LECOM-Pharmacy SchoolImmunology 3 & 4

Dr. Saber Hussein

Innate Immunity & Complement

Objectives1. The early defense against infections and cancer 2. Recognition of microbes by the innate immune system3. Components of innate immunity

– Physical and chemical barriers (e.g. epithelial barrier, stomach acidity)

– Phagocytes: neutrophils and monocytes/macrophages– Natural killer cells– The complement system– Cytokines of innate immunity– Other plasma proteins of innate immunity

4. Microbes evasion of the innate immunity5. How does innate immunity stimulate the acquired

immunity?

Innate Immunity• Natural, none-adaptive• Nonspecific to an antigen but recognize and fight

microbes• Phagocytes: Monocytes, macrophages, PMN

neutrophils• Natural killer (NK) cells• Complement system• Exterior defenses: Skin, Stomach acidity, Mucus,

Cilia, Microflora, Lysozyme in tears, Flushing of urinary tract

Cytokines of Innate Immunity

• Cytokines are small soluble proteins produced by many cells such as macrophages

• They mediate immune and inflammatory reactions

• They are the tool of communication among leukocytes and between those and other cells

Cytokines produced by macrophages

• Tumor necrosis factor (TNF)• Interleukins (IL):

– IL-1

– IL12

– IL-10

– IL-6

– IL-15

– IL-18

• Type I interferons:– IFN-– IFN-

Cytokines of Innate ImmunityCytokine Principal cell

source Principal cellular targets & biologic effect

TNF Macrophage, T cell Endothelial cell: activation Neutrophil: activation Hypothalamus: fever Many cell types: apoptosis

IL-1 Macrophage, endothelial cell, epithelial cell

Endothelial cells: activation

IL-12 Macrophages Dendritic cells

NK cells & T cells: IFN- synthesis, increased cytolytic activity

IFN- NK cells, T cells Activation of macrophages

Type I IFNs

IFN-MacrophagesIFN- Fibroblasts

All cells: antiviral activity, increased MHC I expression NK cells: activation

Innate Immunity• Natural, none-adaptive,

Nonspecific• Phagocytes: Monocytes,

macrophages, PMN neutrophils

• Natural killer (NK) cells• Complement system• Exterior defenses: Skin,

Stomach acidity, Mucus, Cilia, Microflora, Lysozyme in tears, Flushing of urinary tract

Physical Barriers

Functions of activated macrophages• Macrophages may be

activated by signals from many surface receptors.

– Receptor for bacterial endotoxin (LPS), which transduces signals via an attached Toll-like receptor

– Receptor for the most important macrophage-activating cytokine, IFN

•Signals from activating receptors stimulate the production of several proteins, which mediate the important functions of macrophages.

•Different M surface receptors may stimulate distinct or overlapping responses

•Common feature is that they stimulate the production of transcription factors, which result in the production of various proteins

Functions of Natural killer (NK) cells• NK cells kill host cells

infected by intracellular microbes– eliminating reservoirs of

infection.

• NK cells secrete IFN in response to IL-12 produced by macrophages

• IFN activates the macrophages to kill phagocytosed microbes

Reticuloendothelial system & Normal flora

Reticuloendothelial system & Competition by Normal flora

Normal flora on

Oral cavitySkin

Soluble Factors & Physiological responses

• Soluble Factors:1. Lysozyme

2. Complement

3. Antiproteinases

4. C-reactive protein

5. DNAse

6. RNases

• Physiological responses:

– Chemotactic factors from infecting agents• Bacteria F-Met

peptides

• Injured tissue

– Mast cells & PMN

– Leukotrienes &

– Histamines

Mechanisms of killing

• Toxic oxygen– Oxygen radicals

– Hydrogen peroxide

– Hypochlorous acid (HOCl)

Innate & adaptive immunityInnate Immunity

• Pathogen recognized by receptors encoded in the germline

• Receptors have broad specificity, i.e., recognize many related molecular structures called PAMPs (pathogen-associated molecular patterns)

• PAMPs are essential polysaccharides & polynucleotides that differ little from one pathogen to another but are not found in the host (mannose)

• Receptors are PRRs (pattern recognition receptors)

• Immediate response• No memory of prior exposure• Occurs in all metazoans?

