Chapter 7
Why do IMMUNE cells endocytose?
To remove a pathogen Present an antigen Remove dead/dying cells or debris
Non professional phagocytes include lymphocytes, fibroblasts, epithelial cells, endothelial cells, mesenchymal cells, immature cell types
True
True or False: Most nonprofessional phagocytes don't produce ROS in response to phagocytosis
True
True or False: All nucleated cells can endocytose
True. Some may be better than others and some may have receptors.
True or false: PMA oxidizes DHR to make it fluorescent
True. You can then measure the fluorescence using flow cytometry
True or False: Some bacteria avoid being phagocytized - some have found way to survive - exploit and make process work to their advantage
True: phagocytes can get around phagocytosis - this is why we need adaptive immune response
What color do specific granules stain?
Unstained, example are collagenase, gelatinase, lactoferrin, etc
When do monocytes and macrophages arrive to the site of infection?
Within 1-2 days. Often found in more chronic inflammatory conditions.
In endocytosis, a piece of plasma membrane invaginates and close, forming an ______
endosome
What happens to pH as you move further along the compartments of the endocytic pathway?
pH is progressively dropping
What kind of cells can use macropinocytosis?
•Antigen presenting cells (APC) use this
List the 5 steps in ROS-independent killing
•Electrically charged proteins damage bacterial membrane •Lysozyme breaks down cell wall •Lactoferrins withhold essential iron from bacteria •Proteases and proteolytic enzymes digest bacterial proteins •Elastase and cathepsin G cleave elastin
Describe Caveolin-mediated endocytosis
•Flask shaped pits enriched with caveolin form very small (50nm) caveolae •Abundant in smooth muscle, lung, adipocytes, endothelium, fibroblasts. Constitutes up to 1/3 of plasma membrane
How does macropinocytosis work?
•Membrane ruffles or invaginates to ingest large volume of extracellular fluid •Fuses with lysosome for digestion
What are cons to phagocytosis?
•Phagocytes may release ROS and proteases into local tissue, causing damage •Contributes to diseases like chronic obstructive pulmonary disease •Sometimes phagocytosis can't clear a pathogen, so macrophages try to wall it off (leading to granuloma formation)
List and define the three types of endocytosis
•Pinocytosis: drink •Micro: sip •Macro: gulp
Describe Clathrin-mediated endocytosis
•Pits coated with clathrin form small (100nm) vesicles •Uses: endocytose nutrients, LDL, transferrin, growth factors, antibodies
What is the second stage of phagocytosis?
2. Ingestion of microbe by phagosome
What is the third stage of phagocytosis
3. Formation of a phagosome
What is the fourth stage of phagocytosis
4. Fusion of the phagosome with a lysosome to form a phagolysosome
What causes increased production of colony stimulating factors, leading to increased production of neutrophils?
Acute infections
Provide examples of opsonins
Antibodies, complement (C3b), and other soluble PRRs
How does phagocytosis affect adaptive immunity?
Antigen chopped into smaller fragments; fragments bind to MHC proteins; antigen-presenting cells travel to LN and present antigen adaptive immune cells to induce and activate adaptive immune response.
Macrophages serve as?
Antigen presenting cells that •Stimulate TH cells to secrete IFN-γ •Secrete proinflammatory cytokines to direct adaptive immune responses
What is a precursor for NO?
Arginine
What two type of granules do neutrophils contain?
Azurophilic (primary) and specific (secondary) granules
What are immature neutrophils called?
Band neutrophils. older neutrophils have more segmented nuclei.
Where is NOS I located?
Brain; important for neurodevelopment
How does NADPH catalyze the formation of superoxide?
By moving one electron from NADPH to oxygen
How do activated macrophages contribute to tumor destruction?
By producing TNF-α, IFN-γ, NO, ROS, cationic proteins, hydrolytic enzymes
Which enzyme has the highest turn over number
Catalase
What is the purpose of catalase?
Catalyzes the decomposition of hydrogen peroxide into water and oxygen.
What macrophage product do antimicrobial peptides use?
Cathelicidin family - contain defensins (heterogenous - look a lot different) work by damaging and puncturing cell membranes; help destroy lipoprotein membranes inside the phagosomes. Macrophage elastase derived peptide
What does Caveolin-mediated endocytosis use?
Cholesterol and lipid metabolism (highlighted on slide), cell signaling, turnover of adhesion molecules. Continuous uptake of extracellular molecules, some containing protein and cholesterol (lipid rafts) which are important in lipid metabolism and cell signaling
Antigen presentation to TH cells causes what?
Clonal proliferation and cytokine production. This stimulates B cells to make antibodies and Tc cells to kill infected host cells.
What does Myeloperoxidase do?
Combines hydrogen peroxide and chlorine to make hypochlorite
Where are the nonstimulated neutrophils located in?
Components are inactive in cytosol
Which test is preferred, DHR or NBT?
DHR is preferred because it can distinguish between X-linked and autosomal forms. It can also detect carriers
What neutrophil product do antimicrobial peptides use?
Defensins - small, cystine-rich cationic proteins; active against many different pathogens (bacteria, fungi, viruses) can directly kill microbes by disrupting microbial cell membrane; disrupt metabolism for cells to stop processing things.
What are the best professional APCs for activating naive T cells?
