SOLUBLE

MEDIATORS OF
THE IMMUNE
SYSTEM
Presented by Group 3

ImmunoSero 2024 | DMMCIHS

Objectives

Name and compare the three complement activation

pathways.
Describe the mechanisms and consequences of complement
activation.
Explain the biological functions of the complement system.
Name and describe alterations in complement levels.
Briefly describe the assessment of complement levels.
Compare other types of nonspecific mediators of the immune system,
including cytokines, interleukins, tumor
necrosis factor, hematopoietic growth factors, and
chemokines.
Discuss the clinical applications of C-reactive protein.
Compare acute-phase reactant methods.
THE COMPLEMENT SYSTEM

This chapter discusses the other components of the innate immune system:
the complement system and other circulating effector proteins of innate
immunity, including cytokines and acute-phase reactants.

Complement is a heat-labile series of 18 plasma proteins, many of which are

enzymes or proteinases. Collectively, these proteins are a major fraction of the beta-1
and beta-2 globulins. The complement system proteins are named with a capital C
followed by a number. A small letter after the number indicates that the protein is a
smaller protein resulting from the cleavage of a larger precursor by a protease.

The complement system displays three overarching physiologic activities (Table 5-1).
These are initiated in various ways through the following three pathways (Table 5-2):
1. Classic pathway
2. Alternative pathway
3. Mannose-binding lectin pathway

CHAPTER 5 | IMMUNOSERO
 activation of complement

Normally, complement components are present in the

circulation in an inactive form. In addition, the control proteins C1
INH, factor I, factor H, and C4-binding protein (C4-bp) are
normally present to inhibit uncontrolled complement activation.
Under normal physiologic conditions, activation of one pathway
probably also leads to the activation of another pathway.

CHAPTER 5 | IMMUNOSERO
activation of complement

CHAPTER 5 | IMMUNOSERO
THE COMPLEMENT SYSTEM

Enzyme Activation
After complement is initially activated, each enzyme
precursor is activated by the previous complement
component or complex, which is a highly specialized
proteinase. This converts the enzyme precursor to its
catalytically active form by limited proteolysis. The
pathways leading to the cleavage of C3 are triggered
enzyme cascades. During this activation process, a
small peptide fragment is cleaved, a membrane-binding
site is exposed, and the major fragment binds. As a
consequence, the next active enzyme of the sequence
is formed. Because each enzyme can activate many
enzyme precursors, each step is amplified until the C3
stage; therefore, the whole system forms an amplifying
cascade.
THE COMPLEMENT SYSTEM

Enzyme Activation
THE COMPLEMENT SYSTEM

COMPLEMENT RECEPTORS
Various cell types express surface membrane glycoproteins that react with one or more
of the fragments of C3 produced during complement activation and degradation.

Plasmodium falciparum adhesin PfRh4 binds to complement receptor type-1 (CR1) on

human erythrocytes. CR1 is a complement regulator and immune adherence receptor on
erythrocytes required for shuttling C3bC4b-opsonized particles to the liver and spleen
for phagocytosis
THE COMPLEMENT SYSTEM

EFFECTS OF COMPLEMENT ACTIVATION

The activation of complement and the products formed during the complement cascade
have a variety of physiologic and cellular consequences.

The cellular consequences include the following:

Cell activation, such as production of inflammatory mediators.
Cytolysis or hemolysis, if the cells are erythrocytes. The most important biologic role
of complement in blood group serology is the production of cell membrane lysis of
antibody-coated targets
Opsonization, which renders cells vulnerable to phagocytosis.
Alternative
Pathway
The alternative pathway shows points of similarity with the classic sequence. Both pathways generate
a C3 convertase that activates C3 to provide the pivotal event in the final common pathway of both
systems. However, in contrast to the classic pathway, which is initiated by the formation of antigen-
antibody reactions, the alternate complement pathway is pre dominantly a non–antibody-initiated
pathway.

Factors capable of activating the alternative path way include inulin, zymosan (polysaccharide
complex from surface of yeast cells), bacterial polysaccharides and endotoxins, and the aggregated
IgG2, IgA, and IgE. In paroxysmal noctur nal hemoglobinuria (PNH), the patient’s erythrocytes act as an
activator and result in excessive lysis of these erythrocytes. This nonspecific activation is a major
physiologic advantage because host protection can be generated before the induction of a humoral
immune response. A key feature of the alternative pathway is that the first three proteins of the classic
activation pathway—C1, C4, and C2—do not participate in the cascade sequence. The C3a com ponent
is considered to be the counterpart of C2a in the classic pathway. C2 of the classic pathway
structurally resembles fac tor B of the alternative pathway.
Alternative
Pathway
Classic
Pathway

