Immunology Basics Flashcards
What are pathogen-associated molecular patterns (PAMPs)?
characteristic structural molecules pf invading microbes which are recognized by the innate immune system
What are damage-associated molecular patterns (DAMPs) or alarmins?
characteristic molecules released by damaged cells which are recognized by the innate immune system
- can be released when cells die (intracellular)
- can be caused when connective tissue is damaged (extracellular)
- can be released by stimulated sentinel cells
What is the memory of the innate immune system?
minimal
each infection is treated similarly no matter how many times an invader is encountered
Which are cytokines are considered the most important for initiating and mediating innate responses and inflammation?
IL-1
IL-6
IL-8
TNF-a (most potent inducer of inflammation)
What are the components of cellular innate immunity?
Sentinel cells
- Macrophages
- Mast cells
- Dendritic cells
- Neutrophils (not always considered one)
Non-specific leukocytes
- Macrophages
- Neutrophils
- Lymphoid cells (NK)
- Basophils
- Eosinophils
What are the components of the humoral innate immune response?
Complement system
Natural antibodies
Acute-phase proteins
Antimicrobial peptides
Soluble lectins
What is innate immunity?
early, rapid response to block invasion and minimize tissue damage
- primarily within minutes to hours of exposure
generic response to common structures with no memory
sentinel cells detect then recruit other cells
- helps direct later adaptive immune response
main process by which invading microbes are destroyed
What is the potency of the innate immune system?
It can be overwhelmed (whereas the adaptive is rarely overwhelmed)
What do PAMPs and DAMPs bind to?
Preformed pattern recognition receptors (PRRs)
- mostly on cell membranes, cytosol, or in cytoplasmic vesicles
- some are soluble and in blood
What are the PAMPs associated with gram positive bacteria?
peptidoglycans and lipoteichoic acid in their walls
(CD14, PGRPs, NOD1)
What are the PAMPs associated with gram negative bacteria?
lipopolysaccharides and peptidoglycans in their walls
(CD14, TLR4)
What are the PAMPs associated with acid-fast bacteria?
the glycolipids covering them
(CD1, TLR4, NOD)
What are the PAMPs associated with yeast?
mannan or b-glucan in their walls
What happens when PRRs are activated
Cytokines are released –> recruits more WBCs, activates adaptive immunity
Changes to blood flow –> more emigration of WBCs –> kill invader
Activate anti-microbial molecules –> kill invader
What are the soluble PRRs?
Collectins
Ficolins
Complement
Pentraxins
What are the PRRs found within vesicles (endosome)?
TLR 3, 7, 8, 9
What are the cytoplasmic PRRs?
Rig-1
NOD-like
Peptidoglycan receptors
DNA receptors
What are the membrane-bound PRRs?
TLR 1, 2, 4, 5, 6
Lectins
Mannose receptor
Langerin
Dectins
Integrins
Scavenger receptors
What are Toll-like receptors?
Most significant family of PRRs
Mammals have 10-12 of them
Can be on neutrophils, macrophages, mast cells, dendritic cells, epithelial cells, and T/B cells and, as a rule, are present:
- cell membranes for bacteria/fungus/parasites
- inside the cells for viruses
Also found in bone marrow
What type of receptor is a Toll-like receptor?
A transmembrane glycoprotein
- most are homodimers but can form heterodimers
- can form chain pairs so can bind almost all known PAMPs
What happens when a bacterial PAMP binds to a TLR?
Activate MyD88 –> activate NF-kB and IRF3 –> gene activation
*in all except TLR3 which does the TRIF pathway
What activates the inactive precursors to cytokines?
Caspase-1 (triggered by the inflammasome)
Where is TLR 1 and what does it do?
Location: cell surface
Ligand: lipoprotein
Pathogen recognized: bacteria (all kinds)
Where is TLR 2 and what does it do?
Location: cell surface
Ligand: lipoprotein
Pathogen recognized: bacteria (esp G+), viruses, and parasites
* ex for Demodex
* very important!
Where is TLR 3 and what does it do?
Location: intracellular
Ligand: double-stranded RNA (also some ssRNA and dsRNA viruses)
Pathogen recognized: viruses (ex. Reoviridae)
Where is TLR 4 and what does it do?
Location: cell surface
Ligand: lipopolysaccharides of G- bacteria
Pathogen recognized: bacteria (G-), viruses
- very important!
- binding to TLR4 in the bone marrow stimulates production of WBC
Where is TLR 5 and what does it do?
Location: cell surface
Ligand: flagellin of bacteria
Pathogen recognized: bacteria
Where is TLR 6 and what does it do?
Location: cell surface
Ligand: lipoprotein
Pathogen recognized: bacteria and viruses
Where is TLR 7 and what does it do?
Location: intracellular
Ligand: single-stranded RNA, guanosine
Pathogen recognized: viruses (ex. Caliciviridae, Flaviviridae, Arteriviridae and Coronaviridae) and bacteria
*targeted by imiquimod
Where is TLR 8 and what does it do?
Location: intracellular
Ligand: single-stranded RNA
Pathogen recognized: viruses (ex. Caliciviridae, Hepeviridae, Flaviviridae, Arteriviridae and Coronaviridae) and bacteria
Where is TLR 9 and what does it do?
Location: intracellular
Ligand: double-stranded DNA, CpG DNA (from bacteria and protozoa)
Pathogen recognized: viruses (ex. Papillomaviridae, Polyomaviridae, Adenoviridae, and Herpesviridae), bacteria, and protozoa
Where is TLR 10 and what does it do?
Location: intracellular
Ligand: regulates TLR 2 responses
Pathogen recognized: suppressed inflammation
*found in humans, cattle, and sheep but not mice
Which TLR is considered most important for recognition of gram positive bacteria?
TLR 2
Which TLR is considered most important for recognition of gram negative bacteria?
TLR 4
needs to be linked by MD-2, LPD, and CD14 first
Which TLRs are considered most important for recognition of acid fast bacteria?
TLR 2 and TLR 4
and TLR1/TLR6 that heterodimerise with TLR2
Which Toll-like receptor is considered most important for recognition of fungi?
TLR 2, TLR 4, and TLR 9
Which TLRs are found on the plasma membrane?
TLR 1, 2, 4, 5, and 6
Which TLRs are found on the endosome?
TLR 3, 7, 8, and 9
Which TLR is targeted by imiquimod?
TLR 7
Which TLR is important for Demodex?
TLR2 gene is upregulated (classically the one for remembering them) and might be responsible for Demodex-induced clinical manifestations, while TLR4 and TLR6 gene down-regulations could be the paramount strategy of Demodex mites to elude the host-immune interface
Which TLRs are considered most important for the recognition of single-stranded RNA viruses?
TLR 7 and TLR 8
Which TLR is considered most important for the recognition of double-stranded RNA viruses?
TLR 3
Which TLR is considered most important for the recognition of double-stranded DNA viruses?
TLR 9
Which TLR is considered most important for the recognition of parasites (not protozoa)?
TLR2
TLR 4 may help control growth
Which TLRs are considered most important for the recognition of protozoa?
TLR 9, 11, 12
* TLR 2 and TLR 4 may be involved for cutaneous leishmaniasis
The C-type lectin receptor mannose receptor might help
What are the three major cytokines does NF-kB activate the genes for?
IL-1
IL-6
TNF-a
That is the primary thing that IRF3 activates the gene for?
Type 1 interferons (IFN-b)
What are RIG-like receptors?
Another family of PRRs
Within cells
Detect double-stranded RNA viruses
What are NOD-like receptors?
A family of PRRs that recognize intracellular PAMPs
activates NF-κB pathway → triggers production of proinflammatory cytokines
What does NOD-1 bind?
bacterial peptidoglycans
What does NOD-2 bind?
muramyl dipeptide
is a general sensor of intracellular bacteria
triggers production of pro-inflammatory cytokines and defensin
What does NOD-3 bind?
Viruses and inorganic particles
Which TLR recognizes bacterial DNA?
TLR9 because it recognizes CpG DNA
Which DAMP binds TLR2 and TLR4 to sustain and prolong inflammation?
HMGB1
normally helps fold DNA
released by macrophages and damaged cells
What does the release or administration of HMGB1 do?
sustain/prolong inflammation
trigger septic shock (via TNF-a and IL-8)
stimulates angiogenesis and tissue repair
antimicrobial activity
Which cytokine is stored within cells and is a potent DAMP?
IL-33
What is apoptosis?
“normal” way that unwanted cells are eliminated
- extrinsic pathway = external molecules cause a cascade of caspases that cause mitochondrial permeability
- intrinsic pathway = intracellular events cause release of cytochrome c and activation of caspase 9
What is pyroptosis?
cell death initiated by response to infections or irritants that result in the production of an inflammasome
mediated by caspase 1 and 11
causes release of IL-1 and IL-8 = more inflammation
What is necroptosis?
triggered by death receptors like TLR-signaling
results in release of DAMPs like HMGB1 and IL-33
What are the common extracellular DAMPs?
Heparan sulfate
Hyaluronic acid
Fibrinogen
Collagen-derived peptides
Laminin
Elastin
*all things that form the extracellular matrix of the dermis
What are the common intracellular DAMPs?
HMGB1
Anti-microbial peptides (cathelicidins, defensins)
Uric acid
Chromatin
Heat shock proteins
Adenosine
S100 proteins
Lactoferrin
What TLR does heparan sulfate bind to?
TLR 4
What are soluble pattern recognition receptors?
Function in the extracellular fluid to promote phagocytosis of things that don’t typically trigger cytokines
- include carbohydrate-binding lectins
What are P-type lectins?
Pentraxins
- C-reactive protein
- Serum amyloid P
bind to the bacterial LPS in a Ca2+ dependent way
activate the classical complement cascade (C1q)
interact with cells of the innate immune system to boost effect
What are C-type lectins?
Need Ca2+ to bind to carbohydrates
found on macrophages, mast cells, and dendritic cells
Includes:
- dectins (important for Malassezia)
- mannose-binding lectin: opsonize bacteria and activate complement
- selectins: on endothelial cells and play a role in migration of WBCs
How does pain contribute to innate immunity?
Pain causes sensory nerves to release bioactive peptides
What are the 3 major cytokines released by sentinel cells when PRRs are activated?
TNF-a (produced first)
IL-1 (produced second, in waves)
IL-6 (produced last)
What cells produce TNF-a?
*Sentinel cells of the innate immune system
Endothelial cells
Lymphocytes (T-cells and B-cells)
Fibroblasts
Keratinocytes
What are the two primary pathways in the innate immune response that lead to the production of TNF-a?
Activation of TLRs
Activation from molecules secreted from nerves (ex. neurokinin-1)
How does TNF-a promote inflammation?
*Promotes migration of leukocytes into tissue
- Triggers changes in small blood vessels (vasodilation/leakage)
- Activates endothelial adhesion molecules
- Triggers production of cytokines and chemokines (esp. IL-1, 6, 8)
Induces acute phase proteins
Promotes granuloma formation
- Activates procoagulants
Later facilitates transition from innate to adaptive immunity
Other than production of cytokines and chemokines to recruit additional cells, how does TNF-a amplify and prolong inflammation?
promotes macrophages synthesis of nitric oxide and cyclooxygenase
activates mast cells
What cells does TNF-a activate?
Mast cells
Vascular endothelium
Macrophages
Lymphocytes
Neutrophils
Fibroblasts
What are the effects of TNF-a on collagen synthesis and bone resorption?
Enhances them
What are some of the toxic effects of TNF-a?
Kills tumor cells
Septic chock (via TNFR1 and TNFR2)
Sickness behavior
Altered lipid metabolism
What cells produce IL-1?
Macrophages and many others
*very stimulated by CD14 & TLR4
How does IL-1 promote inflammation?
*Promotes migration of leukocytes into tissue
Stimulates macrophages to make NO2 and COX-2
Enhances Th2 cytokine production
Promotes eosinophil and basophil degranulation
Acts on muscles to mobilize amino acids (pain and fatigue)
Promotes production of acute phase proteins by the liver
Promotes collagen, chondrocyte, fibroblast, and keratinocyte activity
How does IL-1 affect the nervous system?
Induces fever
Sickness behavior (lethargy, decreased appetite, etc)
Which member of the IL-1 family is the most active pyrogenic cytokine?
IL-1b
(IL-1a remains bound to the cell surface of macrophages)
How does IL-1 affect blood flow?
triggers changes in small blood vessels
IFN-y synthesis
Integrin expression
What are the most important IL-1 receptors?
CD121a (signaling receptor)
- can be bound by IL-1RA which blocks effect
CD121b (antagonist)
What cells produce IL-6?
Macrophages
Lymphocytes (mostly T)
Mast cells
also dendritic cells, basophils, eosinophils, fibroblasts, keratinocytes, myocytes
What triggers cells to produce IL-6?
Bacterial
IL-1
TNF-a
How does IL-6 contribute to inflammation?
- Stimulate hepatocytes to produce acute-phase proteins
Affects T cells- stimulates Th2 response
- activates Th17
- suppressed Treg cells
- enhances cytotoxicity
Activates B cells and enhances IgA production
May promote change from early neutrophil to later macrophage response
How does IL-6 have an anti-inflammatory effect?
Can inhibit some actions of TNF-a and IL-1
Promotes production of IL-1RA
Promotes production of IL-10
What are chemokines?
