Lecture 4 - Cytokines, Complement System

- small proteins that bind receptors on target cells
- produced by many immune and non-immune cells

- they generate a form of communication between immune cells to orchestrate immune response
- many of hem are produced in response to infectious stimuli

- autocrine
- paracrine
- endocrine

cytokines that affect them same cells from which they were secreted

cytokines that affect cells within the tissue surrounding their cellular source

cytokines that travel in the blood to distant targets

Hyperstimulation of the immune response or hypercytokinemia due to massive overproduction and dysregulation of Cytokines in extreme situations (may lead to multi-organ damage)

TNF-alpha
IL-1
IL-6
IFN-gamma

tumor necrosis factor

interleukin

interferon

TGF-beta
IL-10
IL-13
IL-35

transforming growth factor

anti-inflammatory cytokines help regulate the inflammatory response

pro-inflammatory cytokine production

IL-1β, TNF-α, IL-6:
are responsible for fever, increased release of immune cells (from BM), recruiting effector cells like neutrophils and monocytes, general subjective feeling of being “ill”

IL-1β

TNFs (tumor necrosis factor) are a family of membrane-bound cytokines, released in times of stimulation
• TNF-α is a major inflammatory cytokine (induced by bacterial LPS)
• TNF-β (lymphotoxin) produced by lymphocytes and causes cell death, or cytotoxicity, in many different types of cells

TNF-α

TNF-β

IL-6

- effect on liver to produce CRP
- release of neutrophils from BM
- activation of B and T cells

- derived from cytokine and chemotaxis
- involved in induction of migration and mobility of their target cells
- they have receptors (CRR)

recruitment of leukocytes to site of inflammation or tissue damage

1. CXC group (one amino acid “X” between cysteines
2. CC group (no amino acid)
3. C group (one cysteine)
4. CX3C group (3 amino acids between the two cysteines)

• Mostly stops cell growth and proliferation, so as a control brake it downregulates immune response when it is not needed anymore
• Inhibits activation of macrophages
• Inhibits release of inflammatory cytokines from T cells (IL-12 and IFN-γ)
• Inhibits proliferation of activated B cells (controls Ab production)

First identified as soluble substances produced by virusinfected cells and interfered with virus replication in the infected cell.
• 3 types: I, II, III, type I are the most important ones in viral infections, and
include IFN-α and IFN-β

IFN-α is mainly produced by DCs and macrophages, but IFN-β by any infected cells

• IFNs inhibit virus replication through several mechanisms, e.g, they induce expression of RNase which degrades viral RNA
• Type I IFNs are strong activators of NK cells: most important innate immune response in viral infections
• IFNs have therapeutic applications too: in MS and malignancies (IFN-α in hairy cell leukemia)

CD4+ T helper (Th) cells
there are 4 Th subsets

Th1
Th2
Treg
Th17

IFN-γ and IL-2
• IL-2: drives the growth and differentiation of both T and B cells and enhances the lytic activity of NK cells
• IFN-γ: enhances cell mediated immune response by increasing the expression of MHC-I, II molecules on APCs

- intracellular bacteria and viruses
- cell-mediated immunity

IL-4 and IL-10
• IL-4: inflammatory cytokine, allergic reactions)
• IL-10: anti-inflammatory and suppressor of Th1 cells and inhibition of IFN-gamma

parasitic worms
allergies

Express CD4, CD25 and FOXP3, involved in establishing tolerance to self antigens especially food antigens

• Produce IL-17 family of cytokines (pro-inflammatory)
• Derived from Th cells by the action of TGFβ, IL-6, and IL-23.
• Play an important role in defense against bacterial and fungal infections at mucosal surfaces

Complement is a series of more than 50 soluble and cell-bound proteins of the innate immune system that interact to enhance host defense mechanisms against foreign cells

LIVER!!
exception:
‒ C1 components (produced by intestinal epithelial cells)
‒ Factor D (made in adipose tissue)

C1
C2
C3
C4

zymogens that are converted to active exnzymes in a very precise order

an inactive protein that is converted into an active enzyme by a biochemical change

- Cause lysis of foreign cells (e.g., B group RBCs in a A group plasma)
- Act as opsonins:
• Specific receptors are present on phagocytic cells
• Enhance clearance of immune complexes
- Increase vascular permeability
- Recruit monocytes and neutrophils to the area of antigen concentration
- Trigger secretion of immunoregulatory molecules that amplify the
immune response

- Classical Pathway
- Lectin Pathway
- Alternative Pathway

YES! - more than one pathway can be activated simultaneously

lysis of the invading cell using MAC

1. Recognition Unit
2. Activation Unit
3. Membrane Attack Complex (MAC)

C1 binds to antigen-antibody complexes or other substances to initiate the pathway followed by activation of C4, C2 and formation of C4b2a (C3 convertase)

C3 will be activated, fragments are created and combine to form C5 convertase (C4b2a3b)

MAC is formed following successive activation of C5 through C9 (C5b6789) that causes cell lysis (creates a gateway into the cell causing it to lyse)

• Antibodies (IgG or IgM) bound to antigen
• C-reactive protein bound to ligand
• Certain viruses*
• Mycoplasmas*
• Certain gram-negative bacteria (E. Coli)*
• Some protozoa*

• First complement component to bind (it is inactive at first)
• Consists of three subunits: C1q, C1r, C1s
• At least two of the globular heads of Clq must be bound to antibody to initiate the pathway

C1q - which recognizes The Fc region of 2 adjacent Ab molecules

IgG or IgM

These antibodies must first be bound to antigen in order for complement (C1q) to bind

• Two IgG molecules must attach to antigen within 30 to 40 nm of each other before complement can bind
• Because IgM is a pentamer, it takes only one IgM molecule attached to two adjacent antigenic determinants to initiate the cascade.

