Immunology III

Question 1

Complement:

Answer 1

A blood-borne molecular defense system that “complements; the immunity provided by antibodies.

Activated through a tightly controlled enzyme-triggered cascade.

Question 2

Complement aids:

Answer 2

Phagocytosis => some complement components are opsonin’s

Destruction of microorganisms => the end result of complement activation is formation of a large, anti-microbial protein complex

Inflammation => some complement components are potent inflammatory mediators

Question 3

Cascade:

Answer 3

enzymatic reactions that activate protein effectors in a sequence:
-These proteins could be enzymes or anti-microbial proteins

Normally only turned on at certain localized sites.

Question 4

3 pathways of complement activation:

Answer 4

Alternative pathway:
-Complement component 3 (C3) acts as a pattern-recognition receptor-binds to the surface of a pathogen

Mannose-binding lectin (MBL) pathway:
-Triggered by binding of MBL (another pattern-recognition receptor) to mannose-containing CHO on bacteria/viruses

Classical pathway:
-When antibodies (Ab) bind to a pathogen, C1q binds to the Fc portion of those antibodies

(MBL and Classical pathway have similar activation mechanism)

Question 5

All pathways lead to the production of a stable C3 convertase:

Answer 5

-C3 convertase cleaves C3 => C3b and C3a

-C3b is an important opsonin and also causes progression through the rest of the complement cascade

Question 6

C3b forms a part of the C5 convertase:

Answer 6

-C5 convertase usually formed when C3b binds to the C3 convertase

-C5 convertase cleaves C5 => C5a and C5b

-C5b becomes associated with the cell wall/membrane of the microbe and causes lysis as it activates other components of complement (C6-C9)

-C5b+C6+C7+C8+C9 all associate and form a large pore in the microbial membrane and cause lysis

Question 7

Note the similarities between the classical and lectin pathway:

Answer 7

-Same C3 convertase

-Same C5 convertase

-C1q and MBL look very similar

Question 8

The alternative pathway:

Answer 8

The first responder:
C3 forms C3a and C3b spontaneously in the bloodstream, but is degraded quickly under normal conditions.
-Factor B, circulating protein, is also spontaneously cleaved to a protein known as Bb

-Bb complexes with C3b to form C3bBb

-C3bBb can convert C3 => C3a + C3b
(it’s a C3 convertase)

-C3bBb is rapidly inactivated in the uninfected host

If a bacterium is present, then C3bBb binds to the bacterial membrane, (C3b is a pattern-recognition receptor)
-C3Bb bound to the bacterial membrane is a stable C3 convertase

As more and more C3b is generated, then more and more C3Bb forms.
-Another circulating protein, properdin, helps stabilize the entire complex to form the stable C3 and C5 convertases.

C5 convertase of the alternative pathway:
-C3bBbC3b + properdin
-Needs to bind to the bacterial membrane to stay stable and keep converting C5=>C5a + C5b

Question 9

The lectin and classical pathways:

Answer 9

C1q is a complement protein that will bind to the Fc portion of an Ab that is activated (bound to an antigen)

Mannose-binding lectin (MBL) is a circulating pattern-recognition receptor.
-Recognizes mannose residues on bacterial membranes

When C1q detects a bound antibody or MBL detects mannose on a membrane: => they bind complement-activating proteins => these proteins cleave C2 and C4

C3 convertase:
-C4b2a
-Formed from cleavage of C4 and C2 upon C1q or MBL activation

As the C3 convertase acts, C3b accumulates and binds to the complex.

C5 convertase: C4b2aC3b
-The C5 convertase cleaves C5 => C5a + C5b

Question 10

Complement => cell lysis

Answer 10

Membrane attack complex (MAC)
-Generates a pore in lipid bilayer membranes

Sequence of events:
-C5b triggers assembly of complex of C5b, C6, C7 & C8
-Upon binding to C7, C8 will insert into membrane
-Induces polymerization of C9 (n=10-16) forming pore in membrane

The MAC and the pro-inflammatory effects of C5a and C3a can be extremely damaging to cells if they are not tightly regulated (and limited to microbes)

There are many proteins that down-regulate or degrade complement components

FYI: decay-accelerating factor, factor H, Factor I all cause destruction of the C3 convertases

Question 11

What else can C3a and C5b do?

Answer 11

They both cause vasodilation, increased vascular permeability, smooth muscle contraction (ex: bronchoconstriction) and histamine release from mast cells.