Family of 50+ small proteins that coordinate migration of leukocytes
4 classes based on structure but most are CXCL or CCL based on whether or not the two cysteines are next to each other
Which chemokines attract and activate neutrophils?
*CXCL8 (aka IL-8)
also attracted by CXCL2 (aka MIP-2) which is made by macrophages
Which cells do CCL4 attract?
CD4+ T cells
Which cells do CCL2 attract?
Monocytes
also activates them by stimulating respiratory burst and lysosomal enzyme release
Which chemokines are structurally similar to defensins and therefore have antimicrobial properties?
CXCL4
CCL20
CCL5
Which genotype in cattle is impaired neutrophil migration associated with?
A specific CXCR2 genotype
This is a receptor for CXCL-8 (also CXCL1 and 7)
What are the 5 cardinal signs of inflammation?
Heat
Redness
Swelling
Pain
Loss of function
What are the 3 main changes that occur in small blood vessels in the area of infection, leading to inflammation?
technically first capillaries constrict so that leukocytes can bind vessel wall
1) arterioles dilate = more blood flow
2) capillaries become more permeable = transudate (immediate)
3) leukocytes migrate through venule walls = exudate (several hours later)
lymph will accumulate due to this and take contents to regional nodes
What are the vasoactive amines?
Histamine
Serotonin
How does histamine contribute to inflammation?
- released from mast cells
- has receptors on many cells
- stimulates endothelial cells to produce NO2
–> dilates vessels and causes leakage - increases TLRs on sentinel cells
How does serotonin contribute to inflammation?
vasoconstriction to increase blood pressure
can increase vascular permeability in rodents (but not others)
What are the vasoactive peptides?
Kinins (esp. bradykinin)
includes substance-P and neurokinin
How do kinins contribute to inflammation?
Increase vascular permeability
Also trigger pain receptors and stimulate neutrophils
May have antimicrobial properties
What are the vasoactive lipids?
Aka eicosanoids
-Leukotrienes
- Prostaglandin
- platelet-activating factor (PAF)
How do eicosanoids contribute to inflammation?
Act as locally active hormones
Have many diverse biological effects, most are pro-inflammatory
How are leukotrienes formed in response to inflammation?
Inflammasome activates phospholipases (PLs) –>
PLA2 breaks down phospholipids from cell wall => arachidonic acid
5-lipoxygenase (5-LOX) breaks down arachidonic acid => leukotrienes
What are the main ways that leukotriene contributes to inflammation?
LTB4: attracts/activates neutrophils and eosinophils
LTC4, D4, and E4: increase vascular permeability and smooth muscle contraction
IL-13 upregulates LTD4 which then upregulates IL-13 in a pro-inflammatory feedback loop
How do neutrophils affect the arachidonic acid pathway?
Neutrophils contain 15-lipoxygenase (15-LOX)
This breaks down arachidonic acid => lipoxins
Lipoxins are anti-inflammatory
- inhibit neutrophil activity
- inhibit 5-lipoxygenase = less leukotrienes
How are prostaglandins formed in response to inflammation?
Inflammasome activates phospholipases (PLs) –>
PLA2 breaks down phospholipids from cell wall => arachidonic acid
cyclooxygenase 1 and 2 (COX-1/2) breaks down arachidonic acid => prostaglandins
What are the main ways that prostaglandins contribute to inflammation?
Activity of the 4 main groups caries and net effect on inflammation is complex
What are the 4 main groups of prostaglandins?
PGE2
PGF2
Thromboxans (TxA2, PGA2) - come from platelets
Prostacyclins (PGI2) - comes from vascular endothelial cells
What is platelet-activating factor (PAF)?
phospholipid produced by neutrophils, eosinophils, platelets, and mast cells
makes endothelial cells stickier
- enhances neutrophil migration
makes platelets and neutrophils release vasoactive molecules
What are the primary antimicrobial molecules involved in killing of microbial invaders?
Antimicrobial peptides
Lysozyme
Complement
What are antimicrobial peptides?
Defensins
Cathelicidins
C-type lectin
S100 family (ex. calprotectin)
Lactoferrin
Serprocidins (serine proteases found in neutrophils)
Granulysins (produced by C8+ T-cells and NK cells)
Bacterial permeability-increasing protein (in neutrophils)
What are the ways that antimicrobial peptides affect microbial invaders?
Direct killing
1) insert themselves into lipid membranes –> form pores –> death
2) cover the membrane to cause disruption –> death
*all can kill or inactivate some bacterial, fungi, and enveloped viruses
- canine cathelicidin K9CATH has broad spectrum against G+ and G-
Some can neutralize microbial toxins
- esp defensins
Some sequester important metals needed for bacterial growth
Other than killing of microbial invaders, how can antimicrobial peptides affect inflammation?
can also serve as immune-modulators and regulate cytokine production
some attract monocytes, immature dendritic cells, and T cells
What cells produce antimicrobial peptides?
Neutrophils
Macrophages
Epithelial cells (in response to IL-1, IL-17, and IL-22)
Lymph nodes
How does lactoferrin kill bacteria?
Binds iron, depriving bacteria of this essential nutrient
Increases the permeability of bacterial cell membranes
- via cation-binding region
- binding to bacterial lipopolysaccharide on G-
- but may be more effective on G+ according to some sources
prevents bacteria from adhering to and invading epithelial cells
releasing lactoferricin
What cytokines to cathelicidins stimulate the production of?
IL-6
IL-8
IL-10
How does calprotectin work against bacteria?
sequesters Zn and Mn
What is lysozyme?
An enzyme that destroys peptidoglycans in G+ cell walls
found in all body fluids except CSF and urine
found in neutrophils (except in cattle)
can opsonize bacteria
What are the functions of the complement system?
- opsonization and lysis of microbial invaders
- alerts immune system about invaders
- regulate inflammation (cytokines and chemokines)
- removes abnormal cells
- regulates adaptive immune response
- clears antibody-antigen complexes
- influences angiogenesis, stem cells, tissue regeneration, lipid metabolism
What are positive acute-phase proteins?
Made in the liver in response to IL-1, IL-6, TNF-a
Increase in inflammation
- C-reactive protein
- mannose-binding protein
- complement factors
- ferritin
- ceruloplasmin
- serum amyloid A
- haptoglobin
- coagulation factors
- serpin
What are negative acute-phase proteins?
Decrease in inflammation
- albumin
- transferrin
- transthyretin
- retinol-binding protein
- antithrombin
- transcortin
What is the complement system?
A network of 30+ proteins, proteases, receptors, and regulators
Elaborate antimicrobial defense system
Main effector pathway of the innate humoral response
Where are complement factors synthesized?
Mainly in the liver
secondary source is macrophages
small amounts made in adipose tissue, granulocytes, intestine
What are the 3 ways that complement activation can occur?
1) classical pathway (most recent, needs adaptive response)
2) alternative pathway (most ancient)
3) lectin pathway
How is the alternative complement pathway triggered?
C3 spontaneously breaks down into C3a and C3b
microbial cell wall meets then meets C3b in the blood
* 80-90% of all complement activation
Why does C3b remain bound to bacterial cells walls?
They lack sialic acid (in mammalian cells) so FH can’t bind and FB binds instead
- primarily bound to lipopolysaccharides
What type of binding occurs with the main complement factors?
covalent/irreversible
How does the product of the alternative pathway C3bBbP affect the cascade?
Creates a positive feedback loop so more C3b is produced to cover the surface of the invader
*is the alternative C3 convertase
How is the lectin complement pathway triggered?
binding of a soluble pattern recognition molecule (lectin) to microbial carbohydrates (PAMPs)
–> activates C4 by cleaving it into C4b
—> forms a protease C4b2b (classic C3 converatse)
–> increases the breakdown of C3
- includes mannose-binding lectin and ficolins
How are the lectin and classical pathways similar?
Triggered slightly differently but mannose-binding lectin is structurally similar to C1q so after that they become the same
How is the classical complement pathway triggered?
C1q meets a antibody-antigen complex (so can take 7-10 days to activate)
–> activates C4 by cleaving it into C4b
—> forms a protease C4b2b (classic C3 converatse)
–> increases the breakdown of C3
Where is C3 convertase supposed to be located?
on the surface of the microbial organism
What happens when the classical, alternative, and lectin pathways reach the common amplification pathway?
C5 binds to C3b forming C3b5b and releasing 5a
goes on to form membrane attack complexes (using C5,6, 7, 8, and 9) which insert a hole into the intruder and cause lysis
What does C5a do?
attracts and activates macrophages
anaphylotoxin (mast cell degranulation)
neutrophil chemotaxis
lysosomal enzyme secretion
increased vascular permeability
smooth muscle contraction
T-cell development
enhance production of TNF-a, IL1b, IL-6
interacts with TLR4 and 9
What anaphylotoxins are produced in the complement cascade?
C5a and C3a
How does complement contribute to the removal of apoptotic cells?
Apoptotic cells lose complement inhibitors so are affected
Which parts of the complement cascade are primarily responsible for WBC chemotaxis?
C5a and C5b67
Which parts of the complement cascade are primarily responsible for opsonization?
C3b and C4b
- many bacteria have evolved mechanisms to neutralize this
How is the complement system regulated?
Cells express receptors to C3 or its fragments
C1 inhibitor
C4-binding protein
FH and FI (binds and inactivates C3b)
What happens when there is a deficiency in complement receptor 1?
results in too many circulating immune complexes
- dogs will develop immune complex-mediated nephritis
What happens when there is a deficiency in complement receptor 3?
individuals are prone to infections
What is canine C3 deficiency?
Seen in Brittany spaniels
AR
leads to infections, amyloidosis, and immune complex-mediated nephritis
How is skin a defensive organ?
Physical barrier
- desquamation, desiccation, and low pH (bc of fatty acids in sebum)
Resident microbiota excludes pathogenic bacteria & fungi
- disruption of skin microbiota -> risk of microbial invasion
- skin infections tend to occur in areas where pH & humidity are high
(ex: axilla & inguinal)
Hair prevents desiccation, may protect against some fungal infection
Each layer of skin has its own defensive mechanisms
- Keratinocytes are active
- main source of cathelicidins & β-defensins)
- express MHC II, act as APC
- Resident sentinel cells (mast cells, macrophages)
- Resident Langerhans cells
- Resident T cells (all 3 Th subsets)
- B cells circulate the skin
What are scavenger receptors?
large family of cell-surface receptors
diverse in their structure and biological function
bind to a range of ligands
enhance the elimination of altered-self or non-self targets
What does CD14 do?
recognizes LPS on GN bacteria, peptidoglycans on S. aureus, mannuronic acid polymers on Pseudomonas, and lipoarabinomannans on mycobacteria
What are peptidoglycan recognition proteins (PGRPs)?
peptidoglycans, LPS, & lipoteichoic acids on bacteria
What are the clinical features of immune-mediated vasculitis?
Cutaneous lesions: Purpura (petechiae, ecchymoses), ulcerations, and necrosis, often affecting the extremities (e.g., ear tips, tail, footpads)
What is the most common cause of immune-mediated vasculitis in horses?
Seen frequently in purpura hemorrhagica secondary to infections like Streptococcus equi (strangles), where immune complexes deposit in vessel walls and lead to neutrophilic vasculitis and hemorrhage
What is the most common causes of immune-mediated vasculitis in dogs?
Vasculitis is commonly associated with drug reactions
infectious diseases (e.g., Ehrlichia, Rickettsia)
autoimmune diseases
What is the most common causes of immune-mediated vasculitis in cats?
vasculitis is less commonly recognized compared to dogs and horses
It may occur secondary to feline infectious peritonitis (FIP), systemic lupus, or drug hypersensitivity
What is the pathogenesis of immune-mediated vasculitis?
Immune complex-mediated vasculitis occurs when immune complexes (antigen-antibody complexes) deposit in blood vessels, activating the complement system (primarily via the classical pathway)
Results in the production of C3a, C5a, and other anaphylatoxins, which attract neutrophils and cause endothelial damage
How is SLE related to problems with complement?
autoantibodies form immune complexes activating complement
leads to type III hypersensitivity reactions
complement components (C3b, C4) for immune complex clearance but also contributing to tissue inflammation when overwhelmed
may have decreased serum C3 and C4 levels due to consumption
in humans deficient in C1q, C2, and C4 are associated with SLE
What are the two primary phagocytic cells of the innate immune system?
Neutrophils (just “professional killers”)
Macrophages (“professional killers” and antigen presenting cells)
What are the steps that a hematopoietic stem cell goes through to become a red blood cell?
hematopoietic stem cell –>
myeloid progenitor –>
megakaryocyte/erythroid progenitor –>
red blood cell
What are the steps that a hematopoietic stem cell goes through to become a platelet?
hematopoietic stem cell –>
myeloid progenitor –>
megakaryocyte/erythroid progenitor –>
megakaryocyte –>
platelet
What are the steps that a hematopoietic stem cell goes through to become an eosinophil?
hematopoietic stem cell –>
myeloid progenitor –>
eosinophil/mast cell progenitor –>
eosinophil
What are the steps that a hematopoietic stem cell goes through to become a basophil?
hematopoietic stem cell –>
myeloid progenitor –>
eosinophil/mast cell progenitor –>
basophil
What are the steps that a hematopoietic stem cell goes through to become a mast cell?
hematopoietic stem cell –>
myeloid progenitor –>
eosinophil/mast cell progenitor –>
mast cell
What are the steps that a hematopoietic stem cell goes through to become a a neutrophil?
hematopoietic stem cell –>
myeloid progenitor –>
neutrophil/macrophage progenitor –>
neutrophil
What are the steps that a hematopoietic stem cell goes through to become a a macrophage?
hematopoietic stem cell –>
myeloid progenitor –>
neutrophil/macrophage progenitor –>
monocyte (circulating) –>
macrophage (in tissue)
What is the most common immune cell in mammals?
neutrophils (~2/3 of hematopoietic activity is dedicated to making them)
60-75% of leukocytes in carnivores
50% of leukocytes in horses
20-30% of leukocytes in cattle, sheep and rodents
What is the life of a neutrophil like?
