Lecture notes on Complement system and Cytokines

1. 1 Complement system
Lecture notes in Immunology
Topic: Complement system and cytokines
By,
Mrs. K. P. Komal
Assistant professor in Biochemistry
Government Science College, Chitradurga
Karnataka. 577501

2. 2 Complement system
Complement System:
The complement system consists of a series of heat-labile serum proteins that are activated
in turn. The complements exist as soluble inactive precursors which once activated, a complement
component may then act as an enzyme. Enzymatic chain reactions of this type are known as
cascade reactions and usually require a “trigger” to initiate the reaction chain.
Complement is a chain of enzymes whose activation eventually results in the disruption of
cell membranes and the destruction of cells or invading microorganisms. Complement is an
essential part of the body defense system.
History of Complement System:
The name “complement system” is derived from experiments performed by Jules Bordet.
Experiments:
Conclusion:
1. Antibody (Ab) activity did not decay. Therefore Abs are heat stable.
2. Heated serum is capable of agglutinating bacteria.
3. Therefore serum with some heat labile components are capable of agglutinating bacteria.
These components—are called as complement by J. Bordet.
Biological Functions of Complement System:
Complements perform different biological functions like:
1. Cytolysis:

3. 3 Complement system
2. Opsonization:
2. Activation of inflammation:
4. Solubilization and phagocytic clearance and immune complex:
Mechanism of actions are as follows:

4. 4 Complement system
Components of Complement System:
The complement system is made up of a number (mostly 30) of distinct serum (blood plasma) and
membrane proteins which mostly assist the humoral branch of the immune system. As after initial
activation, the various complement components interact sequentially to generate reaction
products that facilitate antigen clearance and inflammatory response.
Different pathways of complement finally generate a macro-molecular membrane- attack complex
(MAC) which helps to lyse a variety of cells, bacteria and viruses.
The complement products amplify the initial antigen-antibody reaction and convert that
reaction into a more effective defense mechanism. Continuous proteolytic cleavage and activation
of successive complement proteins lead to the covalent bonding or fixing of complement frag-
ments to the pathogen surface. Each precursor of complement is cleaved into two major
fragments- named as larger fragment (designated as ‘b’) and smaller fragment (designated as ‘a’).
The major or larger ‘b’ fragment has two biologically active sites—one binds to cell
membranes to the target cell towards the site of activation and the other for enzymatic cleavage of

5. 5 Complement system
the next complement component. The smaller ‘a’ fragments diffuse from the site and play a role in
initiating a localized inflammatory response (chemotactic activity).
1. The proteins and glycoproteins composing the complement system are synthesized largely by
liver hepatocytes, some by blood monocytes, tissue macrophages and epithelial cells of the gastro-
intestinal and genitourinary tracts.
2. The proteins that form the complement system are labelled numerically with the prefix C (e.g.,
C1 –C9).
3. Some complement components are designated by letter symbols (e.g., factor B, D, P) or by trivial
names (e.g., homologous factor).
4. There are at least 19 of these components; they are all serum proteins and together they make
up about 10% globulin fraction of serum.
5. The molecular weights of the complement components vary between 24 kDa for factor D and
460 kDa for C19.
6. Serum concentration in humans varies between 20 μg/ml of C2 and 1300 μg/ml of C3.
7. Complement components are synthesized at various sites like C2, C3, C4, C5; B, D, P and I are
from macrophages, C3, C6, C8 and B from liver (Table 7.1 and 7.2).

