MTE120: Immunology & Serology

Midterm
Ms. Liel M. Reyes
molecules of the next component in the sequence.
Lesson 6: The Complementary System Each precursor is cleave into two or more
Complement fragments.
• It is a humoral mechanism of no specific • Usually, the larger fragment is
immune response consisting of more than designated as “b” and the smaller fragment
14 soluble and cell bound protein that as “a.” The EXCEPTION is the
proceed in a cascading sequence of designation of the C2 fragments; the
activation resulting in enhance cause larger fragment is designated C2a and
defense mechanism against foreign cells and the smaller fragment is C2b. The rest
cell lysis. C1, C3, C4, C5 to C9 the larger fragment
• Any pathway always end product of is “b” and the smaller fragment is letter
complement or cascade is CELL LYSIS “a”, EXCEPT for the C2 fragment.
• Active sites
• Most plasma complement proteins are - enzymatic site – cleaving
synthesize in liver exception of C1 - attachment site – binding into
components endothelial
• C1q , C1r, C1s mainly produced by intestinal
epithelial cells. There are Two fragments:
Factor D- mainly made of Adipose Tissue • Major fragments
Paul Erhlich- the term complement is coined the • Minor fragments.
3rd complement
• Because in complement action of Antibodies 1.MAJOR FRAGMENT: has two biologically
in destroying organisms active sites.
Joules Bordet- study the nature of complement - One for binding to cell membrane or the
he got the novel price in 1919 triggering complex and other for enzymatic
cleavage of the next complement
Functions of Complement component.
1. Promotes opsonization- cell engulfment - Control of the sequence involves
2. Lysis of bacteria, cell and viruses spontaneous decay of any expose
3. Activation of inflammation and secretion of attachment sites and specific inactivation by
immunoregulatory molecule complement inhibitors.
4. Immune clearance- removes immune
complexes from circulation and deposited 2. MINOR FRAGMENTS: it has usually prefixed
them in the. Spleen and liver ‘a’.
o Minor fragments generated by cleavage of
Complement Component components have important biological
• Basic principle underlying the cleavage of properties in the fluid phase, such as the
complement components. chemotactic activity of C5a.
• The complement system proteins are o Generated at almost every step in the
named with a capital C followed by a cascade and contribute to the inflammatory
number. response.
• A small letter after the number indicates o Some increased permeability such as the
that the protein is a smaller protein C3a while others attract neutrophils and
resulting from the cleavage of a larger macrophages for subsequent of
precursor by a protease. opsonization and phagocytosis such as the
• The components normally exist as soluble C5a.
inactive precursor; once activated, a o C5a not only promotes leukocytosis in the
complement component may then act as an bone marrow but mobilizes and attracts
enzyme which cleaves several neutrophils to the inflammatory site where
it increases their adhesiveness. It also up
regulates complement such as Cr1 and Cr3