- made in the bone marrow (regulated by G-CSF)
- migrate to the blood stream
- 12 hours later migrate into tissue (1/2 hour if inflammation and chemotaxis)
- live for ~5 days
- after they die they are eaten by macrophages
What do macrophages release when they consume dead neutrophils?
IL-23
promotes IL-17 production
stimulates G-CSF = most
Where are neutrophils sequestered and what happens to this population when inflammation occurs?
capillaries of the liver, spleen, lungs, and bone marrow
is released so circulating neutrophils increase 10x
blood neutrophils only account for 1-2% of total volume
What is the structure of a neutrophil?
10-20 um in diameter (RBCs are ~5 um)
Have a sausage-like or segmented nucleus
Have 3 types of granules (do not stain on H&E)
1) primary (azurophil)
2) secondary (specific)
3) tertiary
Have a broad range of PRRs (are major mediators of innate immunity)
Why is it important that neutrophils are short-lived?
They are voracious phagocytes that create chemicals that not only damage invaders but nearby tissue as well
What do primary neutrophil granules contain?
Myeloperoxidase
lysozyme
elastase
b-glucuronidase
cathepsin
What do secondary neutrophil granules contain?
lysozyme
collagenase
lactoferrin
What do tertiary neutrophil granules contain?
gelatinase
What happens to endothelial cells in response to inflammation that allows for neutrophils to emigrate into the tissue?
- blood vessels have a large surface area so can sense invasion
- PAMPs (LPS) and DAMPs (histamine and PAF) reach endothelial cells
- in response, endothelial cells express P-selectin (a glycoprotein)
- P-selectin will bind to L-selectin on passing neutrophils
- this interaction is weak and just slows the neutrophils down (“rolling”)
- neutrophils will shed their L-selectin
- in response to IL-1 and TNF-a, endothelial cells will also express E-selectin
–> happens after several hours
–> is more adhesive
What happens to neutrophils in response to PAF, chemokines, and leukotrienes from endothelial cells that help rolling neutrophils adhere to blood vessel walls?
- Neutrophils express LFA-1 (aka CD18)
- LFA-1 can bind to ICAM-1 on endothelial cells
- this is a strong bond so now neutrophils are adhered to the cell wall
What molecules are the primary mediators for leukocyte rolling?
Selectins
- P-selectin (endothelial cells, later also E-selecin)
- L-selectin (leukocytes)
What molecules are the primary mediators for leukocyte adhesion?
Integrins
- ICAM-1 (endothelial cells, also VCAM-1 esp for lymphs and macs)
- LFA-1 (leukocytes)
What molecule is the primary mediator for leukocyte diapedesis?
PECAM-1 (on both)
- ~20% of neutrophils also release proteases to get through the basement membrane
Though neutrophil phagocytosis of bacteria is a continuous process, what 5 steps can it be divided into?
1) activation
2) chemotaxis
3) adherence and opsonization
4) ingestion or NETosis
5) destruction
What happens during neutrophil activation?
Need to be activated before they attack
Neutrophils bind to endothelial cells and are stimulated
Are stimulated by CXCL8 (IL-8), C5a, or f-met peptides (bacterial fragments)
Now neutrophil secrete:
- elastase (promotes adhesiveness)
- defensins
- oxidants (activate tissue proteases and TNF-a release from macrophages)
In addition to activating tissue proteases, what does oxidant release from neutrophils do?
Stimulate TNF-a release from macrophages
TNF-a attracts neutrophils which provides positive feedback amplification
What happens during neutrophil chemotaxis?
Chemoattractants come from sites of invasion and form a gradient which neutrophils climb up using lamellipodia on their leading edge
- formation of lamellipodia is driven by higher concentration of attractants
What are the primary chemoattractants for neutrophils?
Chemokines (ex. CXCL8 and CXCL2)
Chemotactic lipids (ex. leukotriene B4)
Complement anaphylatoxins (ex. C5a)
Bacterial formyl peptides (f-met peptides)
Cathelicidins
Fibrinopeptide B (derived from fibrinogen)
Hydrogen peroxide (tissue-damage gradient is established in 5 min)
What happens during neutrophil adherence and opsonization?
Once a neutrophil encounters bacteria it needs to “catch” it
Opsonization helps with this
- the negative electrostatic charge on bacteria repels them
- need to have bacteria coated by positive charged molecules
- opsonin = sauce, they make bacteria more attractive to neutrophils
Neutrophil PRRs can also bind to their ligands on bacteria
What are some examples of opsonins?
Mannose-binding lectin
fibronectin
complement components (ex. CD35 binding to C3b)
antibodies (most effective opsonin!)
What is type 1 phagocytosis?
Antibody receptor-mediated phagocytosis
Antibody-coated bacteria attaches to receptors on neutrophils
- ex. CD32 on the neutrophil binding to the Fc region of an antibody
- CD32 is also called FcyRII
What is NETosis?
Form of neutrophil cell death that results in release of the web-like neutrophil extracellular traps
- can trap or kill bacteria, viruses, fungi, and parasites
What causes the neutrophil extracellular trap to be released?
Not fully known, but neutrophils may sense the target is too large
CXCL8 or LPS activates them –> neutrophil oxidants release contents of azurophil granules –> chromatin condensation –> release strands of DNA (NETs)
What covers the strands of neutrophil DNA in NETosis?
Antimicrobial proteins
- histones
- granule components (elastase, myeloperoxidase, lactoferrin, etc)
What is a problem that can occur due to NETosis?
Unwanted tissue damage
What does the cytoplasm of lamellipods of neutrophils contain?
actin and myosin
What is type 1 neutrophil phagocytosis?
Antibody mediated phagocytosis
Antibody-coated microbes are bound by neutrophil CD32 –>
trigger polymerization of actin –>
lamellipods extend from the neutrophil and engulf the particle
What is type 2 neutrophil phagocytosis?
Complement-mediated phagocytosis
Particles sink into the neutrophil without lamellipod formation
Once engulfed, the bacteria is drawn into the cell
Then the bacteria is enclosed in a vacuole called a phagosome
- work better on bacteria with lipid/hydrophobic capsules (Mycobacterium)
What is type 3 neutrophil phagocytosis?
Coiling phagocytosis
A single lamellipod wraps itself around the organism several times
What is autophagy?
Cells will destroy particles within their cytoplasm
- form of cellular waste disposal
Microbe or damaged organelle is closed off into an autophagosome
Fuses with lysosome so the contents are digested
- TLR7 or FCyR can initiate targeting
Contents are released back into cytosol for recycling
Disorders are associated with cancer, neurodegeneration, infection, and aging
How do neutrophils kill ingested bacteria?
Respiratory burst or release of intracellular granules
What is a respiratory burst?
A way phagocytes kill ingested organisms
Happens within seconds
makes radical oxygen species which kill bacteria
How does neutrophil hypochlorous acid (HOCl) kill bacteria?
Created by the respiratory burst
unfolding and aggregating proteins
oxidizing lipids
enhance activities of lysosomal enzymes
What are radical oxygen species?
Superoxide, hydrogen peroxide, singlet oxygen, hypohalides, organic peroxides
How do radical oxygen species contribute to inflammation?
act on an atomic level to bind to sulfur atoms (cysteine and methionine)
inhibit many parts of cell function
oxidize bases in DNA (influence transcription)
activate inflammasomes
promote B and T cell activation
How does a neutrophil use lytic enzymes to kill bacteria?
Once a bacteria is ingested, the cell’s granules (lysosomes) migrate through the cytoplasm –> fuse with the maturing phagosome = phagolysosome –> enzymes are released and digest bacteria
- some bacteria like E. coli are resistant to this
When neutophil enzymes are released, they cleave TNF-a from macrophages
What cytokines do neutrophils produce?
Many but most important are
- IL-1
- TNF-a
- IL-6
- CXCL8
- IL-10
- TGF-b
Each neutrophil only makes a small amount, but together can be powerful)
What type of bacteria does lysozyme work best against?
G+
How does the respiratory burst happen in neutrophils?
NADPH oxidase (NOX) assembles on the cell surface due to stimuli like TNF-a
NADPH + O2 is broken down by NOX to NADP + H + 2*02
superoxide dismutase then creates hydrogen peroxide
myeloperoxidase takes hydrogen peroxide and chloride to make hypochlorite and hypochlorous acid (bleach)
How does the respiratory burst happen in macrophages?
nitric oxide synthase catalyzes production of nitric oxide which combines with superoxide or H2O2 to produce peroxynitrite radical
RNS inactivate iron and sulfur containing enzymes, oxidize lipids, and damage DNA
What cell surface receptors do neutrophils have?
Antibody receptors (CD32/FCyRII)
Cell adhesion molecules (LFA-1, CD11c/CD18)
Complement receptors (CD35, CD11b/CD18)
What is the fate of neutrophils?
short-lived cells with limited energy
die as a result of apoptosis
express an “eat me” signal to monocytes
if dendritic cells eat them, they do 1 of 2 things
- if neutrophil contains bacteria: secrete TGF-b, IL-6, IL-1, IL-23
–> IL-23 stimulates Th17 cells which call in more neutrophils
- if neutrophil is unaffected: secrete IL-10 and TGF-b to promote Tregs
What is the primary first-line phagocyte?
Neutrophils
Which phagocyte tends to be of greater importance for fighting extracellular pathogens?
Neutrophils
How does the function of macrophages compare to that of neutrophils?
Both are phagocytic cells and while macrophages have a slower response, they do more:
- greater antimicrobial properties (esp against intracellular pathogens)
- help initiate tissue repair/control inflammation and clean up debris
- process antigens and initiate adaptive immunity
Monocytes are found circulating in the blood, what are they called when they move to connective tissue?
Histiocytes
Monocytes are found circulating in the blood, what are they called when they move to bone?
Osteoclasts
Monocytes are found circulating in the blood, what are they called when they move to skin?
Macrophage
subset develop into dendritic cells (aka Langerhans cells in epidermis)
*some dendritic cells may come from the fetal yolk sac
Monocytes are found circulating in the blood, what are they called when they move to the sinusoids of the liver?
Kupffer cells
*may originate from the fetal yolk sac
Monocytes are found circulating in the blood, what are they called when they move to the brain?
Microglia
*may originate from the fetal yolk sac
What is the structure of a macrophage?
Round and ~15-20 um in diameter (a RBC is ~5 um)
Central nucleus (potato nucleus on histopathology)
Abundant cytoplasm with lysosomes
- may be foamy if activated
What is the life history of a macrophage?
Present in most tissues before birth (bone marrow or yolk sac)
Usually long-lived unless there is inflammation or tissue damage
- usually replace themselves at a rate of 1% per day
Reinforced by monocytes from the blood
- circulate for 3 days before entering tissue
Can form multinucleated giant cells if their DNA is damaged
What are the 3 stages of macrophage readiness?
1) resting
- go around cleaning up dead cells as a garbage collector
- very few MCH II receptors
2) activated/primed by cytokines (ex. IFN-y)
- can take larger bites
- increases number of MHC II receptors for antigen presentation
3) hyperactivated by PAMPs (ex. LPS)
- stops proliferating and grows larger
- can ingest things as large as unicellular parasites
- produce TNF-a
- increases lysozymes and respiratory burst
How do macrophages function as sentinel cells?
They are widely distributed in the body and have many PRRs
Produce many cytokines (IL-1, IL-6, TNF-a, IL-12, IL-18, and HMGB1)
Produce chemokines (ex CXCL8)
How to macrophages contribute to inflammation?
Recognize tissue damage
HMGB1 and DAMPs make macs release TNF-a, IL-6, CXCL8, and ROS
- bring more neutrophils in
Release exosomes (also done by dendritic cells and B cells)
- have immunostimulatory and proinflammatory molecules
- have bacterial particles
- spread through extracellular fluid
- bind to neutrophils and macrophages => release TNF-a and iNOS
Act as antigen presenting cells
What does myeloperoxidase do?
contribute to the respiratory burst
What do elastin, cathespin, and b-glucuronidase do?
Activate TFN-a
Degrade connective tissue
kill bacteria
What does gelatinase do?
Degrade bacteria and tissue
What attracts macrophages?
bacterial products (PAMPs)
complement
DAMPs
neutrophils (make CCL2 aka MCP-1 under the influence of IL-6)
How do macrophages phagocytize organisms?
Rely more on their respirator burst (produces NO2) and protein synthesis
Can have sustained phagocytic activity (unlike neutrophils)
Can have endosomes with ingested neutrophil granules
Can also make METs
When to macrophages arrive to the site of infection?
Several hours after neutrophils (~12 hr vs 3-4 hr)
How do macrophages “soften” connective tissue?
Produce proteases like collagenase, elastase, and a plasminogen activator that generates plasmin
What is macrophage polarization?