-is a Antibody dependent pathway

-Sequence of component activation C1,
4,2, 3,5,6,7,8, and 9
this pathway need IgG and IgM
-There’s 3 stages:
1. Reception
2. Amplification of proteolytic
complement cascade
3. Membrane Attack Complex
Classic
Pathway
Mannose Binding
Lectin Pathway

-is a Antibody Independent pathway

- is a member of a family of calcium-
dependent lectins, the collectins (collagenous
lectins), and is homologous in structure to C1q.
-Similar to Classic Pathway even the sequence
-Initiated by pathogen membrane containing
carbohydrates like mannose
MASP1 (mannose-binding lectin–
associated serine protease 1)
MASP2 (mannose-binding lectin–
associated serine protease 2)
Biological Reaction
of
Complement Proteins
General
Categories

1. Cell lysis by the membrane attack complex

(MAC)
2. Biological effects of proteolytic fragments of
complement
Elevated Complement Levels

inflammatory conditions
trauma
acute illness (myocardial infarction)
Decreased Complement Levels

Complement has been excessively activated

recently.
Complement is currently being consumed.
A single complement component is absent because
of a genetic defect.
Decreased Complement Levels

Hypocomplementemia
from the complexing of IgG or IgM antibodies
Normal turnover of Complement Proteins (1-2 days) = Normal
Complement Level
Increased Susceptibility to Pyogenic Infections:
1. Deficiency of the opsonic activities of complement
2. Any deficiency that compromises the lytic activity of complement
3. Deficient function of the mannose-binding lectin pathway
 DIAGNOSTIC EVALUATION
C1 Esterase Inhibitor (C1 Inhibitor)
-C1 measures the activity and concentration of C1 inhibitor in serum. A deficiency of this protein is
characteristic of hereditary angioedema.

C2
-The most common complement deficiency is of C2. It is an autosomal recessive disorder; the C2 gene is on
chromosome 6 in the major histocompatibility complex (MHC)

C3
-Also an acute-phase protein, elevated C3 levels can indicate an acute inflammatory disease.

C4
-The C4 level often provides the most sensitive indicator of disease activity.
 C5
-A genetic deficiency of the C5 component is associated with increased susceptibility to bacterial infection
and is expressed as an autoimmune disorder. Patients with dysfunction of C5 (Leiner’s disease) are
predisposed to infections of the skin and bowel, characterized by eczema.

C6
-A decreased quantity of C6 predisposes an individual to significant neisserial (bacterial) infection.

C7
-A decreased level of C7 is associated with Raynaud’s phenomenon, sclerodactyly, telangiectasia, and
severe bacterial infections caused by Neisseria spp.

C8
-A decreased quantity of C8 is associated with SLE. A C8 deficiency makes patients highly susceptible
to Neisseria infections.
SELECT COMPLEMENT DEFICIENCIES
Properdin Deficiency
-Properdin acts to stabilize the alternative pathway C3 convertase (C3bBb). A deficiency leads to
bacterial infections, often meningococcemia. This disorder is an X-linked recessive trait.

Hereditary Angioedema
-This disorder is a deficiency in a complement protein. Infections are not usually a significant
problem. HAE is autosomal dominant, unlike other complement deficiencies.
TWO TYPES:
Type 1:low antigen level and low functional protein.
Type 2:normal antigen level with low function

Familial Mediterranean Fever

-This defect in protease in peritoneal and synovial fluid is transmitted as an autosomal recessive trait
on chromosome 16. Patients with this defect experience recurrent episodes of fever and
inflammation in the joints and pleural and peritoneal fluids
 Page 08

OTHER SOLUBLE IMMUNE

RESPONSE MEDIATORS
 Biological response Page 08

modifiers (BRMs)
modulate an individual’s own immune response.
four main sources of major BRMs secreted by mononuclear leukocytes
1. B lymphocytes that secrete specific antibodies.
2. T lymphocytes that secrete soluble mediators, such as interleukin-2 (IL-2) and other
ILs, granulocyte-monocyte colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ)
and tumor necrosis factor-β (TNF-β)
3. Natural killer (NK) lymphocytes that secrete IFN-α
4. Monocytes and macrophages that secrete IFN-α, IL-1, and other ILs, TNF-α, and GM-
CSF and monocyte colony-stimulating factor (M-CSF)
The classes of immunotherapy:
Active—use of microbial or chemical immunomodulators (adjuvants) in a specific or nonspecific form
Adoptive—use of soluble mediators, such as ILs, to regulate components of the immune system
Passive—transfer of preformed antibodies to tumorous recipients, such as monoclonal antibodies
Restorative—application of soluble substances, such as interferons, for a wide range of diseases
Cytokines
are polypeptide products of activated cells that control a
variety of cellular responses and thereby regulate the immune
response.
Migratory inhibitory factor (MIF) was the first cytokine activity
to be described. MIF performs a T cell–derived activity that
immobilizes macrophage migration, which may cause retention
and accumulation of phagocytes at sites of inflammation.
Lymphokines are cytokines produced by activated
lymphocytes.
Cytokines
Cytokines are very potent, even in minute concentrations.
Their action is usually limited to affecting cells in the local area
of their production, but they can also have systemic effects. As
a group, cytokines differ in molecular structure but share the
following actions:
Secrete cytokines in rapid bursts, synthesized in response to cellular
activation.
Bind to specific membrane receptors on target cells
Regulate receptor expression in T and B cells, which drives positive
amplification or negative feedback.
Act on different cell types.
Excite the same functional effects with multiple cytokines (redundancy).
Act close to the site of synthesis on the same cell or on a nearby cell.
Influence the synthesis and actions of other cytokines.
 Interleukins