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BSMT 2B
MTE120: Immunology & Serology
Midterm
Ms. Liel M. Reyes
on neutrophils and macrophages to C1 esterase Binds to Uncontrolled
maximize phagocytosis. inhibitor activated C1r, activation of
C1s uncoupling it classical pathway
from C1q leading to
1.Classical pathway hereditary
- Initiator are antigen antibody binding angioneurotic
- Complement 1-9 oedema
- C1 has subtypes: C1q, C1r, C1s. Factor H Binds C3b Total deficiency
displacing Bb: causes recurrent
• C1q binds to Fc of IgM and IgG cofactor for bacterial infection,
• C1r activates C1s factor I glomerulonephritis
• C1s cleaves C4 and C2 & renal failure;
partial deficiency
• C4 part of C3 convertase (C4b) With (atypical)
• C2 bind to C4b form a C3 convertase haemolytic
• C3 key intermediate in all pathway uraemic
syndrome;
- is the most concentrated complement particular with
component adult macular
• C5 initiates membrane attack complex degeneration
• C6 Binds to C5b in MAC membrane attack familial a allele
complex
• C7 Binds to C5bC6 in MAC Factor I Serine As for factor H
• C8 start pore formation on membrane protease that
• C9 polymerize to cause cell lysis cleaves C3b;
acts
CELL LYSIS: End product of complement synergistically
activation with factor H
Membrane
2.Alternative Pathway
inhibitors
- initiator are pathogens or throma injury
• Factor B- binds C3b to form C3 convertase Complement Receptor for C3b Protect
• Factor D- cleaves factor B receptor 1 mammalian cells.
• Properdin orFactor P- stabilizes C3 (CR1: CD35) Low CR1 numbers
on red cells in SLE
convertase and C5 convertase in the is a consequence of
presence of magnesium fast turnover
Decay Accelerates by DAF deficiency
3. MBL Pathway / Mannose Binding Lectin accelerating displacing B decay of C3b Bb
- initiator lectin factor (DAF: alone does not
CD55 cause disease
• MBL – binds to mannose Protectin Inhibits In combination
• MASP -1 – helps cleave C4 and C2 (CD59) formation with DAF
• MASP-2 – cleaves C4 and C2 of lytic pathway deficiency leads to
complex on paroxysmal
homologous nocturnal
Control of any cascade sequence is extremely cells: widely haemoglobinuria
important particularly when in results of expressed on cell (see Section
production potentially cell damage mediator of membrane 16.2.4)
initiator inhibitors that classified into two
Protein Function Clinical SLE, Systemic
consequences of lupus
Deficiency erythematosus.
Circulating *This is not an
inhibitors exhaustive list.

2 Phases of Complement Activation

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BSMT 2B
MTE120: Immunology & Serology
Midterm
Ms. Liel M. Reyes
1. Activation of the C3 Component • ACTIVATION UNIT: C4, C2, and C3.
2. Activation of the attack or lytic sequence • C3 cleaves in C3a and C3b.
• Always larger fragment is needed to
The critical step is a cleavage of C3 by complement activate pathways.
derived enzymes termed as the C3 convertases. The • C3b combined to C3 convertase will
cleavage of C3 is achieved by 3 routes: Classical, activate the C4b2a3b (C5 convertase)
Alternative and the Lectin. All of which can • C5 convertase for the classical pathway:
generate C3 convertase but in response to different C4b2a3b
stimuli.
• C5 convertase (C4b2a3b) activates the C5
which cleavesinto C5a and C5b.
1.Classical Pathway
• Always the larger fragment are the
• The first complement being studied.
responsible for the activation or the
• It is initiated by antigen-antibody (Ag-Ab) continuation of the pathway except for the
complexes or binding. C2.
• There should be 1 molecule of IgM and 2 • After C5b, it will resulted into C56789
molecules of IgG for activation. which called the MEMBRANE ATTACK
COMPLEX which is responsible for CELL
LYSIS.
• The function of the complement is for cell
lysis so it is expected that when you reached
the C9, the end product is cell lysis.