C5a is a chemotactic agent for a wide variety of cells (neutrophils and macrophages in particular)

Question 12

Can people have a deficiency of complement?

Answer 12

Yes- it’s a rare cause of immunodeficiency

Inadequate complement proteins (C2, C3, C4, C5, MBL, MAC complex) tend to make patients vulnerable to bacterial infection.

Question 13

Deficiencies in C1q highly predispose patients to systemic lupus erythematosus:

Answer 13

Photosensitivity rash, arthritis of small joints, renal failure, neurological vasculitis, neuropathies, and diverse effects on heart and lungs.

Prevalence: 50-100/100,000

Question 14

C1q helps macrophages to clear apoptotic bodies as well as initiate the classical pathway (C1q recognizes phosphatidylserine):

Answer 14

Thought that continual presence of self-antigens (especially nuclear material int he extracellular space) from dying cells increases the likelihood of developing autoimmunity.

C1q may also have complex immunomodulatory roles with Th cells

Question 15

Why is the alternative pathway the first responder, and the lectin/classical pathways more effective later?

Answer 15

C3 is always present in the bloodstream: it’s consistently being productive by the liver, cleaved, and degraded.
-If a microbe is present, C3b instantly binds (non-specifically) to the cell wall/membrane: if properdin and Bb also bind, then the stable C3 convertase forms very quickly.

Mannose-binding lectin does not circulate in high concentrations unless it is secreted by the liver in response to pro-inflammatory signals.

Significant quantities of antibodies take days: weeks to produce

Question 16

Which toll-like receptors should you know?

Answer 16

TLR1: can detect mycobacteria (like TB) and gram-negative bacteria

TLR2: can detect peptidoglycans: major component of cell wall of gram-positive bacteria

TLR3: double-stranded RNA => only found in viruses

TLR4: lipopolysaccharide (LPS), major component of gram negative bacteria

Toll-like receptors tend to recognize PAMPs (pattern recognition receptors) in the ECF or in the endosomes (not in the cytosol)

Question 17

C-type Lectin receptors:

Answer 17

detect carbohydrate components of many microbes (viruses, fungi, mycobacteria, parasites, some bacteria):

-Found on the cell membrane of a wide variety of immune cells (macrophages, dendritic cells, neutrophils, lymphocytes)

-These receptors are also found on the cell membrane, so they don’t detect pathogens in the cytosol

Question 18

NOD-like receptors (NLRs) are present in:

Answer 18

the cytosol of a wide range of immune cells (dendritic cells, neutrophils, macrophages, lymphocytes) and non-immune cells such as epithelial cells.

FYI: NOD-1, NOD-2, NLR-3 as examples

Most detected bacteria or parasite cell wall components that are present in the cytosol:
-Some bacteria and parasites reproduce and spend part of their life cycle inside cells
-Some NLRs seem to be able to detect viruses as well

Like TLRs, activation of NLRs lead to activation of NF-KB and AP-1

Question 19

RIG-like receptors (RLRs) are present in the:

Answer 19

cytosol of many (if not all) immune cells as well as non-immune cells (ex: endothelial, epithelial cells)

RLRs detect viral RNA (particularly double-stranded RNA) and activated:
-NF-KB
-transcription factors (FYI-IRFs) that lead to the production of particular “antiviral” cytokines known as interferons

Question 20

Danger associated molecular patterns (DAMPs):

Answer 20

molecular signals that are present when a cell is damaged.

We’ve talked about many of these signals before:
-Loss of intracellular K+ due to loss of cell membrane integrity
-High concentrations of free radicals
-Extracellular ATP (leakage from cells due to loss of membrane integrity)
-Unfolded proteins (suggested by some studies)

Can also be crystals that shouldn’t be there:
-Cholesterol crystals (cellular damage or oxidized LDL
-Uric Acid crystals (gout)

Question 21

DAMP detection:

Answer 21

Somehow, one NOD-like receptors seems capable of detecting a wide range of DAMPs as well as PAMPs:
-Known as NLRP3, though other NLRs may be able to do this
-How NLRP3 detects so many diverse signals is unclear and an area of intense research

Question 22

When a DAMP activates NLRP3, it associates with caspase 1 and activates it by forming a large molecular complex known as _________

Answer 22

inflammasome

-Caspase 1 activation has two major effects:
-Activation of pro-IL-1-beta to IL-1-beta
-Insertion of a cell membrane pore-gasdermin-into the cell membrane

