CHAPTER 3

Complement
Learning Objectives
Upon completion of this lesson the student will be able to:

1. Understand different pathways of complement activation

2. Identify the enzymatic and non-enzymatic mechanisms if
complement activation
3. Discuss the biologic properties of C activation products

4. Describe the significance of Complement system in host

resistance, inflammation and damage to cells

5. Discuss the mechanism of regulating complement

activation and its products
3.0 Introduction to Complement
 Complement component are proteins and
glycoproteins, about 5% of serum proteins
 Synthesized mainly by liver hepatocytes, blood monocytes,
tissue macrophages, plus epithelial cells of the
gastrointestinal and genitourinary tracts.
 Complement components are designated by
 numerals (C1–C9),
 letter symbols (e.g., factor D), or
 trivial names (e.g., homologous restriction factor).

 Peptide fragments formed by activation of a

component are denoted by small letters.
3.0 Introduction to Complement
 These proteins are important in inflammation

 Are normally present in the circulation in an inactive state –

once activated show enzymatic action on subsequent
components and finally target antigen

 The two major pathways of complement activation are

 the classical pathway which is activated by certain
isotypes of Abs bound to Ags and the alternative pathway
which is activated on microbial cell surfaces in the absence
of antibody.
 At least 30 are activated in the classical pathway by antibody
Overview of Complement cascade
 3.1 Immune Functions of Complement
 Opsonization and phagocytosis
 Aids inflammation by opsonizaton of antigens and causing
membrane damage to pathogens.
 As complement is activated antigens become coated with
C3b, iC3b or C4b and are phagocytosed by attaching to
specific receptors on macrophage and neutrophils.
 Expansion of inflammatory responses
 Complement fragments C5a, C4a, and C3a induce powerful
anaphylatic changes, which can be systemic.
 Complement- mediated cytolysis
 Membrane Attack Complex (MAC) permits complement-
mediated lysis of foreign organism. This appears to be a
important defense against only a few types of microbes such
as Nisseria because genetic defects in MAC components do
not reduce desruction of other pathogens
 Other Functions of the Complement System
 By binding to antigen-antibody complexes, complement proteins
promote the solublizing of these complexes and their clearance
by phagocytes.
 The later function is achieved by the binding of immune
complexes with attached C3b to CR1 on erythrocytes, and
complexes are cleared by phagocytosis in reticuloendothelial
system.
 The C3d protein generated by C3b cleavage binds to CR2 on B
cells, activates the B cells, and provides a signal for inactivating
humoral immune responses.

 Viral neutralization
Classical Pathway of Activation
 Is initiated by binding complement protein
C1 to antibody (IgG or IgM) molecules that
have attached to foreign antigens.
 C1 is composed of 3 parts: C1q which binds
antibody while C1r and C1s are proteases.
 The C1q subunit, an umbrella like radial
array of six chains, each of with a globular
head connected by a collagen like arm to a
central stalk. C1q performs the recognition
function and binds specifically to the Fc
regions of  and  heavy chains that have
bound to antigen sites.
Classical Pathway of Activation
 1C1g-binding site to 1 Ig Fc C1q bound to 2 IgG sites –
region and C1q molecule activated C1r then C1s
needs 2 binding sites to be
activated
 IgG molecules has only one
Fc region so must have at
least 2 IgG molecules close
together before C1q can bind.
 Globular heads of C1q bound
to the Fc regions of IgG or
IgM enzymatically activate of
the associated C1r which
cleaves and activates C1s.
 Classical Pathway of Activation
 Activated C1s cleaves C4 to form C4b (C4a is released.) C4b
binds it self with the antigen antibody complex or with the
adjacent surface of a cell.

 C2 then complexes with the cell surface-bound C4b and is

cleaved by a near by C1s molecule to generate a soluble C2a
fragment and a larger C2b that remains physically associated
with C4b on the cell surface.
Classical Pathway of Activation
Classical Complement Activation
 In the green box on the previous slide, C4b2b complex, C3
convertase, is binding to and proteolytically cleaving C3
 The C4b actively binds the C3 and C2b catalyzes
proteolysis of the C3 making it active for the next step.
 Activation of C3b is an critical point of expanding the
complement activation

 Proteolysis of C3 cleaves
a small C3a fragment,
 leaving C3b’s that may remain bound with the C4b2b on
the antibody or form covalent bonds with the cells surface
near the antigen/antibody complex.
 Classical Complement Activation

The C4b2b3b complex

functions as the
classical C5 convertase
of complement
activation
Alternate Pathway of Complement Activation
 The alternative pathway activation results in
proteolysis of C3 and stable attachment of its
breakdown product C3b to microbial surfaces without
a role for antibody.
 Microbial surfaces (viral, parasite and fungal surface
antigens) and lipopolysaccharides can accomplish this.