Divided into two subsets based on activation state and functions
- M1: promote host defense
- M2: suppress inflammation and promote tissue repair
Is not permanent and can change phenotype based on outside influence
Some consider a 3rd subset
- Mreg: have anti-inflammatory activity and due to IL-10
What makes M1 macrophages and what do they do?
Influenced primarily by presence of IFN-y, PAMPs, and DAMPs
Appear early in the inflammatory process
Generate large amounts of RNS
Are larger and more active with more bactericidal powers
Have increased MHC II expression
Produce TNF-a and IL-12 which activate NK cells which make more IFN-y
What makes M2 macrophages and what do they do?
Influenced primarily by IL-4, IL-10, and IL-13
Make ornithine from arginine instead of NO
Promote tissue repair/remodeling
Have reduced killing activity
Secrete SLP1 to calm down neutrophils and protect TGF-b
Macrophages produce IL-23. What is the primary function of this?
Stabilize Th17 cells
Macrophages produce IL-1. What is the primary function of this?
Very important for inflammation!
Co-stimulate Th2 cells and stimulates acute-phase response
Macrophages produce IL-6. What is the primary function of this?
Very important for inflammation!
Promotes B-cell differentiation and stimulates acute-phase response
Macrophages produce IL-12. What is the primary function of this?
Co-stimulator of Th1 cells
Macrophages produce IL-18. What is the primary function of this?
Promotes IFN-y production by Th1 cells
Macrophages produce TNF-a. What is the primary function of this?
Very important for inflammation!
Cytotoxic
Stimulate T cell growth
Stimulates acute-phase response
triggers inflammation
What are the primary cytokines and chemokines produced by macrophages?
IL-1
IL-6
IL-8 aka CXCL8
IL-12
IL-18
IL-23
TNF-a
What cell receptors do macrophages have?
TLRs and mannose-binding receptor (CD206)
CD64 (bins to FC region of antibodies)
CD35, CD11b/CD18 (for complement)
CD40 (to communicate with lymphocytes)
How is inflammation resolved?
An active process, M2 and Mreg macrophages play a major role
Coordinated process with lipids related to leukotrienes (resolvins, protectins, maresins, lipoxins)
What drugs suppress macrophage activity?
Corticosteroids (and others)
How do macrophages phagocytize neutrophils?
Palpate them via CD31
if damaged, will eat them through efferocytosis
What can happen due to persistent M2 activity?
Formation of granulomas
Potentially formation of proud flesh
Which interleukins are increased in animals with atopic dermatitis and may explain why their macrophages are not as effective for killing?
IL-4 and IL-13
influence macrophages to become M2 (along with IL-10 and IFN-y)
What are acute phase proteins?
Made in the liver
increase 90 min after insult and subside after 48 hours
Can also happen in prolonged stress
How does bacterial septic shock occur?
Excessive triggering of TLRs –> release of HMGB1 and cytokines –> increase NO2 and COX2 –> increase in prostaglandin and leukotrienes –> decrease in blood pressure, acidosis, fever, lactate, increase in catecholamines –> organ failure +/- DIC
How does bacterial toxic shock occur?
bacterial endotoxins stimulate T cell function –> fever, hypotension, collapse, skin lesions, organ failure
What is the primary mediator of graft-versus-host disease?
TNF-a
What is amyloidosis?
deposition of insoluble proteins in body organs as a result of folding errors in protein chains
- can be a reaction to inflammation
- may be transmissible
Erythropoietin
Main source: Endothelium
Target cell: Stem cells
Major function: RBC production
G-CSF
Classification: Pro-inflammatory
Main source: Fibroblasts, endothelium
Target cell: Stem cells in BM
Major function: Granulocyte production
GM-CSF
Classification: Adaptive immunity
Main source: T cells, macrophages, fibroblasts
Major function: Growth and differentiation of monocytes, and eosinophil, granulocytes production
IL-1
Classification: Pro-inflammatory
Main source: Macrophages, B cells, DCs
Target cell: B cells, NK cells, T-cells
Major function: Pyrogenic, pro-inflammatory, proliferation and differentiation, BM cell proliferation
There are two types: IL-1α and IL-1β (more active)
IL-2
Classification: Adaptive immunity
Main source: Produced by Th»Tc, NK, NKT cells
Target cell: Lymphocytes
Major function: growth factor for lymphocytes, proliferation of B cells, activated T cells, NK cell function, function of many cells
IL-3
Classification: Adaptive immunity
Main source: T cells
Target cell: Stem cells
Major function: Hematopoietic precursor proliferation and differentiation
IL-4
Classification: Adaptive immunity
Main source: Th Cells
Target cell: B cell, T cell, macrophages
Major function: Proliferation of B and cytotoxic T cells, enhances MHC class II expression, stimulates IgG and IgE production
IL-5
Classification: Adaptive immunity
Main source: Th2 Cells and mast cells
Target cell: Eosinophils, B-cells
Major function: B-cell proliferation and maturation, stimulates IgA and IgM production, important for eosinophils
IL-6
Classification: Pro-inflammatory
Main source: Th Cells, macrophages, fibroblasts
Target cell: B-cells, plasma cells
Major function: B-cell differentiation
IL-7
Classification: Adaptive immunity
Main source: BM stromal cells, epithelial cells
Target cell: Stem cells
Major function: B and T cell growth factor
IL-8
Classification: Pro-inflammatory
Main source: Macrophages
Target cell: Neutrophils
Major function: Chemotaxis for neutrophils and T cells
IL-9
Classification: Adaptive immunity
Main source: T cells
Target cell: T cells
Major function: Growth and proliferation
IL-10
Classification: Anti-inflammatory
Main source: T cells, B cells, macrophages
Target cell: Th cells, B cells, macrophages, NK
Major function: Inhibits cytokine production and mononuclear cell function
*the “Debbie-downer” cytokine
IL-11
Classification: Pro-inflammatory
Main source: BM stromal cells
Target cell: B cells
Major function: Differentiation, induces acute phase proteins
IL-12
Classification: Anti-inflammatory
Main source: T cells, macrophages, monocytes
Target cell: NK cells, macrophages, tumor cells
Major function: Activates NK cells, phagocyte cell activation, endotoxic shock, tumor cytotoxicity, cachexia
IL-17
Classification: Pro-inflammatory
Main source: Th17 cells
Target cell: Monocytes, neutrophils
Major function: Recruits monocytes and neutrophils to the site of infection. Activation of IL-17 in turn activate downstream of many cytokines and chemokine, such as IL‐1, IL‐6, IL‐8, IL‐21, TNF‐β, and MCP‐1
IL-18
Classification: Pro-inflammatory
Main source: Macrophages, dendritic cells, and epithelial cells
Target cell: Monocytes and T cells
Major function: Recruits monocytes and T lymphocytes. Synergist with IL-12 in the induction of IFN- γ production and inhibition of angiogenesis
IL-22
Classification: Anti-inflammatory
Main source: Activated T-cells and NK cells
Target cell: Stromal and epithelial cells
Major function: Stimulation of cell survival, proliferation
IFN-α
Classification: Pro-inflammatory
Main source: Macrophages, neutrophils, and some somatic cells
Target cell: Various
Major function: Anti-viral
IFN-β
Classification: Pro-inflammatory
Main source: Fibroblasts
Target cell: Various
Major function: Anti-viral, anti-proliferative
IFN-γ
Classification: Pro-inflammatory
Main source: Th cells»_space; Tc and NK cells, also macrophages
Target cell: Various
Major function: macrophage activation, increases neutrophil and monocyte function, MHC-I and -II expression on cells, and others
M-CSF
Classification: Adaptive immunity
Main source: Fibroblasts, endothelium
Target cell: Stem cells
Major function: Monocyte production and activation
TGF-β
Classification: Anti-inflammatory
Main source: T cells and B cells
Target cell: Activated T and B cells
Major function:
1. activates monocytes but deactivates macrophages
2. anti-inflammatory at low concentrations, profibrotic at high concentrations
3. In the absence of proinflammatory cytokines, induces Treg; in the presence of proinflammatory cytokines induces Th17
TNF-α
Classification: Pro-inflammatory
Main source: Macrophages
Target cell: Macrophages
Major function:
- Phagocyte cell activation
- endotoxic shock
- inflammatory response both locally and in the circulation
- triggers the expression of vascular adhesion molecules
- enhances the infiltration of lymphocyte to the site of infection
TNF-β
Classification: Pro-inflammatory
Main source: T Cells
Target cell: Phagocytes, tumor cells
Major function: Chemotactic, phagocytosis, oncostatic, induces other cytokines
What cell produces the most cytokines?
Macrophages
What cell produces the second most cytokines?
Keratinocytes
How big are cytokines and what concentration do they need to be at to be effective?
Lower molecular weight (8-10 kd)
Effective at very low concentrations
How do hormones compare to cytokines?
Producers: Few (hormones), Many (cytokines)
Targets: Many (hormones), Few (cytokines)
Redundancy: Low (hormones), High (cytokines)
Pleotropy: Low (hormones), High (cytokines)
Circulation: Yes (hormones), Rarely (cytokines)
Influence: Widespread (hormones), Local (cytokines)
Inducers; Physiologic variation (hormones), External insults (cytokines)
How is cytokine action restricted to a local site?
Producer cells close to target cells
Small amounts secreted
Directional release
Receptor endocytosis
Bind to extracellular matrix
Quenching in circulation by soluble receptors, other binding proteins
- Basis by which Etanercept (Enbrel) works
Natural antagonists
- IL-1 RA, TNFsRp55 (soluble TNF receptor)
Secretion in brief and limited, produced as needed
- not stored pre-formed, mRNA is short lived
- some need to be processed following synthesis (IL-1 and TGF-b)
What happens when there are high levels of circulating proinflammatory
cytokines?
= “cytokine storm”
The killer in toxic shock, severe influenza
Severe malaise of food poisoning
Post-viral encephalopathies
What is cytokine pleotropy?
one cytokine has multiple activities
What is cytokine redundancy?
multiple cytokines have same or similar activities (may come from different sources)
What are some examples of redundant cytokines?
IL-1a and IL-1b
IL-2 and IL-15
IL-4 and IL-13
TNFa and TNFb
IL-31 and TSLP
What is cytokine synergysm?
Two cytokines acting together to enhance a biologic effect
(ex. IL-4 and IL-13 having B cells make IgE)
What is cytokine antagonism?
Two cytokines oppose each other’s effects
(ex. TFN-y pushes towards Th2 and IL-4 pushes towards Th1, both inhibit Th17)
What are the most important Th1 effector cytokines?
IFN-y
TNF-a
LT
IL-21
IL-2
*most useful against viral and intracellular pathogens
*work on macrophages, neutrophils, cytotoxic T cells, and B cells
How are naive CD4+ T cells influenced to become Th1 cells?
IL-12 (from macrophages with intracellular organisms)
IL-1
IL-6
IFN-y
What are the most important Th2 effector cytokines?
IL-4
IL-5
IL-13
IL-31
*most important against parasites and in allergies
*works on mast cell, basophils, eosinophils, and B cells
*large amount of IL-9 is made by Th9 subset of Th2
What are the most important Th17 effector cytokines?
IL-17
IL-22
(IL-6, TNF-a, GM-CFS, CXCL8, VEGF)
*Important for inflammation and extracellular pathogens
*works on macrophages and neutrophils
What are the most important Treg effector cytokines?
IL-10
TGF-b
How are naive CD4+ T cells influenced to become Th2 cells?
IL-4
in the skin are largely triggered by type 2 dendritic cells and mast cells
How are naive CD4+ T cells influenced to become Th17 cells?
TGF-b
IL-6
IL-23
How are naive CD4+ T cells influenced to become Treg cells?
IL-10
TGF-b
(also the cytokines they produce)
What do skin alarmins do in atopic dermatitis?
innate cytokines that stimulate Type 2
What do Th22 cells do?
important for maintaining barrier function of skin/exposed surfaces
→ promotes antimicrobial immunity, inflammation, & tissue repair
What are the 4 classes of cytokine receptors?
1) channel-linked receptors
2) tyrosine kinase receptors (or those that activate linked tyrosine kinase)
3) G-protein receptors
4) ceramide activates serine-threonine protein kinase (IL-1, INF-a)
*all are cell surface receptors
What is the most common type of cytokine receptor?
ones that act through tyrosine kinase
What is the most important mechanism of cytokine regulation?
opposing effects from other cytokines
(may also have varying amounts of receptors, receptor antagonists, decoys)
What are the 3 signal transduction pathways that play key roles in the immune system?
1) NF-kB
2) NF-AT
3) STAT
Do cytokines have autocrine, paracrine, or endocrine activity?
All three
Which cytokine is most important for lymphocyte proliferation, activation, and differentiation?
IL-2
(mostly from Th1 cells)
IL-12 will activate T and NK cells
Which cytokines are the most important for inducing Th2 cells?
Il-33
IL-25
TSLP
IL-31
*IL-33, IL-25, and TSLP can all be made by keratinocytes
Which cytokine is most important for proliferation, differentiation, and activation of eosinophils?
IL-5
(mostly from Th2 and mast cells)
Which cytokine is most important for isotype switching to IgE?
IL-4
(mostly from Th2 and mast cells)
Which cytokines are most important for isotype switching to IgA?