6. 6 Complement system
Pathways of Complement System
The Classical Pathway of Complement:
The classical pathway of complement is initiated by the interaction of antibody with antigen
directly (soluble antigen-antibody complexes or immune complexes).
The gradual progress of classical pathway can be mediated by these successive stages
called:
(i) Activation of C1 component
(ii) Production of C3 convertase
(iii) Production of C5 convertase and
(iv) Action of membrane attack complex (MAC)
(i) Activation of C1 component:
The initial stage of activation involves C1, C2, C3 and C4. The soluble antigen-antibody
complex induces a conformational changes in the fragment crystalized (Fc) portion of the
antibody molecule that exposes a binding site for the C1 component of the complement system.
1. C1 is a complex macromolecular protein present in serum in inactive condition. It is a complex
of three proteins named—C1q, C1r and C1s, out of which C1q recognizes and binds to the Fc
region of the antibody and C1r and C1s remain as inactive proteases with their two subunits each.
C1q and two molecules of each C1r and C1s held together is a complex called C1qr2s2 which is
stabilized by Ca2+ ions.
2. The structure of C1 is mainly exhibited by C1q; a large molecule composed of 18 polypeptide
chains that associate in such a way that forms six collagen-like triple helical arms. The amino-
terminal two-thirds of the polypeptides form the stalk and the carboxy-terminal one-third of the
polypeptides form the globular flower, which contains the binding site for antibody.
3. Normally, C1r2s2 complex remains in inactive form and never binds with C1q at that time and
shows the configuration ‘S’. Each C1r and C1s includes two domains named catalytic domain and
interaction domain. Due to action of interaction domain in presence of antigen- antibody complex
in the serum it binds with C1q.
4. C1q binds to an antibody Fc region by its globular heads, in terms, activates serine proteases
C1r and C1s which are proteolytic enzymes gives serine residues at the active site after being
activated.
On binding to antibody, one molecule of C1r is induced to cleave itself, becomes
enzymatically active. Gradually it cleaves and activates the second C1r and both C1s molecules.

7. 7 Complement system
The activated serine protease C1s binds, cleaves and activates the next two components of the
classical pathway i.e. serine protease C4 and C2. Ultimately active CI component is called
C1qr2s2 (Fig. 7.9 and 10).
Activation of classical pathway via IgM and IgG:
The cascade reaction of complement system is only initiated when antibody binds to
multiple sites on a cell surface, normally that of a pathogen. When IgM (pentameric) is bound to
antigen on a target surface, it requires at least three binding sites for C1q attachment.
In case of IgG molecule, it contains a single C1q binding site in the CH2 domain of the Fc. As
C1q globular head requires at least two Fc sites for a stable C1-antibody reaction, it indicates that
two IgG are required to be present on a target surface.
The structural differences between IgM and IgG exert the effect on their activation level. At
the activation of Clq binding, IgG requires less amount of time but a good number of IgG molecules
are to be present. Whereas IgM activation is delayed one but it is more efficient, even a single IgM
molecule can initiate the process (Fig. 7.11).

8. 8 Complement system
(ii) Production of C3 convertase:
Active serine protease enzyme C1qr2s2 has two distinct substrates, C4 and C2. C4
component is a large globular glycoprotein containing three polypeptide chains named α, β and γ.
C4 is activated when C1s hydrolyzes a small fragment C4a from the amino terminus of the chain,

9. 9 Complement system
exposed a binding site on the larger fragment C4b. The C4b fragment attaches to the target surface
of the C1 bound to antibody on the pathogen surface.
Besides, active C4 component, the activated C1s protease acts on C2 serine protease, as a
result the smaller fragment C2b will be cleaved away from the site of action and C2a larger
fragment will remain active at the active site. After that C4b2a active complex is formed which in
turn act on the substrate C3 component. C4b2a is called C3 convertase of the classical pathway.
(iii) Production of C5 convertase:
C3 is almost very similar to C4. C3 component is with two types of polypeptide chains — α
and β. C3 convertase (C4b2a) helps to cleave the smaller fragment C3a from the amino terminus of
the a chain of C3 component.
Even a single C3 convertase molecule can accelerate the production of more than 200 molecules of
C3b, and the result is amplification. In due course produced C3b binds with C4b2a to form a tri-
molecular complex called C4b2a3b i.e. C5 convertase.
(iv) Action of membrane attack complex (MAC):
C5 convertase acts on C5 protein component, cleaves C5a from the action site and C5b
attaches to the antigenic surface. This bound C5b initiates formation of membrane-attack complex
(MAC) by taking participation of C6, C7, C8 and C9 components gradually and ultimately forms
C5b6789 (MAC) which makes a large pore in the membrane of the antigen and accelerates lysis of
it (Fig. 7.12).