Dendritic Cells (DCs). •Immature DCs continuously sample the environment using all types of endocytosis or direct contact with apoptotic or infected cells •Mature DCs migrate to lymph nodes or secondary lymphoid organs and present antigens to T/B cells to initiate an adaptive immune response
What is the fifth stage of phagocytosis?
Digestion of ingested microbe by enzymes
What test involves the stimulation of PMA?
Dihydrorhodamine (DHR) test
What is the seventh stage of phagocytosis?
Discharge of waste materials.
Most of the superoxide produced undergoes _________ to hydrogen peroxide and oxygen spontaneously or by_________ ___________
Dismutation; superoxide dismutase
List 2 reasons why nucleated cells endoyctose?
Drug or nutrient uptake: there is something the cell need from the outside Receptor downregulation: "This is needed so that we are able to control signaling molecules . We need to have a way to be able to take them outside of the plasma membrane and bring them inside the cell so that they are not able to receive signals outside" --> Dr. Morrow's words!
Where is NOS III located?
Endothelial cells
In Neutrophil Extracellular Traps (NETs), neutrophils _______ ___ and cellular components to form a NET
Extrude DNA
True or false: Endocytosis is unable to take up polar molecules.
False. It is actually helpful for taking up polar molecules.
True or False: Non-profesional phagocytes have phagocytosis as their primary function
False: most do not have phagocytosis as their primary function. They can be induced, however, to phagocytose under certain conditions (e.g. inflammation). More limited in the type of particles they can pick up
True or False: Specific granules stain positive
False: they are usntained. Examples are MPO, proteolytic enzymes, defensins etc.
What is the sixth stage of phagocytosis?
Formation of residual body containing indigestible material
Where is NADPH oxidase system found?
Found in eosinophils, neutrophils, monocytes, and macrophages. Highest in eosinophils and second highest in neutrophils
What includes neutrophils, eosinophils, basophils, and mast cells?
Granulocytes (aka polymorphonuclear cells/PMN). Note that PMN and neutrophil can be used interchangeably
What is the primary difference between phagocytosis and pinocytosis?
In phagocytosis, SOLID particles are endocytosed, where as pinocytosis deals with liquids
How do we use lactoferrins?
Iron is a growth-limiting reagent - bacteria steal iron from you - we can use lactoferrins to tie up iron for ourselves and limit bacteria growth
Where is NOS II located?
Macrophages; help modulate vascular tone and vasodilation
What is contained in neutrophil granules?
Myeloperoxidase
What is oxidative burst mediated by?
NADPH oxidase
Tests that measure production of ROS are dependent on what?
NADPH oxidase function
What is used to test for chronic granulomatous disease?
NBT
What does a negative NBT test tell you?
NEGATIVE in patients bc testing for production of ROSs (negative --> disease, cells not making ROSs). Positive indicates normal ROS enzyme
What does NET do to pathogens? What can happen if DNA gets exposed?
NET traps pathogen and enzymes kill it. Exposed DNA can lead to autoimmunity
Oxidase activity is generally not present in resting, non-stimulated __________
Neutrophils
Kaitlyn is a very clumsy person and cut her hand with a butter knife while trying to get peanut butter. Which blood cell will be the first to respond to the injury site?
Neutrophils are first to respond and can reach the site in about 30 minutes to release chemostatic signals.
What kind of cells are phagocytic?
Neutrophils, monocytes, macrophages, dendritic cells, etc.
What catalyses the nitric oxide reaction?
Nitric oxide synthase
What are the 2 main tests to measure how neutrophils are producing ROS?
Nitroblue tetrazolium (NBT) test and Dihydrorhodamine (DHR) test
What do you call factors that facilitate or enhance phagocytosis? How do they help phagocytosis
Opsonins. They faciliate phagocytosis by having a coat or marking the target.
Lysosomes degrade material by what two forms?
Oxygen-dependent or oxygen-independent pathways
What induces macropinocytosis?
Pathogens can induce macropinocytosis (e.g. salmonella)
What does NO + O2 form?
Peroxynitrite (ONOO-): This damages DNA and proteins (BAD!)
What does the innate immune system heavily rely on?
Phagocytes
What triggers phagocytosis?
Phagocytes have receptors for opsonins - binding of opsonins to receptors tells phagocyte they need to eat it
What is phagocytosis
The eating of material. Process where cells bind and engulf particles.
What is exocytosis?
The exporting of material. This is the opposite of endocytosis
What is endocytosis?
The ingesting of material. A cellular process in which a molecule from the outside environment is brought into the cell
Which is more reactive, superoxide or hydrogen peroxide?
Superoxide
What does NADPH catalyze the formation of?
Superoxide (free radical)
Describe how Nitric Oxide is made
(NO) is made by converting L-arginine into L-citrulline in the presence of oxygen. NO is an important second messenger.
What is the first stage of phagocytosis?
1. Chemotaxis and adherence of microbe to phagocyte. Something sticks to the phagocyte (receptor-mediated process)
_________ _________ have PRR that detect/bind PAMPs and phagocytose pathogens
Professional phagocytes
What do oxygen-independent pathways rely on?
Proteolytic enzymes that help break protein bonds (lysozymes, etc.)
What do do oxygen-dependent pathways produce?
Reactive Oxygen Species (ROS)
What is another name for Clathrin-mediated endocytosis?
Receptor-mediated endocytosis
Increased consumption of O2 after phagocyte ingests material is referred to as?
Respiratory or oxidative burst