin complement pathway. In humans, MBL and ficolin that are
lectins form complexes with MASPs (MASP-1,MASP-2 and MASP-3) and sMAP.
Note that MBL consists of several sizes of oligomers and that the composition of
MASPs and sMAP of each MBL oligomer has not been fully elucidated. Once the
complexes bind to carbohydrates on the surfaces of microbes, activated MASPs
cleave C4, C2 and C3.
Activation of Pathways
Order of Activation of 3 Pathways
Regulation of Complement
 Activation of complement cascade results in complex series of molecular
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event with potent biologic consequences.
Modulating mechanisms are necessary to regulate complement activation
and control production of biologically active split products.
First mechanism is spontaneous decay of activated components.
Second mechanism involves specific control proteins that modulate the
activity of certain complement components at critical activation steps.
 C1 inhibitor blocks activities of C1r and C1s.
 Other factors inhibit activation of other complement components.
A number of proteins act to control the membrane attack unit.
Bottom line, gotta turn it off!
References
 http://en.wikipedia.org/wiki/Antibody
 Complement:
http://www.medicine.uiowa.edu/martinlab/complement.html