Immunology is the study of the immune system and is a very important branch of the medical and biological sciences. The immune system protects us from infection through

1. Lecture 5

2. • After reading and studying this chapter, you
should be able to:
♦ Describe sequence of events when the classical
pathway and the alternative pathway of the
complement system is activated.
♦ Discuss biological effects of complement.
♦ Describe complement deficiencies and
associated diseases.

3. • Complement: The term ‘complement’ (C) refers
to a system of factors which occur in normal
serum and are activated characteristically by
antigen-antibody interaction and subsequently
(next) mediate a number of biologically
significant consequences.
• The complement is so named because this
proteins assist or complement the immune cells
to destroy the pathogen.

4. • The complement system is an alarm and a weapon
against infection, especially bacterial infection.
• The complement system includes serum and
membrane-bound proteins that function in both
acquired and constitutive (natural) host defence
system.
• These proteins are highly regulated and interact via a
series of proteolytic cascades.

5. • The complement system belongs to the group of
biological effectors mechanisms (called
triggered enzyme cascades) which also includes
coagulation, fibrinolytic and kinin systems. Such
biological cascades have distinct advantages.
• For example, each enzyme in the cascade is
able to activate many molecules of the
succeeding component providing for
amplification of the response at each step.

6. • Every step has its own control mechanisms so
that, the cascade can be regulated with
precision.
• A cascade: is a set of reactions that amplify
(increase) some effects, i.e. more products are
formed in the second reaction than the first, still
more in the third and so on.

7. • 1. Present in the sera of all mammals and of
most lower animals:
• Complement is present in the sera of all
mammals and also in that of most lower animals,
including birds, amphibians and fishes.
 Hepatocytes, blood monocytes, epithelial cells
of the gastrointestinal tract, and tissue
macrophages synthesize complement proteins.

8. • 2. Nonspecific serological reagent: It is a
nonspecific serological reagent in that
complement from one species can react with
antibodies from other species, though the
efficiency of reaction is influenced by the
taxonomic distance between the species.

9. • 3. Serum molecules: The complement system
consists of approximately 30 serum molecules
constituting nearly 10 percent of the total
serum proteins (plasma or blood proteins,) and
forming one of the major defence systems of the
body.
• A series of circulating and self-cell surface
regulatory proteins keep the complement
system in check.

10. • 4. Heat labile: Complement as a whole is heat
labile, its cytolytic activity undergoing
spontaneous denaturation slowly at room
temperature and being destroyed in 30 minutes
at 56°C though some of its components are heat
stable.
• A serum deprived (depressed) of its complement
activity by heating at 56°C for 30 minutes, is
then said to be ‘inactivated’.

11. • 5. Complement fixation, binding or consumption:
Complement (C) ordinarily does not bind to free
antigen or antibody but only to antibody which has
combined with its antigen.
• Various terms such as fixation, binding or
consumption have been used to refer to the
combination of C with bound immunoglobulin,
leading to the activation of the classical C pathway.

12. • All classes of Ig do not fix complement. Only IgM, IgG3, 1
and 2 (in that order) fix complement, but not IgG4, IgA,
IgD or IgE.
 6. Site of complement binding: The site of complement
binding is located on the Fc piece of the Ig molecule
(CH2 — domain on IgG, CH4 on IgM), and is expressed only
when Ig is combined with its antigen.
 The fixation of complement is not influenced by the nature
of antigens, but only by the class of immunoglobulins.

13. • The complement system comprises a group of
serum proteins, many of which exist in inactive
forms.
• The complement system consists of at least twenty
chemically and immunologically distinct serum
proteins comprising the complement components,
the properdin system and the control proteins.
 There are nine components of complement called C1
to C9.

14. • The fraction (small parts) C1 occurs in serum as a
calcium ion dependent complex, which on chelation
(to bind.) with EDT A yields three protein subunits
called C1q, r, and s.
• Thus, C is made up of a total of 11 different
proteins.
• C fractions are named C1 to C9 in the sequence of
the cascading reaction, except that C4 comes after
C1, before C2.

15.  In normal serum C3 is present in the highest
concentration (1.2 mg/ml) whereas C2 in the
lowest concentration (0.015 mg/ml).
 Activation of the complement system can be
initiated either by antigen-antibody complexes
or by a variety of nonimmunologic molecules.

16.  Sequential activation of complement components
occurs via three main pathways. Classical Pathway,
Alternative Pathway (Properdin Pathway) and
Mannan-binding Lectin Pathway
 All have same result, i.e. lysis or damage of target
cell.
 Classical pathway is triggered by specific antigen-
antibody complex; the alternative pathway can be
initiated by endotoxin, lipopolysaccharides or
zymosan (yeast cell wall).