Adaptive Immunity• Pathogen recognized by

receptors generated randomly• Receptors have very narrow

specificity; i.e., recognize a particular epitope

• Most epitopes are derived from polypeptides (proteins) and reflect the individuality of the pathogen

• Receptors are B-cell (BCR) and T-cell (TCR) receptors for antigen

• Slow (3 -5 days) response (because of the need for clones of responding cells to develop)

• Memory of prior exposure• Occurs in vertebrates only

Innate &

AdaptiveImmunity

C-Reactive Protein

• CRP belongs to the pentaraxin family of proteins– so-called because it has five identical

subunits, encoded by a single gene on chromosome 1, which associate to form a stable disc-like pentameric structure

• It reacts with the somatic C polysaccharide of Streptococcus pneumoniae

• In the presence of calcium, CRP specifically binds to phosphocholine moieties– phosphocholine is found in phospholipid

membranes of many organisms

Pentameric structureof CRP

CRP role in innate immunity

• Binding phosphocholine gives CRP a host-defensive role because: – Phosphocholine is found in microbial polysaccharides

CRP-binding activates the classical complement pathway

Opsonizes ligands for phagocytosis– It neutralizes the pro-inflammatory platelet-activating

factor (PAF)– It down-regulates polymorphs (neutrophil, eosinophil,

basophil)

CRP made in the liver• CRP is exclusively made in the liver • Secreted in increased amounts within 6 hours of an

acute inflammatory stimulus (e.g. infection)• The plasma level can double at least every 8 hours,

reaching a peak after about 50 hours• After effective treatment or removal of the inflammatory

stimulus, levels can fall almost as rapidly as the 5-7-hour plasma half-life of labeled exogenous CRP

• The only condition that interferes with the "normal" CRP response is severe hepatocellular impairment

Defensins• Definition:

– A family of abundant, modestly potent cationic proteins that is found • in the primary granules of neutrophils and • in the lysosomes of some mononuclear

phagocytes

• Bactericidal– can kill fungi and viruses too

• One defensin is chemotactic for monocytes – Neutrophils help to attract the "second wave" of

leukocytes- • Mononuclear phagocytes

Defensins• All of the following are protected by defensins

1. our epithelial surfaces

2. skin

3. lining of the GI tract

4. lining of the genitourinary tracts

5. lining of the nasal passages and lungs

Leukocytes extravasation

Blood vessel

Infected tissue

Complement

Alternative Pathway

Classic Pathway

Objectives

1. Define complement

2. Activation of the classical pathway

3. Activation of the lectin pathway

4. Activation of the alternative pathway

5. Describe the three functions of complement in body defense

6. Complement deficiencies and disease

The complement system: (Chapter 2)• The enzymatic cascade• Activation of the complement cascade

1. The alternative pathway2. The classical pathway3. The lectin pathway

• Anaphylactic and chemotactic products of the complement system

• Membrane attack complex• Evasion of innate immunity by microbes• Role of innate immunity in stimulating adaptive

immune responses

What is Complement?

• Non-immunoglobulin serum proteins

• Involved in:

i. Control of inflammation

ii. Stimulation of phagocytosis

iii. Activation of cell lysis

Opsonization (“Eat Me” tag) & phagocytosis• Preparation for eating

• Phagocytosis facilitating process

• Opsonins are deposited on the Ag (bacteria or virus infected cell)

• Important opsonins can label bacteria:

• IgG

• Complement fragments (e.g. C3b)

Sites of synthesis

• Hepatocytes

• Macrophages, various tissues

• Epithelial cells, GI tract

• Monocytes, in blood

Functions of complement

• Direct killing of bacteria & virus-infected cells by lysis mediated by MAC

• Indirect killing by opsonization followed by phagocytosis & intracellular killing

