Mandl - Granulopoiesis Flashcards
What are the different flavours of ganulocytes and of Monocytes/Monocyte-derived cells/myeloid cells?
*Part of the innate immune response
Granulocytes:
Neutrophils
Eosinophils
Basophils
Mast cells
Myeloid cells:
Monocytes
macrophages
Dendritic cells
CMP (common myeloid progenitor) → GMP (granulocyte monocyte progenitor) → Neutrophils/Eosinophils/Basophils + Monocytes
Monocytes → Macrophages + DCs
What is the problem with using blood to study the immune cells?
There are some caveats, as a lot of the action happens in the tissue and blood is imperfect read out as require that cells are recirculating by the blood, which does not consider tissue-resident cells
Which leukocytes are agranulocytes?
Lymphocytes (25-45% of leukocytes)
Monocytes (3-8% of leukocytes)
What is the life cycle of Neutrophils like?
Short life span ~ 5 days in circulation of humans (mice ~ 12hrs)
Adult humans produce ~10^11 cells daily in the bone marrow → fully mature in the BM (G-CSF stimulates production & release) → go into circulation → liver, spleen → apoptosis & degradation by macrophages (some feedback to the bone marrow)
*Strong circadian component!!!
In infection/Inflammation:
Neutrophils also go to other tissues from the blood + feedback signal to the bone marrow → increased production & release from bone marrow
What are the main functions of neutrophils?
What pathogens are you primarily susceptible to if you do not have functional neutrophils?
- Essential for resistance to invasion by microbes → early responders
- Important sentinel cells
No neutrophils → very sensitive to bacterial infections
What are the different steps of Neutrophil response?
- recruitment to the site of infection via blood vessels
- extravasation from blood vessel
- migration through tissue
- activation in response to pathogens, their products or the products of distressed cells
- via various pattern recognition receptors (PRRs) - initiation of mechanisms to eliminate pathogens or infected cells
- interaction with & recruitment of other immune cells to site of infection
- contribution to resolution of inflammation & tissue repair
We have 11,000km of blood vessels, how do neutrophils know where to exit?
What are the steps for them to exit circulation?
- Vascular endothelium changes
- P-selectin, E-selectin (on endothelial cells)
- bound by P-selecting glycoprotein ligand-1 (PSGL1) and ESL1 (E-selectin ligand) and CD44, on neutrophils
- tethering → slow rolling - Adhesion
- Interaction between chemokine receptors on the neutrophil and chemokines coating the vessel
- Activation of integrins (on neutrophils), ligands on the endothelial cells - Transmigration
- Requires integrins
- can involve digestion of basement membrane with secreted proteinases that digest ECM components
What are the chemokines and chemokine receptors involved in the adhesion step of neutrophil recruitment?
Chemokines = chemical messengers which induce directional movement of cells, detected by chemokine receptors
4 families of chemokine receptors → GPCRs
- CXC, CC, CX3C, XC
Adhesion = Interaction between chemokine receptors on the neutrophil and chemokines coating the vessel
1. CXCL2 - CXCR2
2. IL8 (CXCL8) - CXCR1 or 2
Which integrins are activated in the adhesion and transmigration phases of neutrophil recruitment?
Integrins on the neutrophils: LFA-1 and MAC-1
Interact with the ligands ICAM1 and ICAM2 (on endothelial cells)
How do neutrophils get to the site of infection when they are out of blood vessels?
What is the specific name of this process?
Chemotaxis → directional movement/migratory process of cells along a concentration gradient of a chemical stimulus
Neutrophil chemoattractants:
- Lipid mediator LTB4
- bacteria-derived N-formyl-metionyls-leucyl-phenylalanine (fMLP)
- complement component C5a
- CXCL8, CXCL2
What happens if neutrophils do not express integrins?
Leukocyte adhesion deficiency:
LAD1 is an inherited autosomal recessive disorder with mutations int he ITGB2 (b2 integrin, CD18) leading to impaired neutrophil migration, adhesion and recruitment to site of infection, impaired phagocytosis
What are the different immune mechanisms of actions of neutrophils?