Question 23

Inflammasome:

Answer 23

molecular assembly that activates caspase 1 =>
-Conversion of pro-IL-1 to IL-1
-Activation of a cellular pore known as gasdermin (IL-1 tends to leave the cell via gasdermin pores)

If enough gasdermin is produced, then the cell (usually a macrophage) will lyse and die => release of more DAMPs:
-This can lead to activation of neighboring cells
-This type of cell death is known as pyroptosis

Question 24

IL-1-beta is one of the most important pro-inflammatory cytokines and has a wide range of effects:

Answer 24

Released in very large quantities (for a cytokine) by macrophages in response to significant infection or tissue damage.

Question 25

IL-1 is generated and released in a two-step process:

Answer 25

Step 1: (priming): detection of PAMPs or DAMPs => synthesis and storage of large quantities of pro-IL-1

Step 2: (release): continued or larger DAMP or PAMP stimuli => NLRP3 activation => caspase 1 activation => conversion of pro-IL-1 to IL-1 => IL-1 release

Question 26

The inflammasome: priming and release of IL-1beta FYI:

Answer 26

IL-1beta can be its own priming signal:
-activation of the IL-1beta receptor can increase pro-IL-1beta production

Question 27

What cells are important DAMP and PAMP detectors?

Answer 27

Resident macrophages:
In someone without disease or infection, circulating monocytes often enter a wide range of tissues and differentiate to macrophages to serve as a “cellular sentinel”
-Langerhans cells: dermis & epidermis
-Kupffer cell: liver
-Alveolar macrophages: lung
-Microglia: brain
-Resident macrophages also found in all connective tissue
-Spleen (and other accessory lymphoid organs)

These cells express most PAMP and DAMP receptors and can secrete large quantities of pro-inflammatory cytokines (often due to NF-Kbeta activation)

Question 28

Endothelial cells:

Answer 28

blood and lymph vessels
-These lining cells express TLR’s and RLRs: when they detect PAMP they:
-Increase expression of ICAMs and selectins
-Increase the production of pro-inflammatory cytokines and chemokines

Question 29

Epithelial cells:

Answer 29

Skin and mucosal surfaces
-PRRs tend to result in increased production of local anti-microbial peptides
-Chemokines and cytokines can also be produced if more leukocytes need to be recruited from the circulation

Question 30

The major pro-inflammatory cytokines:

Answer 30

Question 31

IL-1 important cellular sources:

Answer 31

-Macrophages/monocytes
-Dendritic cells
-Keratinocytes
-Epithelial cells
-Endothelial cells

Question 32

TNF-alpha important cellular sources:

Answer 32

-Macrophages monocytes
-Dendritic cells
-Mast cells
-NK cells
-Epithelial cells

Question 33

IL-6 important cellular sources:

Answer 33

-Macrophages/monocytes
-Dendritic cells
-NK cells
-Epithelial cells
-Endothelial cells

Question 34

These cytokines have redundant and pleiotropic effects:

Answer 34

Redundant: functions overlap between these cytokines

Pleiotropic: many effects for each cytokine

Question 35

Fever and acute inflammation:

Answer 35

Temp above 37.7 C:
-caused by changing the hypothalamic set point: your hypothalamus now “thinks” that your normal temperature is higher

Mechanisms if higher body temp:
-Peripheral vasoconstriction => blood flow away from the periphery, to the core => less heat loss
-Shivering
-Increased metabolic rate

Benefits of fever:
-Adaptive immune mechanism in general are more effective at higher temperatures

Question 36

The hypothalamic set-point is altered by increased levels of _______________________:

Answer 36

Pro-inflammatory cytokines:
-IL-1beta and THF-alpha can induce fever at low serum concentrations
-IL-6 induces fever at higher (10X) concentrations (less potent)

Pro-inflammatory cytokines cause elevation of prostaglandin E2 production by cells in the 3rd ventricle (FYI: area called the OVLT)
-PGE2 leads to signaling that changes the hypothalamic setpoint
-Why blockers of cyclooxygenase activity (Tylenol, ibuprofen) are effective anti-pyretics)

Question 37

Acute phase proteins:

Answer 37

Elevated levels of inflammatory cytokines-IL-6 in particular: cause the liver to increase the secretion of useful (from an acute inflammation perspective) proteins into the bloodstream.
-acute phase proteins

