University of Balamand

Faculty of Medicine and

Medical Sciences

IMUN402
Lecture#2
Innate Immunity and Complement System

Instructor: Samer Bazzi, PhD

 Characteristics of Innate and Adaptive Immunity

No
memory,
Innate too not long
lasting
Cellular Components of Innate Immunity
Outcomes of Innate Immune Reponses
- Chemotaxis

- Opsonization/phagocytosis
• Opsonization: process where microorganisms and inanimate particles (e.g., liposomes) are coated with
opsonins (immunoglobulins, complement factors), facilitating the binding of the opsonized bacteria or
particle to specific receptor molecules present on phagocytes (i.e., neutrophils, macrophage, DCs).

- Induction of cytokines
 Inflammatory response
 Acute phase response: an orchestrated response to tissue
injury, infection or inflammation. A prominent feature of
this response is the induction of acute phase proteins, which
are involved in the restoration of homeostasis
i.e. C-reactive protein (CRP) is produced by the liver
in response to inflammatory mediators. Its serum
levels are used as indicator of acute infection or
chronic inflammation. Binds to phosphocholine
(PC) moieties on damaged cells, acts as opsonin, and
target damaged cells for disposal by macrophages
having CRP receptors.
Intrinsic Epithelial Barriers to Infection
Effector Mechanisms in Innate Immunity
- Skin: Keratin in the skin presents a tough physical barrier to most microorganisms and is
resistant to weak acids and bases, bacterial enzymes, and toxins. Desquamation of squamous
cells and low pH induced by fatty acids plus other secretions of sebacious glands prevent
microbe colonization. Sebum contains chemicals toxic to bacteria.

- GI Tract: Stomach mucosa secrete gastric acid (HCl) and protein-digesting enzymes.
Peristaltic movement of columnar cells hinders entry of microbes. Mucus traps microbes that
enter the digestive system.

- Nasopharynx and Eye: Saliva and lacrimal fluid contain mucus and lysozyme (hydolyzes
peptidoglycan). Secretions flush out microbes. Mucus-coated hairs in the nose trap inhaled
particles/microbes.

- Lungs: Mucosa of the lower respiratory tract is ciliated and cilia sweep dust- and bacteria-
laden mucus away from lower respiratory passages. Presence of surfactants render alveolar
surfaces hydrophobic preventing filling of the lungs with fluid and microbe entry.
Effector mechanisms in Innate Immunity (Important Definitions)
Sebacious gland: exocrine gland in skin secreting sebum and weak acid.

Sebum: present antimicrobial activity and is composed of triglycerrides, waxester, squalene (30-C organic compound
used in hormone synthesis), and free fatty acids.

Intestinal glands: found in epithelial lining of small intestine. Contains stem cells, paneth cells, goblet cells,
enteroendocrine cells, and enterocytes.

Mucus: viscous colloid containing antiseptic enzymes (lysozyme, Igs, and mucins) and secreted by goblet cells of
intestinal gland.

Mucins: glycoproteins made by goblet cells in a gel-like form. Can be bound to cell membranes or secreted into
mucosal surfaces, present in saliva, and bind microbes.

Serous glands: constituted of serous cells, associated with parotid and lacrimal glands. Secrete pale yellow, protein-
rich serous fluid that fills body cavities containing α-amylase and lipase

Cathepsins: proteases activated by low pH in lysosomes; degrade bacteria.

Defensins: cysteine-rich small cationic proteins (18-45 aa) produced by neutrophils and epithelial cells. Bind to
microbes and form pore-like defects in membranes allowing efflux of essential ions and nutrients → death of
microbe.

Cryptidins: enteric α-defensins secreted by paneth cells.

Cathelicidins: small peptides (12-80 aa) found in lysosomes of neutrophils, epithelial cells and macrophages.
Induced after cell activation with microbes and degrade lipoprotein membranes of bacteria, fungi and viruses in the
phagolysosome.
Effector mechanisms in Innate Immunity

Site Component Functions

Blood & Phagocytes Phagocytosis and
Lymphiod intracellular killing
organs
NK & NKT cells Direct and antibody
dependent cytolysis
Serum & Lactoferrin, Iron deprivation
other cellular Transferrin
secretions
Interferons, Antiviral proteins,
TNF- phagocyte activation

Lysozyme Peptidoglycan hydrolysis

Fibronectin & Opsonization, enhanced

complement phagocytosis, inflammation
proteins
 Innate Immune Cells Stages in the maturation of mononuclear
Phagocytes: Macrophages phagocytes