2.Alternative Pathway
• It was the first complement pathway
discovered
• Classical pathway: first complement
pathway studied.It is originally thought to
• RECOGNITION UNITS: C1qrs= C1q, C1r,
be initiated or activated byProperdin
and C1s. These recognition units requires
(Factor P): Acts as stabilizer for C3 and C5
the presence of calcium to maintain its
convertase in the presence of magnesium.
structure.
Stabilizer for alternative pathway only.
• The C1q has a molecular weight of 410,000 • It is initiated or activated by pathogens:
and is composed of six strands that form six o Cobra venom factor
globular heads with a collagen-like tail o Lipopolysaccharide o Bacterial cell
portion. wall
• C1q recognizes the fragment crystallizable o Yeast
(FC) region of two adjacent antibody o Virally infected cells o Tumor cell
molecules, and at least two of the globular lines
heads of C1q must be bound to initiate the o some parasites
classical pathway. o Aggregation of immunoglobulin A
• C1q initiates the classical pathway. It is • It bypasses the C1q, C1r and C1s or the
where the recognition unit appear C1qrs. recognition units in classical pathway.
• C1s cleaves C4 and C2. • Activation starts with native C3 through
• C4 cleaves C4a and C4b while C2 cleaves water hydrolysis or from classical and lectin
C2a and C2b. pathways
• b is for larger fragment and a for smaller • Begins with C3
fragment. C4b is larger fragment. C2 is • C3 cleaves into C3a and C3b
an exception: its larger fragment is C2a and
• C3b- it activates Factor B with the help of
smaller fragment is C2b.
Factor D.
• C4b and C2a: C3 convertase (activates the
• Factor B cleaves into Ba and Bb
C3)
Clarizza A. Derequito
BSMT 2B
MTE120: Immunology & Serology
Midterm
Ms. Liel M. Reyes
• It represents another means of activating
compliment
without antibody being present.
• Lectins (which are calcium dependent) are
proteins
that bind to carbohydrates.
• Lectin pathway molecules is structurally similar to
those of the classical MBL
• C1q is homologous to MASP-1
• C1r is homologous to MASP-2 and C1s
• MBL pathway: it binds to mannose or related
• Larger fragments will continue the sugars in
pathway.Bb—C3 convertase will turn into a calcium dependent manner to initiate MBL
C3bBb pathway
• Recognition Unit: MASP-1 and MASP-2
• C3 convertase for alternative pathway:
• MASP-1 and MASP-2 cleaves into C4 and C2
C3bBb
• C4 cleaves into C4a and C4b
• C3 convertase: It is stabilized by factor P
• C2 cleaves into C2a and C2b
or properdin
• Get the larger fragment which is C4b combined
• After that the C3 convertase is activated by with C2a
C3. • C3 convertase: C4b2a
• C3 cleaves into C3a and C3b • C5 convertase: C4b2a3b
• C5 convertase: C3bBb3b It is also
stabilized by factor P and it activates C5 INITIATORS OF THREE COMPLEMENT
• C5 cleaves into C5a and C5b ACTIVATION PATHWAYS
• C5b it activates C56789 Pathway Initiators
• From C5 to Cell lysis— it is the same with Classic Immune complexes
classical pathway. They also have • Apoptotic cells
membrane attack complex (starts from C5 • Certain viruses and
to C9 (which is responsible for cell lysis)) gram-negative bacteria
• Difference between alternative pathway and • C-reactive protein
classical pathway:Alternative Pathway: bound to ligand
Activation starts with native C3 and from Alternate Various bacteria, fungi,
C3, it activates into factor B with the help of viruses, or tumor cells
factor D. Mannose binding lectin Microbes with terminal
mannose groups
3. MBL Pathway / Mannose Binding Lectin • Complement can be inactivated by heating
- initiator lectin to 56° C for 30 minutes or, after 4 hours,
reinactivated by heating 56° C for 10
minutes.