IL-5: mostly from Th2 and mast cells
TGF-b
Which cytokine inhibits Th1, Th2, Th17 function and macrophage/dendritic cell activation?
IL-10
Which cytokines are most important for inducing Th1 cells?
IL-12: mostly from activated macrophages and dendritic cells
IFN-y: mostly from Th1 and NK cells
TNF-a
Which cytokine inhibits Th2 differentiation?
IFN-y
Which cytokine is most important for isotype switching to opsonic subclasses of IgG?
IFN-y
Which cytokine inhibits T cell proliferation and differentiation?
TGF-b
Which cytokines are primarily associated with innate immunity?
IL-1
IL-6
TNF-a
IL-10
IL-12
IL-15
IL-18
IL-23
IL-27
INF-a/b
Which cytokines are primarily associated with adaptive immunity?
IL-2
IL-4
IL-5
IL-13
IL-17
IFN-y
In T cells, what signaling pathways do activated calcineurin promote?
NF-AT and NF-kB
Which cytokines use the JAK/STAT pathway?
More than 50 cytokines and growth factors but the most important are:
IL-4
IL-7
IL-11
IL-13
IL-31
leptin
GM-CSF
IFN-y
What are the four main members of the JAK family?
JAK1
JAK2
JAK3
TYK2
(JAK3 is only expressed in the bone marrow, lymphatic system, endothelial cells and vascular smooth muscle cells; others are expressed widely)
Which cytokine receptors use the JAK1 pathway?
(1) Cytokine of the γc receptor subunit (IL-2R, IL-4R, IL-7R, IL-9R, and IL-15R) IL-21R
(2) Class II cytokine receptor (IFNα/βR, IFN-R, and IL-10 family cytokine receptor)
*most Th2 cytokines belong to this family
(3) Receptor with gp130 subunit: (IL-6R, IL-11R, CNTF-R, OSM-R, LIF-R, CT-1 receptor)
Which cytokine receptors use the JAK2 pathway?
(1) Gp130 receptor family
(2) The class II cytokine-receptor family
(3) IL-3 receptor family (IL-3R, IL-5R, and GM-CSF receptor)
(4) Single-chain receptors (GH-R, EPO-R, TPO-R, PRL-R)
*can cause major hematopoietic abnormalities
Which cytokine receptors use the JAK3 pathway?
All of γC receptors: (IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, IL-21R)
*abnormalities lead to defective lymphocyte (esp T cell) production
Which cytokine receptors use the Tyk2 pathway?
IFN-α/β, IL-6R family, IL-10R family, IL-12R, Il-13R, IL-23R
*regulates the balance of Th1 and Th2 cells in mice and regulates the allergic reaction mediated by Th2 cells
*Tyk2-defective humans develop severe allergic phenotypes due to IFN-mediated loss of antimicrobial capacity
How does the canonical JAK/STAT signaling pathway work?
ligand interacts with its receptor to cause receptor dimerization –>
activated JAK causes tyrosine phosphorylation of the bound receptor –>
forms a docking site for STATs –>
JAK phosphorylates STAT –>
STAT dissociates from the receptor –>
translocate to target gene promoters/regulate gene transcription
What type of interferon is IFN-y?
Type II
What type of interferon is IFN-a and b?
Type I
What makes for an ideal antigen?
Large, stable, foreign protein that is susceptible to enzymatic degradation
- particulates are more immunogenic than soluble
- denatured versions are more immunogenic
What are epitopes?
Antigenic determinants
Regions on the surface of large molecules that can bind to lymphocyte antigen receptors and therefore mount an immune system response
May be found on apparently unrelated molecules and cause cross-reactivity
What are haptens?
Small molecules like drugs or hormones are too small to be recognized by the immune system but if it links to a larger protein (carrier), it can become allergenic
- Ex. Penicillin gets bound to albumin and can be a hapten
What is an antigen?
any molecule that can bind specifically to a receptor on a lymphocyte or to an antibody molecule
What is an immunogen?
an antigen that can induce an immune response
What is immunogenicity?
ability of a molecule to stimulate and immune response
What are dendritic cells?
Specialized antigen presenting cells that initiate adaptive immunity
Subset of resident macrophages (100x more effective APC than normal mac)
- though relationship is unclear and may vary between species
What are the three major functions of dendritic cells?
1) serve as sentinel cells and activate innate defenses
2) process exogenous antigens and initiate adaptive immunity
3) regulate adaptive immunity by determining if an antigen will mount a response
Which antigen presenting cell can activate a naive T cell?
Dendritic cells
What is the basic function of Th1 cells?
stimulates cell-mediated immune responses
designed to protect animals against intracellular organisms
T cell responses
What is the basic function of Th2 cells?
stimulates antibody-mediated immune responses
designed to protect animals against extracellular organisms
B cell responses
What are Langerhans cells?
Specialized, long-lived antigen presenting cells in the skin
Have Birbeck granules in many species (not dogs)
Influence skin immune responses
Have multiple PRRs including langerin and DC-SIGN (CD209)
What causes immature dendritic cells to become mature?
IL-1
TNF-a
PAMPs
DAMPs (ex. heparan sulfate binding to TLR4)
HMGB1
How do immature dendritic cells capture antigens and cell fragments?
Phagocytosis (do not fuse with lysosomes but have alkaline pH)
Pinocytosis (uptake of fluid droplets)
Binding to cell surface receptors
*if they ingest bacteria they usually kill them but don’t completely degrade them
What histopathology stain is good for seeing the dendrites of Langerhans cells?
Vimentin
What happens after an immature dendritic cell captures and processes antigens?
They are now mature
DCs carry these to sites where they can be recognized by T cells
- secrete chemokines that attract T cells
- DCs embrace T cells in their dendrites
- one DC can activate up to 3,000 T cells
DC MHC molecules move to the cell surface
The activated DCs are attracted to lymphoid organs
How do dendritic cells induce tolerance?
In steady-state conditions some immature DCs will spontaneously mature
Will migrate to lymphoid organs with normal tissue antigens
If a T cell recognizes the normal antigen, it will undergo apoptosis
Or the DC will produce IL-10, retinoic acid, or TGF-b
- induce differentiation to Tregs
What do cDC1 cells do?
Antigens make the dendritic cells produce IL-12
Activate Th1 cells and type 1 responses
What do cDC2 cells do?
Antigens make the dendritic cells produce IL-1 and IL-6
Activate Th2 cells and type 2 responses
Or they can produce IL-23 and trigger development of Th17 cells
Triggering of which TLRs result in dendritic cells being cDC1 cells?
TLR 3
TLR 4
TLR 5
TLR 7
TLR 9
Triggering of which TLRs result in dendritic cells being cDC2 cells?
TLR1
TLR2
TLR6
What is expressed in canine dendritic cells but not macrophages?
CD40
What are the two most important IHC markers for Langerhans cells?
CD1a and E-cadherin
(also CD1+/CD11c+/MHC II+/ CD4+/ CD90+)
What is the IHC staining of feline Langerhans cells?
CD18+, MCH II+, CD1a+, CD4+
How does antigen-presentation by macrophages differ from that of dendritic cells?
Macrophages cannot have prolonged interaction with T cells so they cannot activate naive T cells
Antigen processing by macrophages is inefficient because much of the antigen has been digested
How does antigen-presentation by B-cells differ from that of dendritic cells?
B cells cannot undergo prolonged interactions with T cells
- can only work with sensitized T cells
Play a minor role in antigen processing in primary immune response
- more of a role when there are more B cells and sensitized T cells
What are MHC I molecules?
expressed on most nucleated cells (not red blood cells)
presents epitopes to CD8+ T lymphocytes
advertises “self”/endogenous proteins
binding site is closed on both ends
If it is problematic and the T-cell receptor binds it = apoptosis
How many genes are there for MHC I molecules in humans?
3: HLA-A, HLA-B, and HLA-C
will have a total of 6 copies because we have 2 chromosome 6’s
can have many haplotyes because the genes are codominant
What are the three signals that dendritic cells can provide when they stimulate T cells?
1) contact with an antigen associated with a MHC molecule
2) costimulation with molecules like CD40 and CD80/86
3) use cytokines to polarize naive T cells
What do thymic dendritic cells do?
may recognize and kill self-reactive T cells
What do follicular dendritic cells do?
are in lymphoid follicles in B cell areas of lymphoid tissue
trigger B cells
What do interferon-producing dendritic cells do?
found in the blood and are able to produce massive amounts of cytokines
early warning system for viral infections
What is the source of antigens presented via the MHC class I pathway?
Cytoplasm (synthesized within the cell) and processed by proteasome
- viruses and neoplasia can affect these
How many genes are there for MHC I molecules in animals?
Hundreds (A1, A5, A11, B16, B19, B23, etc)
Outbred animals are usually heterozygous
Genes are codominant so both alleles are expressed
*dogs and cats have relatively few functional MHC class I genes so rely on allelic polymorphisms
Haplotyes are closely linked with body odors
What is the source of antigens presented via the MHC class II pathway?
Phagolysosome/endosome (internalized from the outside) and processed by endosomal and lysosomal proteases
What are MHC II molecules?
Presents exogenous antigens so only on antigen presenting cells
CLP in the endosome breaks proteins down and the MCH II-peptide is displayed on the surface
Ends of the receptor are not enclosed, so a bit sticks out
Stimulates CD4+ T cells
What is a primary driver in transplant failure?
The transplant having the wrong MCH class I
What are the internal lymphoid organs?
Thymus
Bone marrow
Spleen
Lymph nodes
What are the surface lymphoid organs?
Salivary glands
Respiratory tract
Mammary glands
Intestine
Urogenital system
Where do lymphoid stem cells come from?
Fetus: fetal omentum, liver, and yolk sac
Adult: bone marrow
What are the functions of bone marrow?
hematopoietic organ
in some animals it can be primary and/or secondary lymph organ
large number of antibody producing cells
has many dendritic cells and macrophages to remove material from blood
*organized in layers
What are primary lymphoid organs?
regulate the development of lymphocytes
- immature lymphocytes proliferate and TCRs/BCRs are synthesized
lymphocytes fall into T or B category based on where they mature
develop early in fetal life from ectoendoderm or endoderm
involute after puberty
unresponsive to antigens
Include the:
- Thymus (all T cells mature here)
- Bursa
- Peyer’s patches
- Bone marrow
What is the difference between the thymus in young animals versus adult animals?
Prominent in young animals (may extend as far as thyroid in some animals)
Will be small in adult animals due to involution (replaced by fat)
What is the function of the thymus?
All T cells mature in the thymus
Young animals: very important for producing circulating lymphocytes
Adults: functional but there is a reservoir of long-lived thymus derived cells
Cells that recognize self-antigens or don’t react to MCH IIs are removed
- “negative selection”
Cells that recognize specific MHC II-antigen complexes are stimulated to grow
These T cells exit the thymus
What do thymic epithelial cells do?
Express more than 400 antigens normally expressed in other tissue
Have high levels of autophagy
Results in “promiscuous” antigen presentation
- thymocytes are presented with diverse array of normal antigens
- important for removing self-responsive T lymphocytes
Which mineral is essential for the development of T cells?
Zinc
What does thymic stromal lymphopoietin (TSLP) do?
activate thymic dendritic cells that can stimulate regulatory T cells
What is the Bursa of Fabricius?
A primary lymphoid organ found in birds
Shrinks in adults like the thymus
Involved in maturation and differentiation of B cells
What are Peyer’s patches?
A primary lymphoid organ along the walls of the small intestine
Structure and function vary among species
Most are found in the ileum
Most important for B cell differentiation
- healthy GI microbiota diversity increases B cell diversity
What are secondary lymphoid organs?
Arise late in fetal life and persist in adults
Enlarge in response to antigenic stimulation
Contain dendritic cells and lymphocytes that mediate immune responses
Structure facilitates antigen trapping
Connected to both the blood and lymphoid systems
Include the:
- Spleen
- Lymph nodes
- tonsils
- lymphoid tissue in intestinal, respiratory, and urogenital tract
- bone marrow
What is the structure of a lymph node?
Round or bean-shaped filter on lymphatic vessels
- afferent lymph vessels enter around the circumference
- efferent lymph vessels leave from the hilus
- blood vessels enter and leave from the hilus
Has a capsule over a reticular network of lymphs, macs, and dendritic cells
A subcapsular sinus is present just under the connective tissue capsule
Inside is divided into 3 regions
- peripheral cortex
- ill-defined paracortex
- central medulla
What type of lymph cell predominates in the lymph node cortex?
B-cells
organize in aggregates called follicles
What are germinal centers of lymph nodes?
Specialized structures in the cortex of a lymph node
Arise in response to an antigen
Sites where B cells grow, mutate, and mature
Centroblasts (dividing B cells) that form the dark zone
- on the outside
- B cell proliferation and somatic mutation occur here
Centroblasts mature => centrocytes and migrate to light zone
- on the inside
- where Ig class switching and memory B cell formation occurs
- rich in antigen-trapping follicular dendritic cells and CD4+ T cells
What type of lymph cell predominates in the lymph node paracortex?
T cells (and dendritic cells)
Arranged in cords between the lymphatic sinuses
What is found in the lymph node medulla?
Lymph draining sinuses
Separated by medullary cords containing plasma cells, macrophages, and memory T cells
What is the primary function of secondary lymphoid organs?
facilitate interactions between antigen-presenting cells and antigen-sensitive T and B cells
What is the red and white pulp of the spleen?