- Phagocytose & kill intracellularly

- Identified by CD14 surface Ag
- Adherent on plastic and glass surfaces
- Activated by cytokines
- Kill intracellular organisms (i.e. Mtb)
- Kill malignant and altered self targets
- Tissue repair and Ag presentation
- Produce cytokines that initiate and regulate
inflammation
- Macrophages will develop along one of 2
different pathways
Mechanism of Phagocytosis
- Once chemotaxis of phagocytic cells into the area of antigen entry is accomplished, these cells ingest and
digest particulate debris, such as microorganisms, host cellular debris, and activated clotting factors. This
process, called phagocytosis, involves the following:
• Extension of pseudopodia to engulf attached material
• Fusion of the pseudopodia to trap the material in a
phagosome
• Fusion of the phagosome with a lysosome to create a
phagolysosome
• Digestion
• Exocytosis of digested contents

- Neutrophils release granule contents into extracellular

milieu during phagocytosis
and inflammation in which the neutrophils die, forming what
is known as pus.
- They extrude nuclear contents, histones, neutrophil
extracellular traps (NETs) which function to:
• Trap and kill pathogens
• May damage tissues when enzymes, ROS get released into
tissues
 Oxygen Dependent Intracellular Killing by Phagocytes
Respiratory Burst (known also as oxidative burst)

Defects in Respiratory Burst

Chronic granulomatous disease (CGD): is an inherited deficiency in the production of one of several subunits of NADPH
oxidase. This defect eliminates the phagocyte’s ability to produce many critical oxygen-dependent intracellular metabolites
(·O2–, .OH, 1O2, and H2O2).

- If the patient is infected with a catalase-negative organism, the H2O2 waste product produced by the bacterium can be used
as a substrate for myeloperoxidase, and the bacterium is killed.

- If the patient is infected with a catalase-positive organism (e.g., Staphylococcus, Klebsiella, Serratia, Aspergillus), the
myeloperoxidase system lacks its substrate (because these organisms destroy H2O2), and the patient is left with the oxygen-
independent lysosomal mechanisms that prove inadequate to control rampant infections. Thus, CGD patients suffer from
chronic, recurrent infections with catalase-positive organisms
Oxygen Dependent Intracellular Killing by Phagocytes
Nitric Oxide (NO) Production
- Mainly takes place inside macrophages (M1-macrophages)
- Cytokines (i.e. IFN-γ) induce macrophages to produce NO
which exerts toxicity on microorganisms and tumor cells. NO
is a gas generated from the conversion of arginine to citrulline
by macrophage inducible nitric oxide synthase (iNOS)

Mediators of Oxygen Independent

Killing in the Phagolysosome
The lysosomal contents of phagocytes
contain oxygen-independent degradative
materials:
• Lysozyme digests bacterial cell walls
by cleaving peptidoglycan
• Defensins form channels in bacterial
cell membranes
• Lactoferrin chelates iron
• Hydrolytic enzymes
 Innate Immune Cells
Natural Killer (NK) Cells
- Identified by the presence of CD56 & CD16 (FcRγ) and the absence of
CD3 Direct
- Kill virally-infected and malignant cells
- Activated by interferons (IFNs) α and β, and IL-12 to become lymphokine-
activated killer (LAK) cells that can kill by releasing perforin and granzyme
B → tumor cell apoptosis (killing of transformed and malignant cells)

- NK cells employ 2 categories of receptor: killer activating receptor (KAR) Indirect

and killer inhibitory receptor (KIR).

- If only KARs are engaged, the target cells will be killed. If both the KIRs
and the KARs are ligated, the target cell lives. Therefore the inhibitory signals
rule over the activation signals.
- Their recognition of targets is not MHC-restricted, and their activity does
not generate immunologic memory.

- NK cells can be activated through the FcR (CD16), only one signal is
required because the antibody signals that there is an active infection. This
occurs through a mechanism called antibody-dependent cell-mediated
cytotoxicity (ADCC)
Pattern Recognition Receptors (PRRs)
- Toll-Like Receptor Family (TLR1-10)
• Expressed on the cell surface or intracellularly
• Binding activates “pro-inflammatory” signals and uptake of microbes

- Phagocytic (endocytic) PRR such as Macrophage Mannose Receptor, Scavenger Receptor and f-mlp
receptor (chemotaxis)
• Expressed on the surface of phagocytic cells.
• Mediates uptake of microbes into phagocytes.

- Secreted PRR such as Mannan-binding lectins

(MBL) and Collectins
• Complement proteins and other lectins,
secreted by macrophage, epithelial cells,
and hepatocytes.
• Activate complement, act as opsonins, and
function as accessory proteins for PAMP
recognition and phagocytosis.