17.  Proteins unique to the alternative pathway are
identified by capital letters other than “C” (for
example, B, D, and P).
 Complement is normally present in circulation in
inactive form, but when its activity is induced by
antigen-antibody reaction or other stimuli,
complement components react in a specific
sequence as a cascade either through the
classical or alternative pathway.

18. • Basically, the complement cascade is a series of
reactions in which the preceding components act
as enzymes on the succeeding components,
cleaving them into dissimilar fragments—larger
and smaller.
• The larger fragments usually join the cascade.

21. • The smaller fragments which are released often
possess biological effects which contribute to
defence mechanisms by amplifying the
inflammatory process, increasing vascular
permeability, inducing smooth muscle
contraction, causing chemotaxis of leukocytes,
promoting virus neutralization, detoxifying
(removal of toxic) endotoxins and effecting the
release of histamine from mast cells.

22. • Three principle pathways are involved in complement
activation (classical pathway, alternate or properdin
pathway, and lectin pathway) all of which converge
(unite) on the activation of the third component C3.
• Sequential activation of complement components
occurs via two main pathways, i.e. classical pathway,
alternate or properdin pathway). The final steps
that lead to a membrane attack are the same in all
pathways.

23. • The classical pathway is so called because it was the first
one identified. The chain of events in which C components
react in a specific sequence following activation of C1 and
typically culminate (end) in immune cytolysis is known as
the classical pathway (Fig. 8.1). It consists of the following
steps:
 1. Antigen-antibody binding: The first step is the binding of
C1 to the antigen-antibody complex. The recognition unit of
C1 is C1q, which reacts with the Fc piece of bound IgM or
IgG.

25.  Following binding of antigen to antibody, the C1
complement component, which is composed of three
proteins (q, r and s), attaches to the Fc portion of the
antibody molecule through its C1q subcomponent.
 C1q has six combining sites. Effective activation occurs
only when C1q is attached to immunoglobulins by at
least two of its binding sites. One molecule of IgM or
two molecules of IgG can therefore initiate the process.

26.  C1q binding in the presence of calcium ions leads
to sequential activation of C1r and s.
 In the presence of calcium ions, a trimolecular
complex (C1 qrs–Ag–Ab) that has esterase activity
is rapidly formed.
 2. Production of C3 convertase: Activated Cls is
an esterase (C1s esterase), one molecule of which
can cleave several molecules of C4–an instance of
amplification.

27.  Activated C1 cleaves C4 into two pieces C4a and C4b
(C4 → C4a + C4b). C4a is an anaphylatoxin and C4b
which binds to cell membrane along with C1.
 C14b in the presence of magnesium ions cleaves C2
into two pieces (C2 → C2a + C2b).
 C2a remains linked to cell bound C4b, and C2b which
is released into fluid phase. The pieces recombine,
forming C4b2a has enzymatic activity and is referred
to as the classical pathway C3 convertase.

29. • 3. Production of C5 convertase: C3 convertase
cleaves C3 into two fragments (C3 → C3a +
C3b).
• C3a is soluble, and is an anaphylatoxin, and C3b
which remains cell-bound along with C4b2a to
form a trimolecular complex C4b2a3b which has
enzymatic activity and is called C5 convertase
of the classic pathway.

30. • 4. Formation of the membrane attack complex (MAC):
The terminal stage of the classic pathway involves creation
of membrane attack complex (MAC), which is also called
the lytic unit.
 Initiation of membrane attack complex (MAC) assembly
begins with cleavage of C5 by C5 convertase into C5a and
C5b fragments (C5 → C5a + C5b).
 The C5a the most potent anaphylatoxin in the body and
C5b, which continues with the cascade. C6 and C7 then
join together.

31. • A heat stable trimolecular complex C567 is formed part
of which binds to the cell membrane and prepares it
for lysis by C8 and C9 which join the reaction
subsequently (later).
• This complex (C5b67) inserts itself into the plasma
membrane of the target cell.
• Most of C567 escape and serve to amplify the reaction
by adsorbing onto unsensitized ‘by stander cells’ and
rendering them susceptible to lysis by C8 and C9.

32. • The unbound C567 has chemotactic activity, though the
effect is transient due to its rapid inactivation. C8 and C9
then bind, forming the membrane attack complex (C5b6789)
that creates a pore in the plasma membrane of the target
cell.
• The mechanism of complement mediated cytolysis is the
production of ‘holes’, approximately 100 A in diameter on
the cell membrane. This disrupts the osmotic integrity of
the membrane, leading to the release of the cell contents.