Red pulp = tissue for blood filtering and RBC storage
White pulp = has both B and T cells
Marginal zone = between them and has macs, dendritic cells, and B cells
What are tertiary lymph organs?
Structurally similar to secondary lymph organs
Develop in response to microbial colonization and chronic immune stimulation
What is the basic structure of lymphocytes?
Small, round cell, 7-15 um in diameter (RBCs are ~5 um)
Single, large, darkly staining nucleus
Thin rim of cytoplasm
What can basic B cells go on to form?
Memory B cells
Plasma cells
What can basic T cells go on to form?
Helper T cells
Treg cells
Effector T cells
Memory T cells
What is the primary way one can tell lymphocytes apart?
Based on their cell surface molecules
Named CD = cluster of differentiation
From an immunologic viewpoint, what are the most important structures on lymphocytes?
Receptors that they use to recognize antigens
- T cell antigen receptor (TCR)
- B cell antigen receptor (BCR)
What are the two populations of T cells that can be differentiated by their TCR antigen-binding chains?
One that uses α and β peptide chains (TCR α/β)
One that uses γ and δ peptide chains (TCR γ/δ)
How do BCRs differ from TCRs?
Subpopulations of B cells use 5 different heavy chains (versus 2)
BCRs are shed into the tissue fluid and blood = antibodies
- antibodies are simply soluble BCRs
What are innate lymphoid cells?
Such as NK cells
Do not have variable antigen receptors
Kill target cells that fail to express MCH molecules
What does the CD3 designation of lymphocytes mean?
Collective designation given to the set of proteins found around the TCR
Are a cluster of signal transducing proteins
Therefore CD3+ is found on all T cells
- Is the basic T cell marker
What does the CD4 designation of lymphocytes mean?
A protein closely associated with TCRs
Receptors for the MCH class II molecules on APCs
Found only on helper T cells
What does the CD8 designation of lymphocytes mean?
A protein closely associated with TCRs
Receptors for the MCH class I
Only expressed on T cells that attach and kill abnormal cells
- “cytotoxic” T cells
What is the most common type of T cell found in circulation in humans?
~65% in humans are CD4+/CD8- (helper T cells)
*if CD4+ is elevated it implies increase lymphocyte reactivity
What cytokine receptors are frequent expressed on lymphocytes?
Many but some of the most important ones are:
CD25 (for IL-2)
CD118 (for IFN)
CD120 (for TNF)
CD210 (for IL-10)
What are antibody receptors called?
Fc receptors because they bind to the Fc region of antibodies
- FcγR binds to the γ region of IgG
- FcαR binds to the α region of IgA
- FcεR binds to the ε region of IgE
What is FcγRI (CD64)?
A high-affinity IgG receptor on dendritic cells, monocytes, and macrophages (less so on neutrophils) that promotes phagocytosis
What is FcγRII (CD32)?
A moderate-affinity IgG receptor on B cells, macrophages, and granulocytes that inhibits B cells and promotes phagocytosis and release of cytokines by macrophages
What is FcγRIII (CD16)?
A low-affinity IgG receptor on granulocytes, NK cells, and macrophages but can only bind to immune complexes which promotes cytotoxicity and phagocytosis
What is FcαR1 (CD89)?
An IgA receptor on neutrophils, eosinophils, macrophages, and dendritic cells which mediates endocytosis and recycling
What is FcεRI?
A high-affinity IgE receptor found on mast cells that plays a role in allergies
What is FcεRII (CD23)?
A low-affinity IgE receptor found on activated B cells, platelets, eosinophils, macrophages, NK cells, and dendritic cells which regulate allergic responses
What are the 4 major complement receptors on lymphocytes?
CR1 (CD35) on B and T cells which binds C3b and C4b
CR2 (CD21) on B cells that binds C3d and C3bi
CR3 and CR4 are found on NK cells
Other than helper T cells, where can CD4 also be expressed in dogs?
neutrophils and macrophages (not monocytes)
What are lymphocyte mitogens?
Stimulants that make lymphocytes divide
Can be another way to categorize lymphocytes
What are the four populations of lymphocytes with antigen-binding receptors?
Th
Treg
Effector/cytotoxic T cells
B cells
What is the structure of a TCR?
A heterodimer of α/β or γ/δ chains
has a cytoplasmic and transmembrane domain
Then a constant domain connected by a disulfide bond
The a variable domain that forms a V-shape
- antigen binding groove
- within each V domain there is a region where the amino acid sequence is variable
What is Th lymphocyte co-stimulation?
Binding of a T cell antigen receptor to an antigen-MHC complex is not sufficient by itself to trigger a helper T cell response so additional signals are needed
Adhesion molecules to prolong signaling
Receptor molecules to amplify signals
Cytokines to turn on/off pathways
What are the Th lymphocyte co-stimulatory receptors?
CD154 on lymphocytes binding to CD40 on APCs
- T cell will now express CD28
- APC will now either express CD80 or CD86
- APC will secrete multiple cytokines (IL-1, IL-6, IL-8, IL-12, TNF-a)
Binding of CD28 on lymphocytes will bind to CD80 on debritic cells, macrophages, or activated B cells or CD86 on B cells
- enhances production of IL-2 and other cytokines
- upregulates cell survival genes and facilitates division
If CTLA-4 binds CD80 or CD86 T cell activation will be suppressed
What are supramolecular activation clusters (SMACs)?
A “bull’s-eye” structure that forms when Th1 cell and antigen-presenting cells come into contact
Ring of TCR-peptide-MHC complexes and costimulatory receptors
Is an immunological synapse
*Th2 cells do not form this but form multifocal immunological synapses
How does signal transduction from TCR binding occur?
TCR binds antigen –>
immunologic synapse forms –>
ITAMs on the CD3 chains activate tyrosine kinases –>
forms a signaling complex with calcineurin –>
Activate NF-AT and MAPK to activate NF-kB
Which cells can activate naive T cells?
Dendritic cells
(macrophages and B-cells do not bind strongly enough)
Which cells can activate primed T cells?
Dendritic cells, macrophages, and B-cells
What happens to T cells in the absence of effective co-stimulation?
Don’t divide or produce cytokines
Either become unresponsive or undergo apoptosis
What are superantigens?
microbial molecules that activate T cells with TCR Vb domains
directly link the TCR Vb domain to an MCH II molecule (on the outside)
stimulate a powerful T cell response
- potentially toxic shock syndrome
What transcription factor causes the development of Th1 cells?
T-bet
What transcription factor causes the development of Th2 cells?
GATA3
What transcription factor causes the development of Th17 cells?
ROR-yt
What transcription factor causes the development of Treg cells?
FoxP3
What are the primary functions of IL-2?
Potent stimulator of T cell proliferation
Increases IFN-y production
Increases B cell antibody production
Enhances cytotoxicity of CD8+ T and NK cells
Promotes CD4+ differentiation into Th1 and Th2
Inhibits Th17 differentiation
Essential for survival of Tregs
What cells are the primary targets of IL-2?
Target T, B, NK cells and macrophages
What cells are the primary producers of IL-2?
Produced by activated CD4+ Th1 cells
So also produced by CD8+ T cells, NK cells, dendritic cells, and mast cells
How does IFN-y activate cells?
Through the JAK-STAT pathway
What cells are the primary producers of IFN-y?
Th1, CD8+ T cells, NK cells
lesser amounts from APCs, B cells, and natural killer T cells
How does IFN-y affect macrophages and lymphocytes?
Activate macrophages
Suppress Th2 cells
Enhance NK cell activity
(also is antiviral)
What is Type 1 Immunity?
A newer way of classifying immune reactions (type 1 vs 2)
Directed against bacteria, viruses, fungi, and protozoa
Initiated by macrophages and dendritic cells
Employs IFN-y, IL-12, IL-17, IL-18, IL-21, IL-22
Activates macrophages, neutrophils, CD8+ lymphs, B cells
Encompasses use of Th1, Th17, CD8+ T cells, M1 macs, IgG, IgA, IgM
What is Type 2 Immunity?
A newer way of classifying immune reactions (type 1 vs 2)
Directed against helminths, venom, and allergens
Initiated by epithelial cells
Employs IL-4, IL-5, IL-9, IL-13, IL-25, IL-33, and TSLP
Activates mast cells, basophils, eosinophils, B cells,
Encompasses use of Th2, basophils, mast cells, eosinophils, M2 macs, IgE
What cells are the primary producers of IL-4?
Th2 and mast cells
What cells are the primary targets of IL-4?
T cells, B cells, macrophages
What are the primary functions of IL-4?
Activates macrophages (alternative M2 activation)
Stimulates B cells
- enhance IgG and IgE responses
- promote MCH II expression
Activate GATA3 for Th2 differentiation
Suppress Th1 and Th17 cells
- antagonize IFN-y and IL-2
What is the balance of Th17 and Tregs important for?
maintain homeostasis during immune responses
excessive Th17 activity can lead to development of chronic diseases
What can Th17 cells do when inflammation is resolved?
differentiate into T-regs
What markers are Tregs positive for?
CD4+
CD25+
FoxP3+
What species have a large number of γ/δ T cells?
Ruminants and pigs
What species have a low number of γ/δ T cells?
Humans and mice
Where are innate γ/δ T cells primarily found?
Skin, genital tract (mucous membranes)
- skin draining afferent lymph nodes
Bind PAMPs, lipids, MCH class I
Are activated by IL-23 (like Th17) and secrete IL-17 and IFN-y
Where adaptive γ/δ T cells primarily found?
Secondary lymph organs (and in the blood of some animals)
What are memory T cells?
After interaction with a dendritic cell, T cells divide in an asymmetric fashion
- daughter cell near the synapse is the precursor to the effector T cell
- daughter cell on opposite pole is the precursor to the memory T cell
Can be central, tissue-resident, or effector memory T cells
Are either CD4+ or CD8+
Need to be exposed to the same antigen
Where are central memory T cells found?
Lymph nodes
Lack immediate effector function but have rapid recall responses
Where are effector memory T cells found?
Circulating in the blood
Can immediately hone in on inflamed tissue and attack
Where are tissue-resident memory T cells found?
In organs/tissue
Provide first response to a pathogen entering the body
Where do CD8+ memory T cells tend to be found?
Accumulated under epithelial surfaces
Where do CD4+ memory T cells tend to be found?
Scattered through the tissue in clusters
Slowly divide and replenish their numbers
Can be thought of adult stem cells
Which cytokines are needed for the survival of CD8+ and CD4+ memory T cells?
IL-7 and IL-5 for CD8+
only IL-7 for CD4+
How long do memory T cells last?
Half-life of 8 to 12 (CD4+) or 15 (CD8+) years in humans
How many types of antigens can B cells bind to?
A single antigen
have a large number of identical antigen-binding receptors
How do the numbers of BCRs on B cells and TCRs on T cells compare?
200,000 - 500,000 BCRs
30,000 TCRs
*BCRs can also bind to antigens in solution unlike TCRs
What is the structure of an immunoglobulin/antibody?
Y-shaped
Paired heavy chains held together by a disulfide bond
Two light chains linked to the heavy chains by disulfide bonds
Antigen-binding sites are grooves between the light and heavy chains
- have two identical antigen-binding sites
Has an Fc and Fab region
What is the structure of an immunoglobulin/antibody light chain?
Constructed from two 110 amino acid domains
The C-terminal is contestant between all B cells = constant domain
- number of constant domains varies between immunoglobulin types
- γ, α, δ have 3 constant domains; ε and μ have 4
The N-terminal varies between B cells = variable domain
- the hypervariable region forms the shape of the antigen-binding site
Mammals make λ and κ light chains
- humans have more κ, dogs and cats have 90% λ
What is the structure of an immunoglobulin/antibody heavy chain?
Constructed from 4-5 domains of 110 amino acids
C-terminal has 3-4 constant domains
N-terminal has a variable domain
Mammals make 5 classes of heavy chains with different activity
- γ (IgG)
- α (IgA)
- δ (IgD)
- ε (IgE)
- μ (IgM)
What is a Fab region of an immunoglobulin/antibody?
The arms of the “Y”
formed by a paired light and heavy chain
break off when treated with the proteolytic enzyme papain
What is an Fc region of an immunoglobulin/antibody?
The tail of the “Y”
formed by paired heavy chains
Attaches to the B cell surface
Determines the biologic role of a B cell
What is the hinge region of an immunoglobulin/antibody?
Fab region contains a “hinge region” so they can swing freely
Contains many hydrophilic and proline residues
- Make this region accessible to proteases
Also contain the disulfide bonds
*not present in IgM
What is the signal transducing component of BCRs?
BCRs cannot signal directly to B cells
have associated CD79 heterodimers
Cross-linking of these exposes ITAMs –>
Phosphorylation of phospholipase C and G protein –>
calcium mobilization –>
generates protein kinase C and calcineurin –>
activates NF-kB and NF-AT
What is an idiotype?
idiotype = variants in variable domains
variants - idiotypes
a shared characteristic between a group of immunoglobulin or T-cell receptor (TCR) molecules based upon the antigen binding specificity and therefore structure of their variable region
How does antigen presentation by B cells occur?
After antigen binding, the BCR is internalized –>
either degraded to transported to an intracellular compartment –>
antigen-MHC II complexes are transported to cell surface
How are B cells activated?