10. 10 Complement system
2. The Alternative Pathway:
Besides the classical pathway, complement system can be initiated by another method
called alternative pathway. Unlike classical pathway the alternative pathway is initiated by the
cell-wall constituents of both gram-positive and gram- negative bacteria as foreign particles.
Microbial surfaces directly affect the serine protease C3, gradually cleaving of C3 into C3a
and C3b. This conformational change extends its effect on another factor i.e. factor B. In turn Ba
removed from active site keeping Bb towards the C3b in presence of Mg++; forms C3bBb, and
considered as C3 convertase of alternative pathway.
Binding of C3b exposes a site on factor B that again serves as the substrate for an
enzymatically active serum protein called factor D. Actually factor D cleaves the C3b bound factor
B, and helps to form C3bBb. The action of C3bBb is very unstable, becomes stabilized by the
presence of another exclusive serum protein properdin in this pathway, helps to increase the
convertase activity period.
Formation of C3bBb accelerates the auto- catalyse of more C3 component and forms
C3bBb3b as C5 convertase. Though structural basis of C3 and C5 convertase vary in these two
pathways of complement system but their mode of action is alike.
Here, C3bBb3b subsequently hydropses the bound C5, C6, C7, C8 and C9 respectively,
resulting in Membrane Attack Complex (MAC) formation which binds to the antigenic surfaces of
microbes (antigen). MAC gradually displaces the membrane phospholipids, forms a large trans-
membrane channel and gradually destroys the membrane and lysis of the antigen occurs.
The Lectin mediated pathway:
The third pathway of complement system is lectin-mediated pathway. Lectin-mediated
pathway is activated by the binding of mannose-binding protein present in blood plasma to
mannose containing proteoglycans on the surfaces of the bacteria and yeast, it forms MBP-MASP
(Mannose-binding protein-mannose-associated serum protease). In lectin pathway MBP-MASP
acts on the substrate C4 and C2component protein.
Three different pathways of complement activation is shown in the Fig. 7.13.

11. 11 Complement system

12. 12 Complement system
Cytokines are soluble protein or glycoprotein molecules secreted by a variety of cells in response
to a foreign antigen or other stimulus. They are primarily involved in regulating the immune
responses.
Formerly, the substances secreted by lymphocytes were called ‘lymphocytes’ and the substances
secreted by monocytes/macrophages were called ‘monokines’. Later it was realized that many
lymphokines and monokines are secreted by a number of cell types other than lymphocytes and
monocytes/macrophages.
Hence the term cytokine is preferred now. Cytokine is a generic name referring to factors released
by cells. Based on factors like cytokine producing cell and cytokine functions, the cytokines are
given other names also.
i. Many cytokines are referred to as ‘interleukins’. The term interleukin indicates that they
are secreted by some leukocytes and act on other leukocytes.
ii. Some cytokines are known by common names such as ‘interferon’s’ and ‘tumor necrosis
factors.
iii. Another group of cytokines are called growth factors.
iv. Another group of cytokines are called chemokine’s. Chemokine’s affect chemo taxis and
some other aspects of leukocyte behaviors. The chemokine’s play important roles in
inflammation.
v. Another group of cytokines are called colony- stimulating factors (CSFs). CSFs are
required for growth and differentiation of blood cells from hematopoietic stem cells.
Cytokines act as intercellular messengers because a cytokine secreted by one cell act on
another cell and influences its functions. Cytokine acts as intercellular- signaling protein. Cytokines
regulate local and systemic immune and inflammatory responses as well as wound healing,
hematopoiesis and many other biologic activities.

13. 13 Complement system
Based on the cells, on which the cytokines act, the cytokines are said to have autocrine or
paracrine or endocrine actions (Figs 13.lA to D).
Autocrine action:
The cytokine acts on the cell, which secreted the cytokine.
Paracrine action:
The cytokine secreted by one cell acts on other cells present in the vicinity of the cytokine-
secreting cell.
Endocrine-like action:
The cytokines are said to have endocrine like action when the cytokines produced from cells
enter into circulation and affect cells at distant parts of the body. Cytokine secreted by a cell may
bind to the cytokine- secreting cell and remain as a cell membrane-bound cytokine. The
membrane-bound cytokine binds to the cytokine receptor on a target cell and mediates its effects
on the target cell.
The following terms are also used to describe the actions of cytokines (Figs 13.2A to E).
i. Synergy:
Two or more cytokines may act on one cell. When the combined effect of two or more cytokines is
greater than the additive effects of individual cytokines, the cytokines are said to have synergic
effects.
Figs 13.1 A to D: Diagrammatic representation of paracrine, autocrine, endocrine-like, and cell-to-cell communicative actions of cytokines.