ANAPHYLATOXINS IN DECREASING
ORDER OF POTENCY
• Anaphylatoxins or small fragment which
attract and activate different types of
leukocytes. They increase vascular
permeability and can cause contraction of
smooth muscle: induce the release of
histamine from basophils and mast cells
Lectin/Mannose-Binding Lectin Pathway and also draw-in additional cell to the site of
• It is the most recently described complement infection to help eliminate the microbes.
pathway
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BSMT 2B
MTE120: Immunology & Serology
Midterm
Ms. Liel M. Reyes
Fragment Acts on Actions C3 Recurrent pyogenic infections, SLE-
C5a Phagocytic Increased Key like syndrome, arthralgias,
Most potent cells phagocytosis intermediate skin rash, Severe recurrent
in all infections; glomerulonephritis
anaphylatoxins
complement pyogenic infections: streptococcus
Endothelial Phagocyte activation Most serious pyogenes
cells deficiency
Neutrophils Activation of C3 Recurrent pyogenic infections,
vascular inactivator urticaria
endothelium C4 SLE-likesyndrome,SLE,
Mast cells Attraction/activation dermatomyositis-like
of neutrophils syndrome.vasculitis
Mast cell C5 Neisseria infections. SLE
degranulation C5 Leiner's disease, gram-negative skin
dysfunction and bowel infection
C3a Phagocytic Increased
C6 Neisseria infections, SLE, Raynaud's
cells phagocytosis phenomenon, scleroderma-like
Endothelial Phagocyte activation syndrome, vasculitis
cells c7 Neisseria infections, SLE, Raynaud's
Mast cells Activation of phenomenon, scleroderma-like
vascular syndrome, vasculitis
endothelium C8 Neisseria infections, xeroderma
Mast cell pigmentosa, SLE-like syndrome
degranulation C9 No known disease association
(release of DAF Decay- Paroxysmal nocturnal
cytoplasmic Accelerating hemoglobinuria
Factor;
granules)
MIRL Paroxysmal nocturnal
C4a Phagocytic Increased Membrane hemoglobinuria
cells phagocytosis Inhibitor of
Phagocytic Mast cell Reactive Lysis
cells degranulation Factor H or Recurrent pyogenic infections
Factor I
Deficiency Associated Disease IMBI Pneumococcal diseases, sepsis,
C1g Systemic lupus erythematosus (SLE) Mannose- Neisseria infections
SLE-like syndrome; Binding Lectin
Decreased secondary to Properdin Pneumococcal diseases, sepsis,
agammaglobulinemia Neisseria infections
C1r SLE-like syndrome; MASP 2 Pneumococcal diseasessepsis,
dermatomyositis, vasculitis, Mannose- Neisseria infections
recurrent infections and chronic Associated
glomerulonephritis, necrotizing skin Serine
lesions, arthritis Protease;
C1s SLE, SLE-like syndrome • Elevated Complement Levels
C1 INH Hereditary angioedema, lupus The complement level can be elevated in many
nephritis inflammatory conditions. Increased
C2 Recurrent pyogenic infections, SLE, complement levels are often associated with
Most common SLE-like syndrome, discoid lupus, inflammatory conditions, trauma, or acute illness
deficiency membranoproliferative such as myocardial infarction because separate
glomerulonephritis,dermatomyositis,
synovitis, purpura, Henoch-
complement components (e.g., C3) are acute-phase
Schönlein purpura, hypertension, proteins. However, these elevations are common
Hodgkin's disease, chronic and nonspecific. Therefore, increased levels are of
lymphocytic leukemia,dermatitis limited clinical significance.
herpetiformis, polymyositis and
atherosclerosis