- Phagocytosis → engulfment & internalization of large particles
- Oxidative burst
- Neutrophil extracellular traps (NETs)
- Cytokine secretion
- Production of antimicrobial peptides & proteolytic enzymes (release of their granules)
What are the different steps of phagocytosis?
- attachment
- engulfment
- phagosome formation
- formation of phagolysosome
- digestion of pathogen
- formation of residual body
- discharge of waste
*Requires actin cytoskeleton remodelling
What are the roles of neutrophilic granules?
Granules are highly specialized lysosomes (found inside the neutrophils) which contain many antimicrobial substances:
– prevent growth by limiting availability of essential nutrients inside phagosome or by compromising the integrity of the microorganism
– contents can be released into the phagosomes or extracellularly (degranulation)
*Pro-inflammatory role which can also lead to tissue damage
How can neutrophils induce oxidative bursts?
Take place at cell membrane or phagosome membrane
- Essential microbicial mechanism
- Generates reactive oxygen intermediates (ROI)
Requires an NADPH oxidase complex:
NADPH + O2 → H2O2
*Microbes are killed intracellularly by H2O2 in the phagosome
What happens if neutrophils are unable to make reactive oxygen intermediates (name of the disease)?
How can this be tested?
Chronic granulomatous disease: defective phagocyte NADPH oxidase (PHOX) leading to reduced reactive oxygen production – recurrent & atypical infections
Assess DHR (fluorescent dye) which is produced if cells are able to undergo redox reactions (activated WT neutrophils show more fluorescent signal)
Type of infections these patients get: bacterial and parasitic infections
What are neutrophil extracellular traps (NETs)?
First described in 2004
Neutrophil activation triggers them to undergo a specific type of cell death
→ Nuclei swells, chromatin are dissolved and the decondensed DNA is expelled from the cell (NETosis)
- DNA is coated in histones & antimicrobial proteins from neutrophilic granules
- NETs form sticky traps that can kill pathogens or trap them so they don’t spread
What is a cytokine? Which ones are produced by neutrophils?
Cytokine: a protein secreted by an immune cell that has an effect on nearby cells expressing the apropriate receptor
Cytokines produced by neutrophils:
- IL-a/-b
- TNF-a
- IL-17
- VEGF (induces vascular regeneration)
Which cytokine recruits Eosinophils? In what context?
Recruited form the blood into tissues by Interleukin-5 → produced in allergic reactions, to amplify the inflammatory response
- CCR3 (on eosinophil) binds CCL11 (eotaxin1)
- Role in type II immune responses (including airways dysfunction
What is the role/main mechanisms of active eosinophils?
- Granule content release involved in anti-parasitic responses: contain enzymes, growth factors, cytokines, chemokines
- Produce inflammatory lipid mediators
- Have homeostatic roles as well (wound healing, found in adipose tissue)
What are similarities and differences between basophils and mast cells?
Basophils:
- Bi-lobed nucleus
- Produced in the bone marrow, recruited from the blood
Mast cells:
- Single lobed nucleus
- Seeded in tissues during early development and turnover in that tissue → localized in tissues around blood vessels & nerves
Both:
- Large number of cytosolic granules (dark/blue stain)
- Produce histamines
- Associated with TH2 CD4 T cell responses
- Provide protective immunity to helminths & ticks (larger parasites)
- Implicated in response to allergens
What are lipid mediators?
Eicosanoids (large family including arachidonic acids)
- Locally acting bioactive lipids
- Cleaved from cell membrane phospholipids by phospholipases → Arachidonic acid → (5-LOX or COX-1/-2) → Eicosanoids
Classical eicosanoids:
- Leukotrienes: leukocyte recruitment (LTB4 produced by neutrophils)
- Prostaglandins: inhibition of platelet aggregation, promote vasodilation & vascular leakage (PGE2); platelet aggregation (thromboxane A2)
Produced by eosinophils, neutrophils, mast cells, macrophages, endothelial cells, etc.