A characteristic of ILs is that secreted peptides and proteins mediate local

interactions between leukocytes but do not bind antigen.
ILs include molecules that are made by and act on lymphocytes.
Interleukins have widely overlapping functions :
These molecules modulate inflammation and immunity by regulating growth,
mobility, and differentiation of lymphoid cells.
Each of the ILs has been shown to be a distinct molecule by gene cloning and
sequencing. In addition, each IL functions through a separate receptor system.
 Interferons

a group of cytokines discovered in virally infected cultured cells.

This interference with viral replication in the cells by another virus led to the term
interferon.
The IFNs are one of the body’s natural defensive responses to foreign
components (e.g., microbes, tumors, antigens).
IFNs are among the most broadly active physiologic regulators, enhancing the
expression of specific genes, inhibiting cell proliferation, and augmentin immune
effector cells.
IFNs have been demonstrated to act as antiviral agents, immunomodulators, and
antineoplastic agents.
 Type I IFNs
mediate the early innate immune response to viral infections.

consist of two distinct groups of proteins, IFN-α and IFN-β that are structurally different
but that bind to the same cell surface receptor and induce similar biologic responses.

IFN-γ is the principal macrophage-activating cytokine and serves a critical function in

innate immunity and in specific cell-mediated immunity.
It stimulates expression of MHC class I and class II molecules and costimulates antigen-
presenting cells (APCs), promotes the differentiation of naive CD4+ T cells to the helper T
cell type 1 (Th1) subset and inhibits the proliferation of Th2 cells.
IFN-γ acts on B cells to promote switching to certain IgG subclasses, activates neutrophils,
and stimulates the cytolytic activity of natural killer (NK) cells.

It is also antagonistic to IL-4. IFN-γ is of most immunologic interest because of its diverse
effects on the immune response. Its ability to augment the activity of many cytokines has
resulted in clinical trials in a number of different diseases.
Tumor Necrosis
Factor
Tumor necrosis factor is the principal mediator of the acute inflammatory
response to gram-negative bacteria and other infectious microbes.
TNF is responsible for many of the systemic complications of severe
infections.
The TNF receptor family stimulates gene transcription or induces
apoptosis in a variety of cells.
The gene-encoding TNF-α is located in the HLA region between the HLA-
DR and HLA-B loci.
TNF-α and TNF-β share similar activities.
the principal physiologic functions of TNF are as follows:

1. to stimulate the recruitment of neutrophils and monocytes to sites of infection

2. to activate these cells to eradicate microbes.

In low concentrations, TNF acts on leukocytes and endo�thelium to induce acute

inflammation.
At moderate concentrations, TNF mediates the systemic effects of inflammation.
In severe infections, TNF is produced in large amounts and causes clinical and
pathologic abnormalities (e.g., septic shock).
When TNFs gain access to the circulation during infection, they mediate a series of
reactions that induce shock and can result in death.
The syndrome known as septic shock is a complication of severe gram-negative
bacterial sepsis.
Stem Cell Factor
(c-kit Ligand)

is a cytokine that interacts with a tyrosine

kinase membrane receptor.
The cytokine that interacts with this receptor is
called c-kit ligand, or stem cell factor, because it acts on immature stem cells.

is needed to make bone marrow stem cells

responsive to other CSFs but it does not cause colony formation itself
Stem cell factor may also play a role in sustaining the
viability and proliferative capacity of immature T cells in the
thymus and mast cells in mucosal tissues.
Transforming Growth
Factors

were identified as products of virally transformed cells.