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BSMT 2B
MTE120: Immunology & Serology
Midterm
Ms. Liel M. Reyes
• Decreased Complement Levels C3 lies at the junction of the two pathways, it is
Low levels of complement suggest one of the much more severely depressed when activation
following biological effects: occurs via the alternative pathway. Extremely
▪ Complement has been excessively decreased levels are seen in patients with
activated recently poststreptococcal glomerulonephritis and in those
▪ Complement is currently being with inherited (C3) complement deficiency. This
consumed. component is also decreased in cases of severe liver
▪ A single complement component is disease and in SLE patients with renal disease.
absent because of a genetic defect.
C3B INHIBITOR (C3B INACTIVATOR)
DIAGNOSTIC EVALUATION • The C3b component of complement causes low
C1 ESTERASE INHIBITOR (C1 INHIBITOR) complement C3 levels, the absence of C3PA in
• C1 measures the activity and concentration of C1 serum, and high C3b levels. A deficiency of C3b
inhibitor in serum. A deficiency of this protein is inhibitor is associated with an increased
characteristic of hereditary angioedema (HAE; see predisposition to infection.
later). Some patients demonstrate catalytically
inactive protein. C3PA (C3 PROACTIVATOR, PROPERDIN
FACTOR B)
C1R, C1S, C2, C3, C4, C5, C6, C7, C8 • The factor B component is consumed by activation
• Homozygous deficiencies predispose a patient to of the alternative complement pathway.
autoimmune disease (especially SLE) and to Assessment of C3PA indicates whether a decreased
arthritis, chronic glomerulonephritis, infections, level of C3 results from the classic or alternative
and vasculitis. pathways of complement activation. Decreased
levels of C3 and C4 demonstrate activation of the
C1Q classical pathway. Decreased levels of C3 and C3PA
• The complement component C1q is evaluated in with a normal level of C4 indicate complement
serum. Decreased levels can be demonstrated in activation via the alternative pathway. Activation of
patients with hypocomplementemic urticarial the classic pathway (and sometimes with
vasculitis, severe combined immunodeficiency accompanying alternative pathway activation) is
(SCID), or X- linked hypogammaglobulinemia. associated with disorders such as immune complex
diseases, various forms of vasculitis, and acute
C1Q BINDING glomerulonephritis. Activation of the alternative
• This procedure measures the binding of immune pathway is associated with many disorders,
complexes containing IgG1, IgG2, or IgG3 and IgM including chronic hypocomplementemic
to the complement component C1q. High values of glomerulonephritis, disseminated intravascular
C1q binding are associated with the presence of coagulation (DIC), septicemia, subacute bacterial
circulating immune complexes of the type that endocarditis, PNH, and sickle cell anemia. In SLE,
interacts with the classic pathway of complement both the classic and alternative pathways are
activation. This test can be useful as a prognostic activated
tool at diagnosis and during remission of acute
myelogenous leukemia. C4
The most common complement deficiency is of C2.
C2 It is an autosomal recessive disorder; the C2 gene is
Pneumococcal diseases, sepsis, Neisseria infections on chromosome 6 in the major histocompatibility
Pneumococcal diseases complex (MHC). The incidence is 1:28,000 to
S. pneumoniae, N. meningitidis, and H. influenzae. 1:40,000; the carrier state is 1.2% in the general
Of symptomatic patients, 50% exhibit a lupus-like population. Half of patients with homozygous C2
disorder with photosensitivity and rash. deficiency have no symptoms; those with symptoms
have infections with
C3 • The C4 level often provides the most sensitive
Also an acute-phase protein, elevated C3 levels can indicator of disease activity. C4 is also an acute-
indicate an acute inflammatory disease. Although phase reactant. Elevated C4 levels can indicate an
Clarizza A. Derequito
BSMT 2B
MTE120: Immunology & Serology
Midterm
Ms. Liel M. Reyes
acute inflammatory reaction or a malignant bacterial infections, often meningococcemia. This
condition. Measurement of C4 may demonstrate disorder is an X-linked recessive trait.
inflammation or infection long before it is clinically
evident by standard assessment methods (e.g., total HEREDITARY ANGIOEDEMA
white blood count [WBC] and leukocyte • This disorder is a deficiency in a complement
differential, febrile response, or elevated protein. Infections are not usually a significant
erythrocyte sedimentation rate [ESR]). C4 is problem. HAE is autosomal dominant, unlike other
destroyed only when the classic pathway is complement deficiencies.
activated. A decreased C4 level with elevated anti–
n-DNA and antinuclear antibody (ANA) titers Two types exist:
confirm the diagnosis of SLE in a patient. In these • Type 1 - low antigen level and low functional
cases of SLE, the periodic assessment of C4 can be protein
useful for monitoring the progress of the disorder. • Type 2 - normal antigen level with low
Patients with extremely low C4 levels in the function
presence of normal levels of the C3 component may
be demonstrating the effects of a genetic deficiency FAMILIAL MEDITERRANEAN FEVER
of C1 inhibitor or C4. Reduction of C3 and C4 • This defect in protease in peritoneal and
components implies that activation of the classic synovial fluid is transmitted as an
pathway has been initiated autosomal recessive trait on chromosome
16. Patients with this defect experience
C6 recurrent episodes of fever and
A decreased quantity of C6 predisposes an inflammation in the joints and pleural and
individual peritoneal fluids.
to significant neisserial (bacterial) infections.
LABORATORY DIAGNOSIS
C7 There are several laboratory assays that have been
A decreased level of C7 is associated with Raynaud's Devised to detect abnormal complement levels.
phenomenon, sclerodactyly, telangiectasia, and These techniques to determine complement
severe bacterial infections caused by Neisseria spp. abnormalities generally fall in two categories
1. Measurement or comoonens as antens In
C8 2. Measurement of functional activity
A decreased quantity of C8 is associated with SLE.
AC8 deficiency makes patients highly susceptible to CH50
Neisseria infections • AKA hemolytic titration assay
• The-commonly used assay for Classical
Hemolytic Uremic Syndrome (HUS) pathway
The most common cause of renal failure in children
• Measures the amount or patents serum.
and it is characterized by hemolytic anemia, low
Required to lyse 50% of standardized
platelet count and acute renal failure. A typical
concentration of antibody sensitized sheep
form of the Hemolytic Uremic Syndrome
erythrocytes. The titer is expressed in CH50
(HUS) occurs becauseof complement
units which is the reciprocal of the dilution
dysregulation caused by genetic polymorphism. The
that is able to lyse 50% of sensitized cells.
genetic mutations associated with typical
The degree of hemolysis is proportional to
Hemolytic Uremic Syndrome includes Factor
the hemolytic activity complement
H, MCP (Membrane cofactor protein), Factor I,
AH50 assay -> measures the functioning of the
Factor B. Thrombomodulin, as well as
alternative pathway
autoantibodies toFactor H and Factor I
• Designed for alternative pathways
SELECT COMPLEMENT DEFICIENCIES • Magnesium chloride and ethylene glycol
PROPERDIN DEFICIENCY tetraacetic acid (EGTA) are added to the
• Properdin acts to stabilize the alternative pathway buffer and calcium is left out. This buffer
C3 convertase (C3bBb). A deficiency leads to
Clarizza A. Derequito
BSMT 2B
MTE120: Immunology & Serology
Midterm
Ms. Liel M. Reyes
chelates calcium which blocks classical 3. The tube should be spun down and the serum
pathway activation. That is why is only should be frozen and placed on a dry ice if not
measures the alternative pathway tested with 1-2 hrs