33. • Lysozyme from the blood enters through the pore
and digests the peptidoglycan cell wall causing the
bacterium to lyse osmotically if the cell is a gram-
negative bacterium.
• In contrast, gram-positive bacteria are resistant to
the cytolytic action of the membrane attack complex
because they lack an exposed outer membrane and
the thick peptidoglycan prevents an attack on the
plasma membrane.

34.  Although the classical pathway is generally
activated by the antigen-antibody complex or
aggregated (group) immunoglobulin, the classic
pathway can also be activated to a lesser
degree by heparin, DNA, certain retroviruses,
mycoplasma, C-reactive protein (CRP), mannose
binding protein (MBP), and certain “trypsin-like”
proteases.

35. • In the complement cascade the central process is the
activation of C3, which is the major component of C.
• In the classical pathway, activation of C3 is achieved by
C42 (classical C3 convertase).
• The activation of C3 without prior participation of C142 is
known as the ‘alternative pathway’ (Fig. 8.2).
 The alternate pathway of complement activation (the
properdin pathway) does not require the formation of
antigen-antibody complexes for activation.

38. • The first example of the alternative pathway was the
demonstration by Pillemer (1954) of the ‘properdin
system’ as a group of serum proteins contributing
to antimicrobial defence without requiring specific
antibodies.
• The activator in this system was zymosan, a
polysaccharide from the yeast cell wall, but many
other substances can also activate the pathway.

39. • These activators include bacterial endotoxins,
IgA and D, the cobra venom factor and the
nephritic factor (a protein present in the serum
of glomerulonephritis patients).

40. • 1. Production of alternative pathway C3
convertase. C5 convertase and MAC (Fig. 15.2).
 This pathway by passes both the recognition unit and
the assembly of the activation unit as described for
the classic pathway. Instead, there are at least three
normal serum proteins that, when activated together
with C3, form a functional C3 convertase and a C5
convertase. These are factor B, factor D, and
properdin (P).

41. • The binding of C3b to an activator is the first step in
the alternative pathway. Although C3b is present in
the circulation but in the free state it is rapidly
inactivated by the serum protein factors H and I.
• The bound C3b, in the presence of Mg++, interacts
with plasma protein factor B forming C3bB which is
also known as ‘C3 proactivator convertase’ to form
a magnesium-dependent complex ‘C3b, B’.

42. • Factor B in the complex is cleaved by serum
factor D (also called ‘C3 proactivator
convertase’) into two fragments; Ba and Bb.
• Fragment Ba is released into the medium.
Fragment Bb remains bound to C3b producing
C3bBb.
• C3bBb acts as the alternate pathway C3
convertase, capable of producing more C3b.

43.  This enzyme C3bBb is extremely labile.
 The function of properdin (also called Factor P)
a serum protein is to stabilize the C3
convertase, which hydrolyzes C3, leading to
further steps in the cascade, as in the classical
pathway (Fig. 8.1).

44. • Unchecked complement activity can cause not
only exhaustion (collapse) of the complement
system but also serious damage to tissues.
• A series of circulating and self-cell surface
regulatory proteins keep the complement system
in check.
• Several in built control mechanisms regulate the
complement cascade at different steps.

45. • These are mainly of two kinds:
 A. Inhibitors
 B. Inactivators
 A. Inhibitors: Inhibitors bind to complement
components and halt their further function:
 1. Clesterase: Normal serum contains an
inhibitor of Cl esterase.

46.  This heat labile alpha neuraminoglycoprotein also
inhibits many other esterases found in blood, such as
plasmin, kininogen and the Hageman factor.
 This does not prevent the normal progress of the
complement cascade but checks its autocatalytic
prolongation.
 2. Vitronectin: Vitronectin, also known as the S
protein.

47.  The S protein present in normal serum binds to C567
and modulates the cytolytic action of the membrane
attack complex.
 B. Inactivators: Inactivators are enzymes that
destroy complement proteins:
 1. Factor 1: Control of C3b is necessary for the
regulation of both the classic and the alternate
pathways. Normal serum contains an endopeptidase,
called Factor 1 which cleaves C3b and possibly C4b.

48. • 2. Factor H: Another beta globulin factor H acts in
concert with factor I modulating C3 activation.
• It has a strong affinity for C3b, and, after binding
C3b, it exerts its control.
 3. Anaphylatoxin inactivator: Anaphylatoxin
inactivator is an alpha globulin that enzymatically
degrades C3a, C4a and C5a which are anaphylatoxins
released during the C cascade.

49. • 4. C4 binding protein: It is a normal human
serum protein that binds tightly to activated C4
and enhances C4b degradation.
 Many other regulators of C have been reported
such as the cell surface proteins, decay-
accelerating factor (DAF), homologous
restriction factor, complement receptor-1 (CR-
1).