What are the basic cellular features of monocytes?
Mononuclear cells, kidney-shaped nucleus, 4-10% of WBCs
- Mostly in blood but pools exist in spleen that can be mobilized
- Development in mice dependent on colony stimulating factor 1 (CSF-1/M- CSF)
- Continuously generated in bone marrow
What are the 2 types of Monocytes?
- Patrolling monocytes (mice: Ly6cLow , humans: CD16+) maintain blood vessel integrity, detect pathogens
- In circulation at steady state - Inflammatory monocytes (mice: Ly6c+, humans: CD14+) are rapidly recruited to sites of infection or injury
Both can differentiate into macrophages or dendritic cells upon recruitment into tissues during inflammation (transient populations)
What specific macrophages are found in each the following tissue?
Lungs, Liver, CNS, Bone, Adipose tissue, Lymph node
Lungs: Alveolar macrophages → sit in the epithelial layer between alveolar sacs
Liver: Kupffer cells → clearance of aged erythrocytes
CNS: microglia → synaptic remodelling & removal of dead neurons
Bone: osteoclasts → resorb bone
Adipose tissue: → regulation of metabolism, insulin resistance
Lymph node: CD169+ subcapsular sinus macrophages
What specific macrophages are found in each part of the spleen?
Spleen: white-pulp, red-pulp, marginal zone and metallophilic macrophages → erythrocyte clearance, immune surveillance, iron metabolism
Where are macrophages mostly found?
How large are they?
Large → 15-85 mm
- professional phagocytes
- Resident within tissues
- They arrive at the site of infection after neutrophils and phagocytose the whole plug (with neutrophils + foreign organisms)
- Important role in deciding if the immune system should mobilize an army (recruit neutrophils) or not/sample the environment
Early paradigm → monocytes replenish macrophages in tissues
Recent insight → most macrophage populations are seeded during embryogenesis & replenished by division within tissue
What is the M1/M2 paradigm?
Depending on their exposure to activating stimuli, as well as other signals from host tissues macrophages can become M1 or M2
IFN-y produced by TH1 cells, CD8 T cells or NK cells and TNF-a produced by APC → M1 macrophages
- Have microbicidal activity, produce IL1-, IL-6 and IL-23: pro-inflammatory
IL-4 produced by TH2 cells or by granulocytes → M2 macrophages
- Have wound-healing activity & tissue repair, produce ECM proteins, can be immmunosupressive
What are some important features of the immune system?
PCIUTRE ON PHONE
What is the difference in definition between Immunity, Immune System and Inflammation?
Immunity: The ability to resist infection to a particular pathogen.
Immune System: The tissues, cells and molecules involved in innate immunity and adaptive immunity.
Inflammation: General term for the local accumulation of fluid, plasma proteins, and white blood cells that is initiated by physical injury (sterile dammage?), infection, or local immune response.
Where does the word inflammation come from?
From the latin inflammable: “to set on fire”
- Reaction to injury or infection
- Heat, redness, swelling, pain,
What are the 2 school of thoughts around the general underlying mechanism of Immunity?
How are they associated with?
Cellular vs Humoral (1900s)
- Protective function lies with cells (Elie Mechnikoff)
- By studying starfish and seeing phagocytic activity and it is more present in immunized animals - Protective function lies with humor (cell-free bodily fluid) (Paul Ehrlich)
- More popular, more tools to study it
Today, the controversy has been resolved: cellular & humoral immunity are intertwined systems which are both required for an immune response
Which 2 theories are part of humoral immunity?
Selective theory: Antigen selects the appropriate receptor which has a specificity that is determined before encounters the Ag
*We now know this is the right theory
Instructional theory: Ag serves as a template around which the Ab would assume its configuration
*Antigens = any molecule that binds specifically to an antibody or T cell receptor
What are the 4 classes of pathogens the immune system protects us against?