 Normally small amounts of C3b are activated in the

absence of antigen/antibody reactions or by the alternate
pathway.
Alternate Pathway of Complement Activation continued

 The surface bound C3b binds Factor B, a plasma protein

 Bound Factor B is cleaved by Factor D, a plasma serine

protease to generate a fragment called Bb that remains
attached to C3b forming C3bBb (and Ba is released ).
 C3bBb has a short active life (5 min.) unless stabilized by
Properidin, a serum component, – this extends the active
life up to 30 minutes.
 The C3bBb complex is the alternative pathway’s C3
convertase, and it functions to cleave more C3 molecules
amplifying the reaction.
Alternative Pathway
Alternate Path Begins

Amplification loop
D = Factor D
P = Properdin
B = Factor B
Classical

Feeds into Classical

complement pathway
from here
Complement Continued
 The C3 convertase activity of C3bBb generates the C3bBb3b
complex, which exhibits C5 convertase activity, analogous to
the C4b2a3b complex in the classical pathway.

 The nonenzymatic C3b component binds C5, and the Bb

component subsequently hydrolyzes the bound C5 to generate
C5a and C5b.
The Lectin Pathway of Complement Activation
 Lectins are proteins that recognize and bind to specific
carbohydrate targets. (Because the lectin that activates
complement binds to mannose residues, some authors
designate this the MBLectin pathway or mannan-binding lectin
pathway.)

 The lectin pathway, like the alternative pathway, does not

depend on antibody for its activation.

 However, the mechanism is more like that of the classical

pathway, because after initiation, it proceeds, through the
action of C4 and C2, to produce a C5 convertase
Lectin Pathway Continued
 The lectin pathway is activated by bound mannose-binding
lectin (MBL) to mannose residues found on microorganisms
including certain Salmonella, Listeria, and Neisseria strains, as
well as Cryptococcus neoformans and Candida albicans.

 MBL, an acute phase protein, functions in the complement

pathway similarly to C1q, which it resembles in structure.

 After MBL binds to the surface of a cell or pathogen, MBL-

associated serine proteases,MASP-1 and MASP-2, bind to
MBL.
Lectin Pathway Continued
 The active MBL/MASP-1/MASP-2 complex cleaves and
activates C4 and C2. The MASP-1 and -2 proteins, structurally
similar to C1r and C1s, mimic their activities.

 This means of activating the C2–C4 components to form a C5

convertase without need for specific antibody binding
represents an important innate defense mechanism comparable
to the alternative pathway, but utilizing the elements of the
classical pathway except for the C1 proteins.
Components of mannose-binding lectin
pathway

MBL MASP1
 Mannose-binding lectin pathway

_____
C4b2a is C3 convertase; it will
lead to the generation of C5
convertase
MASP1

MBL
Terminal Sequence Shared by ALL Pathways

 The terminal sequence of complement activation involves

C5b, C6, C7, C8, and C9, which interact sequentially to form a
macromolecular structure called the membrane-attack
complex (MAC).

 This complex forms a large channel through the membrane of

the target cell, enabling ions and small molecules to diffuse
freely across the membrane.

 The end result of activating the classical, alternative, or lectin

pathway
Terminal Steps in Complement Activation
Continued
 C5 convertase cleave C5 into a small C5a fragment that is
released and the large C5b fragment, which binds to the
surface of the target cell and provides a binding site for the
subsequent components of the membrane-attack complex
 The C5b component is extremely labile and becomes inactive
within 2 minutes unless C6 binds to it and stabilizes its
activity.
 C6, C7, C8, and C9, are structurally related proteins with out
enzymatic activity.
 As C5b6 binds to C7, the resulting complex undergoes a
hydrophilic-amphiphilic structural transition that exposes
hydrophobic regions, which serve as binding sites for
membrane phospholipids.
Terminal Steps in Complement Activation
Continued
 If the reaction occurs on a target-cell membrane, the
hydrophobic binding sites enable the C5b67 complex to insert
into the phospholipid bilayer. This complex has limited ability to
lyse cells.

 The formation of a fully active MAC is accomplished by the

binding of C9, the final component of the complement cascade,
to the C5b-8 complex.