50. • 1. Bacteriolysis and cytolysis: Complement mediates
immunological membrane damage.
• This results in bacteriolysis and cytolysis. Cells vary
in their susceptibility to complement mediated lysis.
 2. Virus neutralization: Neutralization of certain
viruses requires the participation of C, e.g.
neutralization of herpes virus by IgM antibody
requires the participation of C1, C4 and possibly C3.

51. • 3. Anaphylatoxins: C fragments released during the
cascade reaction help in amplifying the inflammatory
response.
• The cleavage products of both pathways of
complement activation, C3a and C5a are
anaphylatoxic (histamine releasing) and chemotactic.
• C4a also has anaphylatoxin activity but is less potent
even than C3a. C567 is chemotactic and also brings
about reactive lysis.

52. • 4. Immune adherence and opsonization: An
important function of C is to facilitate the
uptake and destruction of pathogens by
phagocytic cells.
• This opsonic effect is based on the presence on
the surface of phagocytic cells, (macrophages,
monocytes, neutrophils and others) of
complement receptors or CRs.

53.  If immune complexes have activated the
complement system, the C3b bound to them
stimulate phagocytosis and removal of immune
complexes.
 This facilitated phagocytosis is referred to as
opsonization. C3b can act as a bridge to bring
antibody-coated material into intimate contact
with phagocytic cells, inducing their
phagocytosis and destruction.

54. • 5. Chemotaxis: Any substance that attracts
leukocytes to an area of inflammation is a
chemotactic agent.
• Factors Ba (the split product from the alternate
pathway) and C5a are both chemotactic for PMNs
and macrophages, thus contributing to local
inflammation.
• C5b67, the partially formed attack complex, also has
been implicated as a chemotactic agent.

55. • 6. Hypersensitivity reaction: Complement participates in
 i. Type II hypersensitivity (cytotoxic) reactions.
 ii. Type III (immune complex) hypersensitivity reactions.
 7. Autoimmune diseases: Serum C (complement)
components are decreased in many autoimmune diseases
such as systemic lupus erythromatosus and rheumatoid
arthritis.
 They may, therefore, be involved in the pathogenesis of
autoimmune diseases.

56. • C plays a major role in the pathogenesis of
autoimmune hemolytic anemia, paroxysmal nocturnal
hemoglobinuria and hereditary angioneurotic edema.
 8. Endotoxic shock: Endotoxins can efficiently
activate the alternative pathway of the complement
cascade.
 There is massive C3 fixation and platelets adherence
in endotoxic shock.

57.  Large scale platelet lysis and release of large
amounts of platelet factor lead to disseminated
intravascular coagulation (DIC) and
thrombocytopenia.
 In endotoxic shock with gram-negative
septicemia or dengue hemorrhagic fever may
have a similar pathogenesis. Depletion
(reduction) of C protects against the
Schwartzman reaction.

58.  Schwartzman reaction produced in rabbits by
intravenous injection of endotoxin is a good
model of excessive C3 activation.

59. • Various complement components are synthesized
in different parts of the body, e.g. intestinal
epithelium (C1), macrophages (C2, C4), spleen
(C5, C8) and liver (C3, C6, C9).
• The site of synthesis of C7 is not known. The
control mechanism that controls the synthesis of
the complement component is not known.

60. • Some animal and humans have been found to possess
genetic defects that either result in a deficiency in a
complement component or in a deficient regulatory system
for the control of the activated components of
complement.
• These generally lead to enhanced susceptibility to
infectious diseases.
• Indeed, complement deficiencies have been associated
with recurrent bacterial and fungal infections as well as
with collagen-vascular inflammatory diseases.

61. • A deficiency or dysfunction of C1 esterase inhibitor
results in hereditary angiedema, an autosomal
dominant heritable disease.
• Deficiency of C3b inactivator (factor 1), factor D
and properdin predispose to recurrent infections.
 Individuals with defects in the complement
components C1q, C1r, C1s are predisposed to
develop Systemic lupus erythematosus (SLE) and
lupus nephritis.

62. • Deficiency in C3 leads to an increased
susceptibility to bacterial infections and a
predisposition to immune complex disease as
deficiency in C2 and C4, both of which are
located within the MHC region.
• The development of SLE-like symptoms in C1q
knockout mice parallels the human situation.

63. • Several complement components are proenymes
(inactive), which must be cleaved to form active
enzymes.
• The complement system utilizes a unique nomenclature.
Most complement plasma proteins are named with a
capital “C,” followed by a number (for example, C3).
• C is an abbreviation for the complement system.
• The components of the classical pathway are numbered
from C1 to C9.