- Extracellular bacteria, parasite, fungi
Ex: Pneumonia, Tetanus, Spleeping sickness - Intracellular bacteria, parasites
Ex: Leprosy, leishmaniasis, malaria - Viruses (intracellular)
Ex: Smallpox, Chickenpox, Flu - Parasitic worms (extracellular)
Ex: Ascariasis, Scistosomiasis
*Large variations in size
What are the different anatomical barriers to infection? (1st layer)
*All have epithelial cells joined by tight junctions which form a mechanical barrier + normal microbiota
Skin
Mechanical → Longitudinal flow or air/fluid
Chemical → Fatty acids, b-defensins/lamellar bodies/cathelicidin
Gut
Mechanical → Longitudinal flow or air/fluid
Chemical → low pH, Enzymes (pepsin), a-defensins/athelicidin
Lungs
Mechanical → movement of mucus by cilia
Chemical → pulmonary surfactant/a-defensins/cathelicidin
Eyes/nose/oral cavity
Mechanical → Tears, nasal cilia
Chemical → Enzymes in tears and saliva (lysozymes), Histamines/b-defensins
When is an immune response initiated in terms of layer?
When the anatomic barriers are breached
What are the different properties of the Immune System?
- must identify foreign vs. self
- nature of immune response varies depending on the number and type of recognition molecules that are engaged
- Two types of receptors: germline encoded (innate) vs. somatically generated (adaptative, by recombination)
What are the 2 arms of the immune systems involved in Recognizing and reacting to pathogens?
Innate immunity:
Non-antigen specific host defenses that exist prior to exposure to an antigen (before induction of adaptive immunity) and involve anatomic, physiologic, endocytic and phagocytic, anti-microbial, and inflammatory mechanisms. Innate immunity does not increase with repeated exposure to a given pathogen.
- Receptors are germline-encoded → recognize PAMPs via PPRs
- Limited specificity
Adaptative immunity:
Host response mediated by B cells and T cells following exposure to antigen and that exhibit specificity, diversity and memory.
- Receptors are uniquely generated within each cell
- Specificity is unlimited
Where in evolution did innate/adaptative immunity occur?
Every multi-cellular organism has an innate immune system
Only jawed vertebrates have adaptative immunity as we have it
- Jawless vertebrates also have adaptive immune system but different type of receptor and diversification mechanism (they do have B cell and T cell-like cells
Which are the cellular and humoral mediators of innate immunity?
Cellular mediators:
- dendritic cells, monocytes, macrophages, granulocytes (from Common Myeloid Progenitor)
- Natural Killer cells (from common lymphoid progenitor)
Humoral:
- Pre-existing serum proteins known as complement, anti- microbial proteins
What is the purpose of innate immunity? And its kinetic scale?
Goal: early clearance, activation of adaptive response, healing & tissue repair
Kinetics:
- Quick response (min – hours)
- Identical response to secondary infection
How is communication between cells of innate and adaptative immune system made?
Communication between cells of innate and adaptive immune system is via cell-cell contact or via chemical mediators:
- Cytokine: protein secreted by an immune cell that has an effect on nearby cells expressing the appropriate receptor (eg. interleukins, interferons)
- Chemokine: chemoattractant proteins that mediate chemotaxis and adhesiveness of leukocytes
What are the cellular and humoral mediators of adaptative immunity?
What is its goal and its kinetic scale?
Cellular mediators: B and T lymphocytes
Humoral mediators: antibodies
Goal = clearance of pathogen, immune memory
Kinetics:
- Response takes longer to develop (days)
- Rapid response to secondary infections with higher magnitude
What is clonal selection?
it refers to te fact that we have a very broad repertoire or receptors and a single cell encoutering its specific antigen will undergo clonal expansion and produce antibodies
*Tolerance is essential to be maintained through diversity → maintain unresponsiveness to self
What are the 3 phases of an immunen response?
Once the anatomical barrier has been breached:
1. Innate phase
2. Early induced innate phase
3. Adaptative immune phase
*Layering
What are the primary lymphoid organs?