 C9 is a serum protein that polymerizes at the site of the bound

C5b-8 to form pores in plasma membranes.
Terminal Steps in Complement Activation
Continued
 Pathways of complement
activation

CLASSICAL LECTIN ALTERNATIVE

PATHWAY PATHWAY PATHWAY

antibody antibody
dependent independent

Activation of C3 and
generation of C5 convertase

activation
of C5

LYTIC ATTACK
PATHWAY
Regulation of the Complement System

Protein Distribution Interacts With Function

C1 inhibitor Plasma Protein C1r, C1s Serine protease
(C1INH) inhibitor; binds to C1r
& C1s & dissociates
them from C1q
Factor I Plasma Protein C4b, C3b Serine protease;
cleaves C3b & C4b by
using Factor H, MCP,
C4BP or CR1 as a
cofactor
Factor H Plasma protein C3b Binds C3b & displace
Bb Cofactor for factor
I-mediated C3b
cleavage
Regulation of the Complement System
Continued
C4-binding Plasma Protein C4b Binds C4b and
protein (C4BP) displaces C2b

Membrane Leukocytes, C3b, C4b Cofactor for

cofactor protein epithelial cells, Factor I-mediated
(MCP, CD46) endothelial cells cleavage of C3b &
C4b

Decay- Blood cells, C4b2b, Displaces C2b from

accelerating Endothelial cells, C3bBb C4b and Bb from
factor (DAF) Epithelial cells C3b.
CD59 Blood cells, C7, C8 Blocks C9 binding
Endothelia & & prevents
Epithelial cells formation of the
MAC
Laboratory Issues in Complement
 Why measure?
 Complement over utilization (decreases)
 Infection (bacterial, etc)

 Autoimmune (indication of flare or active disease)

 Long list

 Complement increases
 Acute phase proteins

 Any disease associated with increase in acute phase

proteins (ie. Rheumatoid Arthritis, Diabetes, ulcerative
colitis)
• Complement deficiencies (rare)
Laboratory Issues in Complement
 What to measure?
 CH50 (Hemolytic complement Assay)
 Good screen for clinically relevant levels

 Decreased levels/Function

 All parameters taken into account

 Assay description - cautions (labile protein)

 C3, C4 Quantitative Measurements

 Classical vs Alternative pathway indication

 C3 common to both, C4 only Classical

 Highest serum conc. - easy to measure

 Follow disease course - sensitive

 Other components
 Rarely C2, C1q inh quantitative & functional

 C5-9 Extremely rare

Complement Disorder
 Hereditary Angioedema -
C1Q inhibitor deficiency
 Clinical pathology
 unregulated production
of protease enzymes and
mediators of inflamation
 increased vascular permeability and exudation of fluid

 What Lab assays can be useful ???

 Quantitative

 Functional
Complement summary
CHAPTER 4

Cytokines
Learning Objectives
 Upon completion of this lesson the student will be able to:

1. List innate, adaptive and haematopoietic cytokines

2. Identify cytokines according to their function

3. Describe properties of cytokines

4. Discuss cytokine receptors on different cells

5. Differentiate immunological role of cytokines

6. List cytokine related diseases

7. Discuss effect of cytokines on T helper cells

4.1 Properties of Cytokines
 Cytokines, proteins, secreted by cells of innate and adaptive
immunity.

 Cytokines, produced in response to microbes and other

antigens, and other cytokines.

 Cytokines determine and stimulate many diverse responses of

cells involved in immunity and inflammation.

 During immune response activation phase, cytokines stimulate

the growth and differentiation of lymphocytes,
Properties of Cytokines Continued
 In the effector phases of innate and adaptive immunity, they
activate a different effector cells to eliminate microbes and
other antigens.

 They also stimulate the development of hematopoietic cells.

 In clinical medicine, cytokines are important as therapeutic

agents or as targets for specific antagonist in numerous
immune and inflammatory disease
Properties of Cytokines Continued
 The term cytokine is a general term used to describe a large
group of proteins but there are other terms that are commonly
used to describe particular kinds of cytokines. These include:

1) monokines, cytokines produced by mononuclear phagocytic

cells;

2) lymphokines, cytokines produced by activated lymphocytes,

especially Th cells; and

3) interleukins, cytokines that act as mediators between

leukocytes.
Properties of Cytokines Continued
Properties of Cytokines Continued
Properties of Cytokines Continued
Cytokine Antagonist
 A number of proteins inhibit biological activity of cytokines.

 These proteins act in one of two ways:

 bind directly to a cytokine receptor but fail to activate the
cell,
 bind directly to a cytokine, inhibiting its activity.

 Best-characterized inhibitor is IL-1 receptor antagonist (IL-

1Ra), which binds to the IL-1 receptor but has no activity.
 This receptor blocks binding of IL-1’s
 Production of IL-1Ra is thought to play in regulating
intensity of inflammatory response.
 These inhibitors are in both blood and extracellular fluids.
Cytokine Antagonists continued
 Soluble antagonists arise from enzymatic cleavage of the
extracellular domain of cytokine receptors.

 Soluble cytokine receptors that have been detected are those

for IL-2, -4, -6, and -7, IFN TNF, and LIF.

 Proteolytic cleavage forms a soluble IL-2 receptor. This shed

receptor binds IL-2 and prevent its interaction with the
membrane-bound IL-2 receptor.
Cytokine Antagonists continued
 Some viruses also produce cytokine-binding proteins or
cytokine mimics.