- Nuclear Oligomerization Domain receptors NOD

1 and 2 proteins:
• Bind bacterial cell wall structures (LPS and
MDP) and transduce signals leading to
macrophage activation. Present cytoplasmic
location and mediate NF-κB activation
Innate Immunity Sensors
PRRs Location, Ligands and Downstream Effects
Innate Immunity Sensors
Inflammasome
- Important component of the innate immune system
- Expressed in innate immune cells as a signaling system for detection of pathogens and stressors
- Activation of the inflammasome results in the production of IL-1β and IL-18, which are potent
inflammatory cytokines.
Role of Innate Immunity in Stimulating Adaptive Immune Responses
 Complement System
- A set of interacting proteins (> 30) released into the blood after production in the liver. The components act together as
zymogens, activating one another in cascade fashion after initiation from a variety of stimuli (i.e. presence of pathogens or of
antibody bound to pathogens). In the absence of infection, complement proteins circulate in an inactive form

- It represents the activity of fresh serum to lyse Ab-coated cells. The lytic activity is destroyed when heated at 56 °C for 30
min. The lytic activity of complement is decreased in certain diseases, e.g. SLE and some chronic infections.
- Complement activation: alteration, in sequence, of complement proteins such that they interact and cleave the next
complement component into a larger moiety (suffix b; binding to the activation complex or membrane) and a smaller peptide
(suffix a) released in the microenvironment (C3b and C3a) [For C2, the larger fragment was referred to as C2a and it is still
used by some references].
- Complement fixation: utilization/consumption of C by Ag-Ab complexes
- Complement inactivation (screening assay for the activation of the classical complement pathway: denaturation (usually by
heat) of an early complement component resulting in loss of hemolytic activity (dilution of serum resulting in 50% lysis of
Ab-coated RBC, referred to as CH50)
- There are 3 pathways of complement activation:
Alternative pathway Complement System
- It is phylogenetically more primitive pathway compared to the “classical pathway”, but was named
“alternative” probably because it was discovered later among the two. This pathway consists of proteins
known by the term “Factors” like Factor B, Factor D.
- Spontaneous hydrolysis of C3 upon sensing of microbe surface molecules
- C3b product is very reactive and can bind to invader’s cell surface.
- If C3b cannot find the cell surface to bind within 60 microseconds - it is hydrolyzed.
- Factor D cleaves Factor B, leaving “Bb” which has a “chainsaw mechanism” and it can interact bind to C3b
to form a complex C3bBb
- C3bBb acts as C3 convertase which cleaves C3, this creates a continuous loop and many C3b can be
deposited on the invader’s cell surface.
- C3bBb together with another C3b molecule can act as C5 convertase which cleaves C5
- C5b together with C6, C7, C8 and C9 forms the membrane attack complex “MAC”
- C5b, C6, C7, and C8 form a “stalk” that anchors the complex in the bacterial cell membrane. Then C9
proteins are added to make a channel that opens up a hole in the surface of the bacterium.

Bacterial
membrane
Classical pathway
Complement System
- First protein in this pathway is C1 which is activated by the binding of 2 C1q heads
to the Fc portion of Ab molecules (IgG or IgM) on the surface of bacterium, inducing
a conformational change in the serine proteases C1r/C1s, leading to the activation of
the autolytic enzymatic activity of C1r which then cleaves C1s
- Activated C1s cleaves C4 into C4b and C4a. C4b either binds to antigen-antibody
complex or to the adjacent cell surface.
- C1s also cleaves C2 (into C2b and C2a) and then C4bC2b complex is formed which
serves as the C3 convertase of classical pathway.
- Once C3b is formed, it can bind to C4bC2b (C3 convertase) to form C4b2b3b (C5
convertase).
- Later steps are similar to the alternative pathway, MAC is formed.
Lectin pathway
Complement System
- Mannose binding lectin (MBL) recognizes specific carbohydrates such as d-mannose, l-fucose and N-
acetylglucosamine that are represented on the surface of a wide variety of infectious agents
-In the blood, MBL binds to another protein called mannan associated serine protease (MASP)
- MBL is similar in structure to C1q, having an amino terminal domain with a collagen-like structure that
binds the MASP proteins. There are three MASP proteins, called MASP-1, MASP-2, and MASP-3, which
are very similar in structure to C1r and C1s
- MASP-2 can cleave both C4 and C2, forming a C4b2b (C3 convertase), while MASP-1 can target C2 and
MASP-2 to help accelerate C3 convertase formation.
- Once C4b2b (C3 convertase) is formed, the subsequent events are similar to the classical pathway
- MBL pathway is particularly important in children who do not have much antibody
 Functions of Complement

C3aR
C3a and C5a (the small cleavage
fragments) released by complement
activation, are potent mediators of
inflammation. They are anaphylatoxins C5aR
and act as cell activators exerting their
functions through binding to specific
receptors (C3aR and C5aR)
Plasma and Membrane Proteins Regulating Complement Activation

24
Inhibition of the Formation Regulation of Formation of MAC
of C3 Convertases
b
b b

Regulation of Complement Activation

Complement Functions
Host benefit
 Opsonization to enhance phagocytosis
 Phagocyte attraction and activation
 Lysis of bacteria and infected cells
 Regulation of antibody responses (C3d, C3dg)
 Clearance of immune complexes (C3b)
 Clearance of apoptic cells (C1q)
Host detriment
 Inflammation, anaphylaxis
Distribution and Function of Complement Receptors