Co-stimulation by activated T-helper cells
- increase expression of IgM BCR and MCH II
- increase receptors for and expression of cytokines
- can trigger immunoglobulin class switching
T cell-independent
- complement help (CD21/CD19)
- TLR and PAMP help
- mostly IgM isotype with little switching to IgG
- usually results in less memory
What are the primary cytokines that Th cells produce to activate B-cells?
IL-4
IL-5
IL-6
IL-13
IL-21
What does IL-4 do to B cells?
Stimulates growth and differentiation
enhances expression of MCH II and Fc receptors
Induces class switching (IgA and IgE production)
What does IL-5 do to B cells?
Promotes differentiation of activated B cells
Stimulates IgG and IgM production
Enhances IL-4 mediated IgE production
Stimulates IgA production in mucosal B cells
What does IL-6 do to B cells?
needed for differentiation into plasma cells
What does IL-13 do to B cells?
Has biologic activities similar to IL-4 because their receptors are structurally similar
What does IL-21 do to B cells?
induces differentiation into plasma cells and memory B cells
Stimulate IgM –> IgG class switching
What is a T cell or B cell clonotype?
A clonotype designates a collection of T or B cells that descend from a common, antecedent cell, and therefore, bear the same adaptive immune receptors and recognize the same epitopes
Which cytokines control class switching?
- IL-4 (but needs CD40 and CD154)
IFN-y
TGF-b
Which two cell-stimulating cytokines are crucial survival factors for B cells?
B-cell activating factor (BAFF)
- on the cell membranes of antibody producing cells
- can be cleaved and become a soluble cytokine
A proliferation-inducing ligand (APRIL)
- only functions as a soluble cytokine
*Both promote B cell division and inhibit apoptosis
What are plasma cells?
develop from antigen-stimulated B cells
Ovoid, 8-9 um in diameter
Round, eccentrically placed nucleus
Unevenly distributed chromatin
Extensive cytoplasm with rough endoplasmic reticulum
Large Golgi apparatus
Can secrete up to 10,000 molecules of Ig/sec
- the Ig is identical to specificity of BCRs in parent B cell
What is a Mott cells?
A mature plasma cell that produces excessive Igs
The defective antibodies accumulate = Russel bodies
- usually are large, clear to light blue cytoplasmic inclusion bodies
What are memory B cells?
Long-lived antigen sensitive B cells
Several classes based on Ig class, location, and passage through germinal centers
How can you separate types of immunoglobulins?
Electrophoresis
Which immunoglobulin is found in the highest concentration in the serum of mammals?
IgG
plays a major role in antibody-mediated defenses
Which immunoglobulin is found in the second highest concentration in the serum of mammals?
IgM
Which immunoglobulin is the predominant immunoglobulin in saliva, milk, and intestinal fluid?
IgA
*predominate in mucous membranes
Which immunoglobulin is the primary mediator of allergic reactions?
IgE
*seldom detected in blood except in parasitize or allergic anima
Which immunoglobulin is not present in all animals (ex. is missing in cats)?
IgD
Where and when during inflammation is IgG produced?
Produced by plasma cells in spleen, lymph nodes, and bone marrow
Second isotype secreted during inflammation
- sustains the response
Exists as different subclasses in many animal species
Which is the smallest immunoglobulin?
IgG
can escape from blood vessels more easily than others
- especially important in inflammation
What are the functions of IgG?
Functions in extravascular fluid as well as blood
Can agglutinate insoluble (particulate) antigens
- good for bacteria
- not as effective as IgM
Can activate complement when bound to antigen (but not very efficiently)
Clumps soluble antigens to make then easier to eliminate (=precipitation)
Opsonizes antigens
- Fc region binds to phagocytes
Binds to toxins or viruses to neutralize them
Which is the largest immunoglobulin?
IgM
*in plasma cells it forms a pentamer before secretion
- linked via a J chain
When in inflammation is IgM predominantly found?
If found on naive B lymphocytes (even after IgD disappears)
- is a monomer at this point
Is the first isotype to be secreted during the primary immune response
What are the functions of IgM?
Stays in the blood (cannot pass through vessel walls or placenta)
Pentameric structure provides good agglutination ability
The multiple adjacent Fc structures efficiently activate complement
- when bound to antigen
- via classical pathway
What is IgD?
Isotype of BCR found on naive B lymphocytes in some animals
Disappears when naive B cells are stimulated by antigens
Might be important for regulating GI and microbiota
- may also be important for respiratory disease in humans
Which is the main antibody of primary immune responses?
IgM
Which is the main antibody of secondary immune responses?
IgG
How does the shape of IgA differ between serum and secretions?
Is a monomer in serum
Is a dimer (sIgA) ins secretions
- linked end-to-end by a J chain and secretory component peptide
- protects it from digestion by enzymes
How does IgA get from the B cells to the epithelial surface?
Passes through the epithelial cell
What are the functions of sIgA?
Prevent attachment of microorganisms to mucosal epithelium
Capable of agglutinating particulate antigens
Can also neutralize toxins and viruses
Provides passive protection in intestine of neonates
*is not bactericidal and does not activate complement
What are the functions of IgE?
Binds to the Fc receptor on mast cells and basophils
- release of granule contents
Attaches to parasite nematodes and target for attack by eosinophils
Which is the shortest-lived immunoglobulin?
IgE
Super short-lived with half-life of 2-3 days
Very easily killed by heat
What are immunoglobulin subclasses?
Have arisen as a result of heavy chain gene duplication
New gene is gradually changed by mutation
What are immunoglobulin allotypes?
heritable immunoglobulin differences in heavy chains
How many subclasses of IgG are usually present?
4 (IgG1, IgG2, IgG3, IgG4)
What are the two DNA recombination events that B cells undergo as they mature?
1) V (D) J recombination
- creates the antigen binding site of the BCR
Once the antigen binds the BCR and activate the B cell the second happens
2) class switching recombination
- does not change the antigen-binding site
What is “class switching”?
During the course of a B-cell response, the class of Igs produced changes
Genes for constant regions of heavy chains are clustered on a chromosome
Required IGH gene is spliced directly to the IGHV gene
Unwanted, unused IGH genes are excised
- looping out-deletion and enzyme called recombinase
B cell first uses IGHM gene to make IgM
As the immune response changes the IGHG, IGHA, or IGHE genes turn on
- determined by Th type and cytokines
How does antigen recognition by T cells occur?
Distinguish by primary amino acid sequences
Antigens must be processed by antigen presenting cells
Antigens must contains a protein or peptide
The fragment must be displayed in an MCH II
How does antigen recognition by B cells occur?
Distinguished by its 3-dimensional structure
Antigens are not processed by antigen presenting cells first
Proteins are not a necessary component of antigens
Epitope binds directly to the BCR
What are the methods of generating antibody diversity?
VJ and VDJ gene recombination
- VJ is for light chains, VDJ is for heavy chains
Base deletion
Base insertion
Somatic mutation (B cells only)
Combinatorial association
Gene conversion (B cell only)
Receptor editing
What type of bond is formed between antigens and BCRs or TCRs?
Non-covalent so bond is not permanent
Strongest binding is when the shapes of antigen and receptor match
- can form multiple non-covalent bonds
- collectively increase strength
Mostly hydrophobic (exclude water molecules) but can also be via hydrogen or electrostatic bonds
How does antibody light chain gene recombination occur?
Randomly selected V genes and J genes are combined
–> joined to form a complete variable region gene
–> mRNA is generated
–> unwanted genes are excised (usually via looping and recombinase)
–> the V-J section is attached to the constant region gene
–> V-J-C mRNA is translated to form a light chain
How does antibody heavy chain gene recombination occur?
IGHD is joined to IGHJ
–> IGHV (determines Ig type) is attached to form complete V-region gene
–> unwanted J genes are deleted
–> IGHC gene mRNA is attached
–> V-D-J-C mRNA is translated
What is somatic mutation?
Happens in B cells
- not T cells because that increases risk of recognizing self
Doesn’t start until B cells have switched from IgM to IgG or IgA
Mutations in V genes are generated by same enzymes for class switching
Triggered by crosslinking of two BCRs, CD40-CD154/CD80-28 binding
Creates uracils which are recognized as errors
Uracils are deleted which leaves a gap
DNA polymerases repair gaps with random short nucleotide sequences
So V gene sequences gradually change as B cells respond to antigens
- ~1 amino acid changes each time a B cell divides
How many “tries” do pre-B cells get to make functional immunoglobulins?
4, if they don’t manage by then they undergo apoptosis
- if unproductive at checks, they undergo rearrangements
- if they successfully bind they undergo clonal expansion
What are the 3 major mechanisms of epigenetic regulation and how are they involved in B cell activation?
1) adding (turns off) or removing (turns on) DNA methylation
2) histone modification
3) production of microRNA that bind to mRNAs and influence function
*B cell activation is associated with genome-wide hypomethylation, increase in histone acetylation, and appearance of specific miRNAs
- class switching and somatic hypermutation are regulated by all 3
- if dysregulated can result in autoimmunity
What are the two major pathways of apoptosis?
1) Extrinsic/death receptor pathway
2) Intrinsic/mitochondrial pathway
How does the extrinsic pathway of apoptosis occur?
Cytokines like TNF-a bind to the death receptors CD95 or TNFR
Activates initiator caspases 8 and 10
activate effector caspases –> cell death
*triggered by CD8+ T cells through CD95 pathway
How does the intrinsic pathway of apoptosis occur?
Noxious stimuli cause mitochondrial injury
- oxidants, radiation, etc
Causes release of cytochrome C
This triggers formation of the apoptosome
The apoptosome activates the initiator caspase 9
activate effector caspases –> cell death
*triggered by CD8+ T cells through perforin pathway
What do initiator caspases do?
Activate a cascade of effector caspases (3, 6, 7) which degrade proteins and lead to apoposis
How are CD8+ T cells activated?
1) Dendritic cell process MCH I molecules and take them to lymph organ
2) CD8+ T cells are co-stimulated by CD4+ T cells recognizing same antigen
- helper T cell activates dendritic cell, increasing MCH I and IL-12
3) dendritic MCH I-linked peptides bind to CD8+ T cell
What are the 3 signals that CD8+ T cells need in order to respond to an infected cell for complete activation?
1) IL-12 from activated dendritic cells
2) antigen-specific signal from the antigen-MCH I complex on target cell
3) IL-2 and IFN-y produced by Th1 cells
How do different levels of stimulation trigger different activation responses in CD8+ T cells?
Like with Th cells, duration of stimulus is important
Naive CD8+ T cells to be stimulated for hours
- once activated, they begin to divide
Activated CD8+ T cells can be triggered by brief exposure
What happens to CD8+ T cells once they are activated?
move to peripheral sites and differentiate into effector and memory cells
- short-lived effector cells are most of population, die when infection is cleared
- cells that may have received less stimulation survive and become long-lived memory cells
How do cytotoxic T cells kill their target?
1) Bind to peptide-MCH I complex
- only 1 may be sufficient to kill the cell
- 100-1,000 complexes are needed to stimulate cytokine production
2) form a synapse at the point of contact
- the center attracted lysosomes to kill cell
- outside is a pSMAC rich in adhesion molecules (LFA-1, ICAM-1)
3) Target cell is dead <10 min
- use 2 pathways (preforin and CD95)
4) Cytotoxic T cell can disengage and move on to kill other targets in 5-6 min
What is the perforin pathway used for and what are the 3 steps?
Involves the secretion of perforins and granzymes from lysosomes
Kill cells through intrinsic apoptosis
Used for virus-infected cells
The 3 steps are:
- adhesion –> attaching to target
- lethal hit –> perforin makes holes and granzymes/granulosin comes in
- cell death
What is the CD95 pathway in used for and how is it triggered by CD8+ T cells?
Mostly used to kill unwanted, surplus or self-reactive T cells
Binding of MCH I to TCR causes CD95L on T cell to CD95 on target
Trigger extrinsic apoptosis
Other than cytokines, what do activate macrophages secrete?
Proteases that activate complement
Interferons
Thromboplastin
Prostaglandins
Fibronectins
Plasminogen activator
Complement components
What are the types of innate lymphoid cells?
NK cells
ILC1
ILC2
ILC3
NKT cells (innate-like T cells with limited diversity TCRs)
What do group 1 innate lymphoid cells do?
defend against viruses, intracellular bacteria, and parasites
What do group 2 innate lymphoid cells do?
mediate the innate arm of type 2 responses
- lots of crosstalk with Th2
defend against helminths and involved in allergies
make IL-5, IL-13, IL-4, IL-9
- control eosinophilia
What do group 3 innate lymphoid cells do?
Promote immunity to intracellular bacteria
What is the characteristic surface phenotype of NK cells?
CD3-, CD56+, NKp46+
How do NK cell distinguish normal from abnormal cells?
They do not have a ton of antigen receptors
Use two types of receptors
One monitors the expression of MHC class 1 antigens
- binding of an NK cell to a normal MHC I = cell is ok
- if NK cell fails to bind to a MHC I (“missing self”) = cell death
Other detects “stress” molecules on unhealthy cells
- if present = death
Which cytokine enhances NK survival?
IL-3
Which cytokines enhance NK cytotoxicity?
IL-2 and IL-4
How do activated NK cells kill their targets?
Perforin/granulysin/NK-lysin pathway or CD95L pathway
What are the functions of NK cells?