 Molecules produced by viruses that mimic cytokines allow the

virus to manipulate the immune response in ways that aid the
survival

of the pathogen.
Cytokine Secretions by TH1 or TH2 cells
 Differences in cytokine-secretion patterns among TH-
cell subsets produce different responses to different
types antigens in immune response.

 CD4+ TH cells exert most of their helper functions

through secreted cytokines
 acting on the cells, or

 modulating the responses of other cells.

Cytokine Secretions by TH1 or TH2 cells
 Two CD4+ TH-cell subpopulations designated TH1
and TH2, can be distinguished in vitro by the
cytokines they secrete.
 Both subsets secrete IL-3 and GM-CSF but differ in the
other cytokines they produce

 TH1 and TH2 cells are characterized by the following

functional differences:
 TH1 subset

 Responsible for
 many cell-mediated functions (e.g., delayed-type
hypersensitivity and activation of TC cells),

 production of opsonization-promoting IgG antibodies (i.e.

antibodies binding to phagocyte high-affinity Fc receptors
and interact with the complement system), and

 the promotion of excessive inflammation and tissue injury.

 TH1 subset
 Secretes the important cytokine, IFN-
 Activates macrophages, to increase microbicidal activity,
 up-regulate the level of class II MHC, and
 Induces antibody-class switching to IgG classes enhancing
phagocytosis and fixation of complement.
 Secretes IL-12, inducing TH cells to differentiate into the TH1
subset.
 Secrete TNF- and IFN- mediating inflammation, especially
TH1’s activity in delayed hypersensitivity.
 Produce IL-2 and IFN- cytokines promoting differentiation of
fully cytotoxic TC cells from CD8+ precursors.
 This cytokine makes the TH1 subset particularly suited to
respond to viral infections and intracellular pathogens.
 IFN- inhibits the expansion of the TH2 population.
 TH2 subset

 Responsible for
 eosinophil activation and differentiation,

 providing help B cells that specifically produce increased

amounts of IgM, IgE, and non-complement-activating IgG
isotypes, and also

 supports allergic reactions.

TH2 Subset continued
 The secretion of IL-4 and IL-5 induces production of IgE and
supports eosinophil-mediated attack on helminth
(roundworm).

 IL-4 promotes class switch from IgM to IgG subclass that does
not activate the complement pathway.

 IL-4 also increases class switch from IgM to IgE.

 Combined IL-4 and Il-5 action increases Fc receptors on

eosinophils.
TH2 Subset continued
 IL-4 and IL-10 suppress the expansion of TH1 cell
populations.

 Many helper T cells do not show either a TH1 or a

TH2 profile;
 individual cells have shown striking heterogeneity in the
TH-cell population.

 One of these is the TH0 subset, which secretes IL-2, IL-4,

IL-5, IFN-, and IL-10, as well as IL-3 and GM-CSF.
Comparison of TH1 And TH2 Cytokine functions
Cytokine/function TH1 TH2
 CYTOKINE SECRETION
 IL-2 + –
 IFN- ++ –
 TNF- ++ –
 GM-CSF ++ +
 IL-3 ++ ++
 IL-4 – ++
 IL-5 – ++
 IL-10 – ++
 IL-13 – ++
 FUNCTIONS
TH1 TH2
 Help for total antibody production + ++
 Help for IgE production – ++
 Help for IgG2a production ++ +
 Eosinophil and mast-cell production – ++
 Macrophage activation ++ –
 Delayed-type hypersensitivity ++ –
 TC-cell activation ++ –
SOURCE: Adapted from F. Powrie and R. L. Coffman,
1993, Immunol. Today 14:270.
Cytokines by Functional Category
 Mediators of natural/innate immunity

 Type I IFN

 TNF-a

 Regulators of lymphocytic growth, activation and

differentiation

 IL-2, IL-4, IL-5, IL-12, IL-15

Cytokines by Functional Category continued

 Activators of inflammatory cells

 Type II IFN
 IFN-g

 Stimulators of hematopoiesis
 IL-3, GM-CSF, IL-7
Cytokine-Related Diseases
 Defects in the complex regulatory networks governing the
expression of cytokines and cytokine receptors have been
implicated in a number of diseases.

Bacterial Septic Shock

 The role of cytokine overproduction in pathogenesis can be

illustrated by bacterial septic shock

 It develops because bacterial cell-wall endotoxins stimulate

macrophages to overproduce IL-1 and TNF- to levels that
cause septic shock
Cytokine Activity Is Implicated in Lymphoid and
Myeloid Cancers
 Abnormal production of cytokines or their receptors have been
associated with some types of cancer. For example.

 In myeloma cells, IL-6 appears to operate in an autocrine

manner to stimulate cell proliferation.

 When monoclonal antibodies to IL-6 are added to in vitro

cultures of myeloma cells, their growth is inhibited