These factors were found to induce phenotypic transformation in non-
neoplastic cells and subsequently were termed transforming growth
factors.
TGF-β is a group of five cytokines released by many cell types, including
macrophages and platelets.
TGF-β is known to be a potent inhibitor of IL-1–induced T cell
proliferation.
The principal action of TGF-β in the immune system is to inhibit the
proliferation and activation of lymphocytes and other leukocytes.
Chemokines

are a large family of structurally homologous cytokines that stimulate

transendothelial leukocyte movement from the blood to the tissue
site of infection and regulate the migration of polymorphonuclear
leukocytes (PMNs) and mononuclear leukocytes within tissues.
The largest family consists of CC chemokines that attract
mononuclear cells to sites of chronic inflammation, such as monocyte
chemoattractant protein 1 (MCP-1).
 Chemokines

A second family of chemokines consists of CXC chemokines, of which

IL-8 (CXCL8) is the prototype.
CXCL8 attracts PMNs to sites of acute inflammation, activates
monocytes, and may direct the recruitment of these cells to vascular
lesions.
The third family, CX3, forms a cell adhesion receptor capable of
arresting cells under physiologic flow conditions.
TNF-α–converting enzyme can cleave CX3CL1 from the cell
membrane.
Other functions of various chemokines include the following:
1. Increasing the affinity of leukocyte integrins for their ligands on
endothelium (e.g., intercellular adhesion molecule-1 [ICAM-1], ICAM-2,
vascular cell adhesion molecule-1 [VCAM-1])
2.Regulating the traffic of lymphocytes and other leukocytes through
peripheral lymphoid tissues
3. Maintaining normal migration of immune cells into lymphoid organs or
other specialized cells to particular sites
Assessment of
Cytokines

Defects in cytokine production can lead to

autoimmunity. Traditional methods for assessment
of cytokines
include the following:
• Bioassays
• Enzyme-linked immunosorbent assay (ELISA)
• Intracellular staining
• Ribonuclease protection assay
• Polymerase chain reaction (PCR)
ACUTE-PHASE PROTEINS
 Page 10
ACUTE-PHASE PROTEINS
A group of glycoproteins associated with the acute-phase response
include
C-reactive protein (CRP)
inflammatory mediators
fibrinogen
transport proteins
inhibitors
α1-acid glycoprotein.
Persistent increases in CRP can also occur in chronic inflammatory disorders
(e.g., autoimmune disease, malignancy).
 Page 10
C-Reactive Protein
Traditionally, CRP has been used clinically for monitoring infection,
autoimmune disorders and, more recently, healing after a myocardial
infarction (MI).
the fastest responding and most sensitive indicator of acute
inflammation.
CRP increases faster than ESR in responding to inflammation, whereas the
leukocyte count may remain within normal limits despite infection
CRP is the method of choice for screening for inflammatory and malignant
organic diseases and monitoring therapy in inflammatory diseases.
α1-Antitrypsin is an acute-phase protein that increases in acute
inflammatory reactions.
Defects in the complement components C3a and C5a and the opsonin
C3b result in serious infections.
immune complex disease and gram-negative bacteremia result in low
levels of complement components, particularly C3 and C4, because the
components are consumed during complement activation.
Lymphomas may result in a marked increase in C1 esterase inhibitor,
with little other change.
Ceruloplasmin, often measured as serum copper, is used to monitor
Hodgkin’s disease; increases are considered specific indicators of relapse.
 Page 11
Assessment Methods

C-Reactive Protein Rapid Latex Agglutination Test

is based on the reaction between patient serum containing CRP as the
antigen and the corresponding antihuman (CRP) antibody coated to the
treated surface of latex particles.
The coated particles enhance the detection of an agglutination reaction
when antigen is present in the serum being tested.

Reference range :
0.01 mg/dL in newborns
less than 0.05 mg/dL in adult men and nonpregnant women.
Positive Reaction
Agglutination of the latex suspension is a positive result. A positive
reaction is reported when the undiluted specimen or the 1:5
diluted specimen demonstrates agglutination, or when both
exhibit agglutination.

Negative Reaction
The absence of visible agglutination and the presence of opaque
fluid constitute a negative reaction. A negative reaction is
reported only when both the undiluted specimen and the 1:5
diluted specimen exhibit no visible agglutination
Specimen collection and handling are important to the quality
of the test.
Strict adherence must be paid to technique, with a special
emphasis on drop size, complete mixing, reaction time, and
temperature of reagents.
The strength of a positive reaction may be graded as follows:
1+ Very small clumping with an opaque fluid background
2+ Small clumping with a slightly opaque fluid background
3+ Moderate clumping with a fairly clear fluid background
4+ Large clumping with a clear fluid background
Sources of Error
False-positive results
lipemic
hemolyzed
heavily contaminated with bacteria
reaction time is longer than 2 minutes
from a drying effect.
False-negative results
undiluted serum specimen

LIMITATION
The strength of the agglutination reaction is not always indicative of the
CRP concentration.
Weak reactions may be produced in samples with elevated or low CRP
values.
Results may vary, depending on the patient’s condition.
This 2-minute slide latex agglutination test has a detection level of 1 mg
CRP/dL; therefore, patients with CRP values less than 1 mg/dL may go
undetected.
The sensitivity of the procedure has been assessed at 93%.
 THANK
YOU!
Any Questions?
Chapter 5 / Group 3