RID (RADIAL IMMUNODIFFUSION) MUST KNOW

Methods most frequently used individual • Complement -> promotes opsonization and
components include the Radial Immunodiffusion lysis of foreign cells and immune complexes.
(RID)and neophelmetry. • Chronic activation of these systems can lead to
• Nephelometry access complement inflammation and tissue damage.
component such as C3 and C4 • Most plasma complement proteins are
• RID and Nephelometry are both useful in synthesized in the liver with the exception of C1
the diagnosis and monitoring of patients components which are mainly produced by
intestinal epithelial cells, Factor D which is made
ELISA (ENZYME-LINKED in adipose tissue
IMMUNOSORBENT ASSAY) • ORDER OF DISCOVERY = C1-C9
• Can be used to measure individual • ORDER OF ACTIVATION = C1,C4, C2, C3, C5-
complement components C9
• Proper handling of specimen is essential to • Acts as a powerful opsonin -> C3b
ensure correct interpretation of laboratory • Acts as chemotaxin -> C5a
testing. Complement fixation test use • Acts as anaphylatoxin (most potent to least) ->
complement as a reagent to detect the C5a, C3a, C4a
presence of antigen or antibody. Antigen or • Ions involved
antibody are allowed to combine to measure o Classical Pathway = calcium ions
the amount of complement and indicator o Alternative Pathway = magnesium ions
sheep red blood cells are added, these are • C3 convertase
coated with hemolysin (anti sheep cell a. Classical and Lectin Pathway -> C4b2a
antibody). If complement has not been tied b. Alternative pathway -> C3bBb
up in the first reaction, then lysis of the cells • C5 convertase
occur a. Classical and Lectin -> C4b2a3b
o Lysis = negative test -> indicates antigen- b. Alternative pathway -> C3bBb3b
antibody does not present.
• As in all complement testing, the use of
standard and control is necessary for
accurate results

Complement fixation test – the indicator cell

used is the sheep RBC coated with antisheep-
antibody (hemolysin AKA amboceptor)
- The source of complement -> guinea pig
- Positive or Active result -> no hemolysis
- Negative result -> presence of hemolysis

INTERPRETATION OF LABORATORY
FINDINGS
1. The decrease levels of complement components
or activity may be caused by decrease production,
consumption or In vitro consumption
2. Specimen handling is extremely important.
Blood should be collected in a clot tube with no
serum separator

Clarizza A. Derequito
BSMT 2B