• Immunization does NOT increase complement concentration in the serum

Role of C3 in bacterial clearance and killing

1. C3 bound to bacteria as C3b or iC3b binds to CR1 on erythrocytes, which transport bacteria through circulation

2. C3 acts as focus for the deposition of lytic MAC on bacterial cell membrane

3. It ligates complement receptor on phagocytes

4. Complement, in turn, activates the phagocyte leading to

• Bacterial phagocytosis,• Respiratory burst

generation and• Bacterial killing

intermediate

Activators of the alternative pathway (AP)

• Lipopolysaccharides (LPS)

• Bacterial cell walls

• Cell walls of yeasts

• Aggregated IgA

• Cobra venom

• NO need for Ab-Ag complex or IgM for activation

Activation cascade of the

Alternative Pathway

• C3b binds foreign substance• C3b + Serum’s Factor B C3bB• Factor D cleaves Factor B C3bBb• C3bBb acts as C3 convertase: C3 C3a + C3b

– Properdin regulates the process; stabilizes C3bBb. – Factor I & H inactivate free C3b

AlternativePathway

Activation of Classical Pathway (CP)

• CP is activated by:– Antigen-IgG complex– Pentameric IgM

• Non-activators of CP:

– IgG4

– IgA– IgE– They lack C1q

receptors they cannot bind C1q

Classical pathway

Steps of CP activation• Step 1: Activation of C1

– C1q-FcIgG activates C1r &C1s

– C1s activates C4 C4a + C4b

• Step 2: Activation of C4– C4 C4a + C4b

• Step 3: Activation of C2 by C4b– C4b + C2 C4b2a (C3-

convertase) + 2b• Step 4: Activation of C3 • Step 5: Activation of C5,

C6 & C7• Step 6: Activation of C8 &

C9

Classical pathway

Complement

Classic Pathway

AlternativePathway

Common Pathway

MAC & Cell Lysis

Split Products and Their Activity

Activities of complement fragments

The Lectin Pathway (LP)

• Activator: Mannose-binding lectin (MBL), a plasma protein that is similar to C1q

• MBL binds to terminal mannose (sugar) on the surface glycoprotein of microbes

• This lectin activates the classical pathway proteins

• Difference between LP and CP:– LP activation does not require antibodies

Therefore, LP activation is part of the innate immune system

Three functions of complement in host defense

1. Opsonization: – C3b binds to microbes and so enables the

phagocytes to grab the microbe, via their C3b receptor, and phagocytose it

2. Chemotactic activity of C5a: – This complement fragment attracts neutrophils

and monocytes to the site of infection– C5a promotes inflammation at the site of

complement activation3. Formation of membrane attack complex(MAC)

– MAC forms a pore (hole)in the membrane of the microbe leading to the loss of cellular contents, lysis of the microbe and death of the microbe

Complement Proteins’ Deficiencies

• Alternative pathway defects Susceptibility to Haemophilus influenzae

• Defects of Factor D & Properdin increase Neisseria infection

• C5 deficiency, less severe consequences, but increases susceptibility to Neisseria gonorrheae & N. meningitidis

Complement & disease pathogenesis

• Complement may cause disease pathogenesis by:

1. Systemic production of anaphylaxis such as after Gram-negative sepsis

2. Insertion of MAC into host cell membrane leading to cellular activation and stimulation of membrane arachidonic acid metabolism

3. Fixation of C3b to immune complexes located in tissues causing recruitment and activation of tissue and circulating WBCs

Heritable angioedema (HAE)

• C1-inhibitor deficiency

• Autosomal dominant trait inheritance

• Edema in various organs & tissues

• Especially bad: – Edema of intestine and throat

• Treatment– Epinephrine for emergency treatment

Acquired angioedema

• Caused by an autoantibody to C1 inhibitor,

• This leads to inactivation of C1 inhibitor – A result similar to HAE:

• C1 inhibitor becomes deficient