14. 14 Complement system
(A) Autocrine action:
The cytokine secreted by the cell acts on the secreting cell itself,
(B) Paracrine action:
The cytokine secreted by one cell acts on another nearby cell,
(C) Endocrine-like action:
The cytokine secreted by a cell enters into the circulation and acts on a cell far away from
the cytokine secreting cell, and
(D) Cell-to-cell communication:
The cytokine produced by cell X remains bound to the cell membrane of the cell X. Another
cell Y having receptors for the cytokine bind to the cytokine on the membrane of cell X. This
binding sends signal into the cell Y
ii. Antagonism:
Two or more cytokines may act on one cell. When the effects of one cytokine inhibit or offset the
effects of another cytokine, the cytokines are said to have antagonistic effects.
iii. Pleiotropy:
A cytokin has different actions on different cell types.
For example, interleukin-4 (IL-4) produced by TH cell has the following effects on different cell
types:
a. Effect on B cell- Activation, proliferation, and differentiation of B cells.
b. Effect on mast cell- Proliferation of mast cell.
c. Effect on memory B cell- Induction of class switching to IgE.
iv. Redundant:
Two or more cytokines that moderate similar functions on a cell are said to be redundant.
v. Cascade induction:
The cytokine secreted by one cell type activates a second cell type; the second cell type, in turn,
secretes a cytokine that acts on another cell type.

15. 15 Complement system
Figs 13.2A to E: Diagrammatic representation of pleiotropic, synergic, antagonistic, redundant, and cascade induction actions of cytokines.
(A) Pleiotropy:
The cytokine IL-4 has different actions on different cells: IL-4 causes activation, differentiation,
and proliferation of B cells; IL-4 causes proliferation of thymocyte; and IL-4 induces memory B
cell to class switch to IgE,
(B) Synergy:
The cytokines IL-4 and IL-5 acts on one cell simultaneously. The combined effects of IL-4 and IL-
5 are greater than the additive effects of individual cytokines IL-4 and IL-5,
(C) Antagonism:
Cytokines IL-4 and IFNγ acts on a B cell. The effect of IFNγ on the B cell prevents the action of
IL-4 on B cell,
(D) Redundancy:

16. 16 Complement system
The cytokines IL-2, IL-4, and IL-5 acting on a B cell have similar functions, and
(E) Cascade induction:
The cytokine IFNγ secreted by an activated TH cell acts on macrophage. The macrophage in turn
secretes the cytokine IL-12, which acts on activated TH cell. Upon binding to IL-12 the activated
TH cell secretes IFNγ, TNF, IL-2, and many other cytokines
For example, activated TH cell secretes interferon gamma (IFNγ)
↓
IFNγ acts on macrophages
↓
The macrophages in turn produce interleukin-12
↓
The IL-12 cytokine in turn acts on TH cells to produce many other cytokines Over 100 cytokines
have been identified so far. Most cytokines are peptides or glycoproteins with molecular weights
(MW) between 6,000 and 60,000. They act by binding to specific cytokine receptors present on
the surface of cells. Cytokines are extremely potent at very low concentrations (10-9 to 10-M).
The half-life of cytokines is usually very short. Hence they act for a very limited period after being
secreted; consequently, the cytokines actions are usually limited to shorter distances from the
secreting cells and most cytokines act over short distances in an autocrine or paracrine manner.
Although a variety of cells can secrete cytokines, the TH cells and macrophages are the two most
important cell types with respect to cytokines secretion. The cytokines secreted by THcells and
macrophages influence almost the entire network of interacting cells during an immune response
(Table 13.1).

17. 17 Complement system

18. 18 Complement system

19. 19 Complement system
One important point should be kept in mind. A particular T cell is activated only by
specific antigen. Upon specific antigen stimulation, the activated T cell secretes the cytokines. The
cytokines bind to other cell types (such as macrophages) and influence the activities of the cells.
Though the T cells, which secreted the cytokines, are specific to an antigen, the cells on which the
cytokines act are not specific to the antigen, which initiated the cytokine secretion, i.e. the
cytokine-activated cell will act against any other antigen also. (For example, the Policeman pulls
the trigger of the gun with an intention to hit an enemy. But the bullets coming out of a gun
don’t discriminate anyone as friend or enemy and the bullets hit whoever comes across their way.
Likewise the cytokine and the cell activated by the cytokine don’t differentiate between the
antigen, which induced the cytokine secretion, and other antigens).
Now many cytokines (like IL-2, and IFNγ) are produced in large quantities by recombinant
DNA technology and are used as therapeutic agents to treat patients.