Always “on-call” versus T and B cells that need several days for activation
- Rapidly activated by IFNs or IL-12
Destruction of tumor and virus-infected cells
Destruction of bacteria and fungi
Destruction of parasites
Autoimmunity/graft rejection
Control of hematopoiesis
What is central T or B cell tolerance?
the process of eliminating any developing T or B lymphocytes that are autoreactive
What is peripheral T or B cell tolerance?
second branch of immunological tolerance, after central tolerance
takes place in the immune periphery
- after T and B cells egress from primary lymphoid organs
self reactive cells are subject to clonal deletion, diversion, or anergy
mediated by Treg and tolerogenic dendritic cells
What happens following oral administration of an antigen that may play a large role in tolerance to food antigens?
pTreg production
How do Tregs suppress other immune responses?
1) direct contact with T effectors
2) secretion of suppressive molecules
3) interference with antigen presentation
What suppressive molecules do Tregs secrete?
IL-10
TGF-b
IL-35
Prostaglandin E2
Which important cytokines do IL-10 reduce production of?
Th1 cytokines
IL-2
IFN-y
TNF-a
Th2 cytokines
IL-4
IL-5
Th17 cytokines
IL-17
Others
IL-8
IL-12
GM-CSF
C-CSF
What are some of the major functions of TGF-b?
Reduce T cell proliferation
Antagonize IFN-y and IL-12
Regulate cell proliferation, growth, differentiation, and motility
Enhance macrophage phagocytosis but decrease cytotoxicity
Reduce B cell proliferation
Promote IgA production
Promote B cell apoptosis
Required for optimal dendritic cell development
What is indoleamine 2,3-dioxygenase (IDO)?
An enzyme made by Tregs, dendritic cells, few other cells
Degrades and locally depletes tryptophan
Inhibits T cell activation, proliferation, and survival
- Th1 are more sensitive than Th2
Enhances peripheral tolerance
Can also act as a defensive enzyme against some invaders
How does stress affect the immune system?
Depresses T cell responses, NK activity, IL-2 production, IL-2R expression
Decreases neutrophil phagocytosis and respiratory burst
Changes TLR signaling pathways
Changes Th1 and Th2 response
How is the stress effect on the immune system mediated?
1) autonomic nervous system producing neurotransmitters
- epinephrine, norepinephrine, acetylcholine
2) hypothalamic-pituitary-adrenal cortical axis
- glucocorticoids
What nerves innervate Langerhans cells?
Autonomic nerves
- release of peptides by these can depress APC capabilities of these cells
- why hotspots are worse in dogs with anxiety?
Which interleukins act on the hypothalamus and pituitary to increase ACTH and therefore cortisol?
IL-1 and IL-6
How does cortisol suppress T cell function?
Blocking NF-kB pathway
What Th subset does Staphylococcus epidermidis activate?
Th17
What are adjuvants?
substances added to vaccines to increase response
- Aluminum salts
- Saponin-based
- water-in-oil
- Particulates
- microbial products
- combination
What is the structure and location of mast cells?
Large, round cells 15-20 um in diameter
Cytoplasm has large granules
Do not circulate
Are found in largest numbers in skin, intestine and airways
Tend to be close to blood vessels
- regulate blood flow and cellular migration
Also found in connective tissue, under mucosa, and around nerves
Are long-lived and may divide
What stimuli trigger mast cell degranulation?
IgE-linked allergens (or IgG linked)
Cytokines and chemokines
Chemical agents
Physical stimuli
Insect and animal venom
Viruses
DAMPs (defensins, anaphalotoxins, IL-33, neuropeptides, etc)
What do bacterial peptidoglycans do to mast cells histamine release?
Trigger TLR2 = histamine release
What do bacterial lipopolysaccharides do to mast cells histamine release?
Trigger TLR4 = no histamine release
What are the primary cytokines that mast cells produce?
Primarily produce Th2 cytokines
- IL-4, IL-5, IL-6, IL-10, IL-13, and TNF-a
Capture/store IL-17 and release it during allergic reactions
How does degranulation of mast cells occur via IgE cross-linking?
Antigen cross-links IgE on two mast cell FcεRI –>
Activates their tyrosine kinases –>
activates phospholipase C –>
through mediators increases intracellular Ca2+ –>
more protein kinases –>
phosphorylation of myosin, creates “degranulation channels” –>
granules move to cell surface, fuse with membrane, and release contents
Other than causing degranulation, what does IgE cross-linking do to mast cells?
activates phospholipase A –>
membrane phospholipids produce arachidonic acid –>
increase transcription of LOX, COX, and cytokines –>
make leukotrienes, prostaglandins, and cytokines
What do mast cell granules contain?
- They are not homogenous
histamine and TNF or serotonin and cathepsin D
dopamine
leukotrienes and prostaglandins
lysosomal enzymes (including chitinases, chitin becomes a PAMP)
cytokines and chemokines (including Th2 cytokines and PAF)
heparin-containing granules rich in TNF-a (heparin stabilizes it)
What does IL-33 do?
Is a potent DAMP
- not secreted normally but released from nuclei of damaged cells
Promotes production of Th2 cytokines, IFN-y, TNF-a, and IL-2
Recruits eosinophils and promotes degranulation
Involved in M2 polarization and tissue repair
Enhances neutrophil phagocytosis
Activates mast cells and basophils
Binds to neurons to cause pruritus
What are the primary functions of mast cells?
Regulate local blood flow and influence cellular migration
Sentinel cells – can act as APC during ACD (get MCHII from DCs)
Control innate immunity
Promote wound healing, can kill bacteria in wounds
- IL-6 tells keratinocytes to make defensins
Influence eos fx
- IL-5, SCF, histamine, PAG, PGD2, leukotrienes, VEGF, adenosine, tryptase
What does the development and survival of mast cells depend on?
SCF signaling via KIT receptor on mast cells (c-kit gene)
Other than SCF, what cytokines/chemokines regulate mast cell development?
IL-3
IL-4
IL-9
IL-31
IL-33
CXCL 12
TNF-b
nerve growth factor
How do connective tissue and mucosal mast cells differ?
Connective tissue mast cells are constitutive and T cell-independent, whereas mucosal mast cells must be induced and are T cell-dependent
What is FcεRI?
a high-affinity IgE receptor
occurs in two forms
tetrameric form is predominant
- high numbers found on mast cells, basophils, and neutrophils
trimer form is found on
- dendritic cells, eosinophils, neutrophils, and monocytes
- function on APCs enhanced by IL-4 so creates positive Th2 feedback loop
What is FcεRII aka CD23?
low-affinity IgE receptor
on mast cells, B cells, NK cells, macrophages, DC cells, eos, and platelets
Help take IgE-allergen complexes to the spleen
Enhance antibody reponse
What can the house dust mite allergen Der p 1 do to FcεRII aka CD23?
Cleave it from the surface of mast cells
more soluble CD23 = promote IgE response
Which proteases do mast cells contain that can be used to classify them into different subpopulations in humans?
tryptase and chymase
In addition to “compound exocytosis”, how can mast cell release their granules?
small vesicles bud off from the cytoplasmic granules
- are shed as exosomes
- take contents, including miRNA and MHC II to distant cells
- may also take substance P to sensory nerve fibers
generate tunneling nanotubes that break off
can generate extracellular traps
- stimulated by IL-23 and IL-1b, releases IL-17
What are the ways that mast cells degranulate without using IgE?
IgG mediate through FcγRII
G-protein coupled receptors (MRGPRX2)
- neuropeptides, substance P, AMPs, eosinophil peroxidase
- compound 48/80
Complement mediated (C3a and C5a)
Pre-mediated activation (PRRs)
Dendritic cell-mediated activation
What are the two catecholamine receptors that mast cells have?
a- and b-adrenoceptors
What do molecules that stimulate the a-adrenoceptors (such as norepinephrine and phenylephrine) or block the b-adrenoceptors (such as propranolol) do to mast cells?
enhance mast cell degranulation
What do molecules that stimulate the b-adrenoreceptors (isoproterenol, epinephrine, and salbutamol) or block the a-receptors do to mast cells?
inhibit mast cell degranulation
How do basophils compare to mast cells?
Basophils are least common blood leukocyte (<1%)
Are functionally similar to mast cells
- basophils can be activated by IgE or TSLP
Origin and gene expression profiles are very different
- basophils do not need KIT but do need IL-3
basophils are short-lived, non-dividers
Basophils have smaller and fewer granules
- contain histamine and a restricted protease profile
What drives the development/production of eosinophils?
*IL-5, IL-3, and GM-CSF activating GATA transcription factors
What is the structure of an eosinophil?
slightly larger than neutrophils or basophils (12-17 um in diameter)
large segmented nucleus
many red eosin staining cytoplasmic granules
- in some adult sighthound they do not stain with eosin (pale grey or clear)
shape of granules vary by species
- cats are rod-shaped
- horses have very large granules
number of granules can vary
What are the functions of eosinophils?
Extracellular killing of helminths
Phagocytosis (less good than neutrophils)
Antigen presenting cells (MHCII, CD80/86)
Prime B cells to make Abs
Support plasma cell survival (APRIL & IL-6)
Activates mast cell and promotes histamine release
What are the 3 types of eosinophil granules?
large specific (crystalloid) granules
small primary granules
small dense vesicles
What are in eosinophil primary granules?
arylsulfatase
eosinophil peroxidase
acid phosphatase
What are in eosinophil crystalloid granules?
major basic protein (MBP)
eosinophil peroxidase (mainly responsible for killing ingesta)
eosinophil cationic protein
eosinophil-derived neurotoxin (endogenous ligand for TLR2)
cytokines and growth factors
enzymes
Which epithelial cytokines induce IL-5 production and therefore promote eosinophilia?
TSLP
IL-25
IL-33
(also IL-23)
What does IL-33 do to eosinophils?
Promotes them via induction of IL-5 production
also directly activates eosinophils and stimulates their adhesion, degranulation, chemotaxis, and surface protein expression
What do Th2 cells do to eosinophils?
mobilize eosinophils and promote IgE responses
What are some of the main eotaxins?
Released by mast cells:
- histamine (and breakdown product imidiazoleacetic acid)
- leukotriene B4
- 5-hydroxytryptamine
- platelet-activating factor
- numbered eotaxins (ex. eotaxin 1)
CXCL8 (IL-8) complexed to IgA
anaphylatoxins
What are “sombrero vesicles”?
“docking complex” forming in eosinophil plasma membrane by exocytosing granules during piecemeal degranulation
What are the two ways eosinophils can degranulate?
release of entire granule or piecemeal degranulation
How do eosinophils kill large extracellular parasites?
employ their Fc receptors to bind to antibody-coated parasites
–> release their granule contents directly onto the worm cuticle
–> damage the cuticles
How does paraneoplastic eosinophilia occur?
production of IL-5 or eotaxin 1, either directly by tumor cells or indirectly by tumor-infiltrating Th2 cells
Which drugs can cause eosinopenia?
GC, catecholamines (β-adrenergic receptor)
What are the primary cytokines produced by eosinophils?
IL-1, IL-6, IL-31, TNFα, GM-CSF, IL-5
What are direct fluorescent antibody tests used for?
identifying antigen in a tissue sample
*uses the patient’s tissue
What are indirect fluorescent antibody tests used for?
measuring antibodies in serum or identifying specific antigens in tissue
*uses the patient’s serum
How does an indirect ELISA work?
Wells are precoated with antigen
Test serum is added so antibodies bind
A labeled antiglobulin is added and binds to antibodies
An enzyme substrate is added and color changes
*Most common form in experiments
How does a direct ELISA work?
Wells are precoated with antigen
Substance with label antibody is added
An enzyme substrate is added and color changes
How does a sandwich ELISA work?
Wells are precoated with specific capture antibody
Test antigen is added
Detection antibody is added
A labeled antiglobulin is added and binds to antibodies
An enzyme substrate is added and color changes
*good for things like circulating viruses
How does a labeled antigen ELISA work?
Well is precoated with antigen
Test serum is added
Enzyme-labeled antigen is added
An enzyme substrate is added and color changes
*most common form in test kits
How does a competitive ELISA work?
Well is precoated with an antibody
Test antigen and labeled antigen are added
An enzyme substrate is added and color changes
*good for haptens and virus molecules
How does Western blotting work?
Antigen is added
electrophoresis
blot/transfer to a nitrocellulose paper
add a labeled antibody
visualize bands
How does a multiplex assay work?
it is a derivative of an ELISA using beads for binding the capture antibody
microspheres are precoated with antibodies and added to wells
Add the sample
Add the detection antibody
Add the reporter dye
results can be read by flow cytometry
What is a microassay?
aka gene chip or DNA chip
detect the expression of thousands of genes at the same time
analyze gene expression and detect DNA sequences
What is an inflammasome?
large cytosolic multiprotein complexes that assemble in response to detection of infection- or stress-associated stimuli and lead to the activation of caspase-1-mediated inflammatory responses
typically consist of a sensor protein, the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and the proinflammatory caspase, caspase-1
Which adhesion molecule does histamine increase?
P-selectin
What is canine leukocyte adhesion disorder type 1?
AR mutation in CD18 (b-2 integrin gene = ITGB2) on leukocyte surfaces seen primarily in Irish Setters and GSDs, characterized by recurrent bacterial infections
Usually fatal by 6 mo of age
What is canine leukocyte adhesion disorder type 3?
AR mutation in FERMT3 (kindlin-3) which is seen on leukocytes and platelets and regulates integrin activity seen in GSDs