Major acute phase proteins include:
C-reactive protein (CRP):
-Opsonin that binds to phosphorylcholine: a component of bacterial cell walls
-It can also activate C1q, and thus trigger the classical complement cascade when it binds to phosphorylcholine
-CRP is a common lab measurement ordered to diagnose inflammatory disease

Ferritin:
-Binds to serum iron with high affinity: many microbes depend on iron for their metabolism, and ferritin sequesters it from these microbes

Hepcidin:
-Interferes with intestinal transport or iron into the bloodstream: this also sequesters iron from microbes

Mannose-binding lectin (MBL):
-We’ve seen this as the PRR that initiates the lectin complement cascade

Serum amyloid protein A (SAA): complicated molecule
-Modulates (usually increases) the activation of the inflammasome and TLRs
-Opsonizes some gram-negative bacteria

Question 38

Interferons and the antiviral response:
2 major groups:

Answer 38

Group 1: interferon-alpha (IFN-alpha) and interferon- beta (IFN-beta)
-Secreted by macrophages, dendritic cells, and cells that have detected viruses via PRRs
-Role: to “interfer” with viral replication in a wide variety of cells
-Receptors for these group 1 IFN’s found on most cells

Group 2: Interferon-gamma (IFN-gamma)
-Secreted by Th cells and NK cells
-Role: activates macrophages and “pushes” the adaptive immune system to a cell-mediated response

Question 39

_________ interferons act in an autocrine and paracrine fashion to provide a rapid, innate protection to viral infection.

Answer 39

Type I interferons:
-If Typ-1 interferons and the NK response is inadequate, then we rely on the adaptive immune response

Question 40

How do interferons “interfere” with viral replication inside cells?

Answer 40

Inhibition of protein translation in the presence of viral RNA
-FYI: mediated by protein kinase R (PKR)

Degrading viral mRNA

Inhibition of viral protein assembly
-FYI: mediated by Mx proteins

The net result is that type 1 IFNs reduce the ability of infected cells and the virus to synthesize and assemble proteins.

Question 41

The natural killer (NK) cells:

Answer 41

A specialized cell that is derived from the lymphoid lineage but has a relatively non-specific way of detecting cancer cells or virally infected cells.
-No highly specific, unique TCR or BCR

Large lymphocytes that are activated by Type 1 IFNs and IL-12:
-Kill cells that are infected by viruses
-Secreted cytokines that predispose the adaptive immune system to adopt a response that relies on cellular effectors rather than antibodies

Question 42

NK cells survey the body for ________

Answer 42

Infected or “stressed”, abnormal cells

Question 43

NK cells have ________ and _________ receptors

Answer 43

“activating” and “inhibitory” receptors.

Question 44

NK activating receptors (NKARs):

Answer 44

detect molecules expressed on the membrane of cells that are infected by viruses or have developed into cells that may be malignant.

Question 45

NK inhibitory receptors (NKIRs):

Answer 45

detect molecules that are typically expressed by “normal” cells

Question 46

NKARs examples:

Answer 46

NKG2D: an NK receptor that detects “strange” MHC-I molecules on cells.
-These abnormal MHC-I proteins cannot present intracellular antigens and are more typically expressed by virally infected cells or cells with damaged genomes (ex: cancer cells)

Question 47

NKIRs examples:

Answer 47

KIR: an NK receptor that detects normal MHC-I molecules on a cell membrane.
-These MHC-I molecules can present antigens

Question 48

Before NK cells can be activated, they need to be licensed to make sure their NKIRs are functional:

Answer 48

-Likely done by dendritic cells: protects the body from indiscriminate NK-killing and tissue damage

Question 49

Once NK cells are activated and licensed, they travel to areas of damage/inflammation and “survey” the cells:

Answer 49

-NKIRs “over-rule” NKARs: if a cell expresses mostly normal MHC-I proteins, for example, then the NK cell does not kill it.

If there are many abnormal MHC-I proteins, then the NKARs “over-rule” the NKIRs, and the NK cell kills the abnormal cell.

Question 50

When NKARs are activated, it kills the cell by:

Answer 50

Receptor-mediated apoptosis via Fas-Fas ligand interaction

Secretion of the protein’s perforin and granzyme:
-Perforin pokes holes in the membrane
-Granzyme directly activates the BH3-only apoptotic protein Bid as well as executioner caspase 3