Where immune cells develop
Bone marrow:
– site of self-renewal and differentiation of
hematopoietic stem cells (HSCs)
– site of B cell development and of mature plasma cells & T cell memory
Thymus:
– site of T cell development & generation of T cell receptors
What are the secondary lymphoid organs?
Sites of initiation of adaptive immune response (priming)/hubs for lymphocytes:
- Spleen
- Lymph nodes
→ Interconnected by vascular and lymphatic system
How is the spleen organized as a secondary lymphoid organ?
Role in mounting immune responses to blood born antigens (systemic infections)
- Blood enters through the splenic artery and leaves by the splenic vein
- White pulp = where T and B cells sit (marginal zone w/T cells + PAL/B follicles)
- Red pulp = removes waste from the blood and gets rid of old or damaged blood cells, lymphocytes and macrophages (surrounds the white pulp)
What is the organization of lymph nodes?
Highly organized SLO where immune cells meet: DCs, B cells, T cells, macrophages
Lymphocytes entry is through high endothelial venules (HEV) → Similar steps of entry then entry into a tissue (Rolling, Activation, Adhesion, Transmigration)
Lymph node infrastructure = stromal cell network of fibrous mesh (ECM) → layed down by fibroblastic reticular cells, T cells move along this network and DC positioned along this network to present antigens
What is the purpose of the lymphatic system?
- Movement of interstitial fluid that is derived from plasma = lymph
- Super highway for lymphocytes (continuous recirculation)
- Drains antigen to lymph nodes
- Remove wastes and drain fluid in tissues
What are the basic features of the dendritic cells?
What are the 2 main types of DCs?
- Stellate/dendritic morphology
- Speicalized antigen-processing & presenting cells
- Express MHC-I and MHC-II, as well as PRR which enable their activation in response to microbial stimuli
Heterogeneous population with many subsets defined by distinct phenotypes/anatomic location:
- cDC – classical/myeloid: APCs initiating T/B immunity
- pDC – plasmacytoid: secrete large amounts of type I interferon (IFNa) during viral infections
What are important functions of DC?
How do they traffic around?
- Initiate, coordinate and regulate adaptive immune responses, maintain tolerance to self
- Resident population in tissue take up antigens
- Migrate to lymphatics and then traffic to 2ndary lymphoid organs where initiate adaptative immune responses
What are general treatments for disorders of granulopoiesis?
- Prophylactic methods to reduce exposure to infections
- Antimicrobial drugs to prevent and treat infections
- Administration of colony stimulating factors:
G-CSF & GM-CSF - Bone marrow transplantation:
Ablation of bone marrow of affected individual with cytotoxic drugs, followed by transplantation of healthy donor cells – bone marrow, cord blood or enriched hematopoietic stem cells
What are important differences between dendritic cells and macrophages?
Antigen presentation → DCs are highly specialized in antigen presentation to T cells, migrating to lymph nodes to activate naive T cells, whereas macrophages mainly present antigens locally within tissues
Tissue distribution → DCs are strategically located in tissues where pathogens are likely to enter (like skin and mucosal surfaces), while macrophages are more widely distributed throughout the body.
Immune response → DCs are considered the primary initiator of adaptive immune responses by activating naive T cells, while macrophages contribute more to the innate immune response by eliminating pathogens and debris.
What type of pathogens do the following examples belong to?
- Leishmania
- Pneumonia (Streptococcus pneumonia)
- Tetanus (Clostridium tetani)
- Smallpox
- Flu (influenza)
- Schistosomiasis (Schistosoma)
- Leishmania → intracellular bacteria/parasite
- Pneumonia (Streptococcus pneumonia) → extracellular bacteria/parasite
- Tetanus (Clostridium tetani) → extracellular bacteria/parasite
- Smallpox → virus (intracellular)
- Flu (influenza) → virus (intracellular)
- Schistosomiasis (Schistosoma) → parasitic worm