Within the IgG group, IgG3 is the most effective, followed by IgG1 and then IgG2

IgG4, IgA, or IgE cannot activate complement

1. The zymogens C1r and C1s are converted into active enzymes as binding of C1q occurs.
2. Autoactivation of C1r results from a change that takes place as C1q is bound.
3. When activated, C1r cleaves a thioester bond on C1s, which activates it.
4. C1s has a limited specificity, with its only substrates being C4 and C2.

1. C1s cleaves C4 to create C4a (released into circulation) and C4b, and it cleaves C2 to create C2a and C2b. C4b and C2a combine to form C4b2a which is called C3 convertase.
2. C3 convertase cleaves C3 to C3a (released into circulation) and C3b which is bound to C4b2a to form C4b2a3b known as C5 convertase.
3. C5 convertase cleaves C5 to C5a (released into circulation) and C5b

1. C5 convertase splits C5 into C5a and C5b.
2. C5b attaches to the cell membrane and binds to C6 which is followed by sequential binding of C7, C8, C9 to initiate formation of the MAC (C5b6789).
3. MAC produces a pore of 70 to 100Å that allows ions to pass in and out of the membrane. Destruction of target cells actually occurs through an influx of water and a corresponding loss of electrolytes

Rather than activating complement through antibody binding (Classical), the Lectin pathway is activated by surface moieties on microbial cell walls (pathogens)

- lectins
- ficolins
- collecins

C4 and C2 are shared between this pathway and classical, after they are cleaved the rest of the pathway is like classical

Mannan-binding lectin (MBL)

- an acute-phase protein - produced in liver and normally present in serum, but increases during initial inflammatory response
- binds to mannose or related sugars on microbes (bacteria, yeasts, viruses, and some parasites) to initiate the complement pathway

• Acts mainly as an amplification loop for classical or lectin pathways
• Can be activated directly by bacterial or fungal cell walls, viruses, tumor cells, or some parasites
• Involves C3, C5 – C9, Factors B, D, and P (properdin)

decay accelerating factor

membrane inhibitor of reactive lysis

monocyte chemotactic protein

• Amplifies inflammatory response
- Anaphylatoxins (C5a, C3a)
- Chemotaxins (C5a)
- Opsonins (C3b, C4b, iC3b, C3dg)
• Role in uptake and presentation of antigens in adaptive immune response
• Facilitates B-cell activation
• Role in neural development

• It is activated systemically on a large scale, as in gram-negative septicemia (systemic rxn in blood - causes high inflammation unlike apoptosis).
• It is activated by tissue necrosis and results in obstruction of the blood supply, as in myocardial infarction.
• Lysis of RBCs occurs, as in cold agglutinin disease or autoimmune hemolytic anemia.

Lupus-like syndrome; recurrent infections

Lupus-like syndrome; recurrent infections; atherosclerosis

Severe recurrent infections; glomerulonephritis

Lupus-like syndrome

Neisseria infections

No known disease association

Hereditary angioedema

Paroxysmal nocturnal hemoglobinuria (PNH)

Paroxysmal nocturnal hemoglobinuria (PNH)

Recurrent pyogenic infections

Pneumococcal diseases, sepsis, Neisseria infections

Neisseria infections

- Atypical hemolytic uremic syndrome (aHUS)
- C3 glomerulopathy

• Rare kidney disorder that results in acute renal failure in children.
• Caused by mutations in genes coding for Factor H, MCP, Factor I, Factor B, C3 or thrombomodulin; or to autoantibodies against Factor H.

• Measurement of components as antigens in serum
• Measurement of functional activity of complement pathways

• Automated nephelometry or immunoturbidimetry
• Radial immunodiffusion (RID)

‒ Hemolytic titration (CH50) assay
‒ CH50 test for lysis of liposomes
‒ ELISA to detect C9 epitopes exposed after binding in MAC

• AH50
• ELISA to detect C3bBbP or C3bP

A CH50 blood test, also known as a total complement test or CH100, measures the level and activity of complement proteins in the blood.

- serum to be tested is diluted seriall yand added to sensitized sheep red blood cells
- the tubes are incubated at 37C and then centrifuged to pellet the unlysed cells
- the CH50 is defined as the reciprocal of the dilution that causes lysis of 50% of the cells used in the assay
- in the example shown, the CH50 would be about 80 U/mL

Classical: Low
Lectin: Normal
Alternative: Normal

Classical: Low
Lectin: Normal
Alternative: Normal

Classical: Normal
Lectin: Low
Alternative: Normal

Classical: Normal
Lectin: Normal
Alternative: Low

Classical: Low
Lectin: Low
Alternative: Low

Classical: Low
Lectin: Low
Alternative: Normal

Classical: Low
Lectin: Low
Alternative: Low

Classical: Low
Lectin: Low
Alternative: Low

they target key points in the complement pathways to treat disease

Recombinant C1-INH

monoclonal antibody to C5 (eculizumab)

- used to treat aHUS and PNH
- leads to decreases in soluble C5b-9 levels, CH50, and AH50