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