Explain the process of phagocytosis.
What is a phagosome?
What are 2 consequences of phagocytosis?
Phagocytosis is the process by which certain cells (e.g., neutrophils and macrophages) of the innate immune system engulf large particles (>0.5 Pm). In this section we will cover the steps involved in elimination of microbes by phagocytic cells as well as to discuss how the innate immune system ultimately triggers the adaptive immune system to respond to a microbe.
- The process involves extension of the plasma membrane of the phagocyte surrounding the particle.
- The internalization of the particle by the phagocyte leads to the formation of a phagosome, which surrounds the particle and leads to its ultimate destruction and removal from the body.
- The destruction of the particle also allows for its components to be presented to T-cells in the form of small peptides (8-15 amino acids) by major histocompatibility molecules on macrophages and specialized phagocytes known as dendritic cells.
Phagocytes are non-replicative cells of the innate immune response that are capable of phagocytosis
What are the two major phagocytic cells of the immune system? What part of immunity (innate or adaptive) are they a part of?
What progenitor do they develop from?
What kind of antigens do they respond to?
Neutrophils [also known as polymorphonuclear leukocytes (PMN)] and macrophages (discussed in Innate Immunity lecture notes) are major phagocytic cells of the innate immune response to microbial pathogens.
Neutrophils and macrophages originate from a common myeloid progenitor. These two cells interact with and internalize microbes such as bacteria, fungi and small parasites.
Where are neutrophils stored? What is their life span in blood? What recruits neutrophils into infected areas? What is this process called? What is the life span of neutrophils once in infected tissue?
Where do macrophages reside? What are they derived from? What is the lifespan of macrophages?
What is the difference btwn the consequences to macrophages and neutrophils after phagocytosis?
What is the difference in response to extracellular and intracellular pathogens btwn macrophages and neutrophils?
- Neutrophils are stored in the bone marrow and released into the peripheral blood (40-70% of all leukocytes are neutrophils) where they migrate for approximately 5 days before they die.
- Neutrophils will rapidly move from the blood into the inflamed/infected tissue if recruited by chemokines.
The movement of neutrophils in between blood vessel epithelial cells into inflamed or infected tissue occurs though a process known as diapedesis. Once in the tissue the neutrophil will live for 1-2 days.
Macrophages are tissue-localized and do not migrate in or out of the tissue they reside.
Macrophages originate from monocytes, which are present in the blood. Monocytes move into tissues and differentiate into macrophages. Once a macrophage is present in tissue they can survive for months.
Neutrophils die after they destroy microbes whereas macrophages are not prone to dying in the process of killing microbes.
Macrophages target extracellular and intracellular microbes. Neutrophils target extracellular bacteria
What receptors are involved in diapedesis of neutrophils into infected tissues? Explain the role of chemokines in this process.
Neutrophils bind chemokines (IL-8) on endothelial cells secreted by macrophages and other cells within the infected tissue. Upon binding IL-8, neutrophils bind ICAM-1 (intracellular adhesion molecule 1) on the endothelial cell with their LFA-1 receptors (leukocyte functional antigen 1).
What receptors are present on neutrophils and macrophages that allow them to bind pathogens? What do they bind to on pathogens?
Step 1: Recognition of PAMPS and complement fragments on microbes by PRRs and complement receptors on phagocytes:
Macrophages express receptors (PRRs) which recognize PAMPs and complement on pathogen surfaces and leads to the internalization of pathogens into the phagocytes (innate immunology lecture). These receptors are also found on neutrophils and act like they do on macrophages.
Explain what occurs next in neutrophils and macrophages after ingestion of pathogens. What is the difference btwn the fate of macrophages and neutrophils after completion of this step?
Step 2: Fusion of release of phagosome with granules containing proteins and enzymes that kill microbes.
Once the phagosome (internalized vesicle containing microbe) is formed, it fuses with specific and azurophilic granules which contain anti-microbial peptides, enzymes and toxic protein. These peptide/proteins attack specific components in the microbe and “poke holes” in the bacterial membrane. The pH of the phagosome changes to neutral pH and enzymes become active. Eventually the phagosome’s pH drops and also fuse with lysosomes containing acid hydrolase. Acid hydrolases digests the microbe completely.
Note that neutrophils undergo apoptosis after degradation of the pathogen and are phagocytized by macrophages (see last box in attached pic).
In addition to things discussed in the previous card (granules with peptidases, pH, etc.) what else is used by macrophages and neutrophils to destroy pathogens?
Step 3: Toxic oxidative products and nitric oxide are also involved in the destruction of ingested microbes.
What is the main ROS produced in defense against pathogens? What are enzymes, substrates, and products?
What are the most potent ROS produced from the main ROS?
How is NO produced? What enzyme catalyzes this rxn?
How do ROS and NO lead to destruction of pathogens?
Reactive oxygen species: O2 is converted to reactive oxygen species (VERY UNSTABLE OXYGEN SPECIES) through the initial action of NADPH oxidase in which NADPH is converted to NADPH H+, which leads to the transfer of electrons to oxygen to form superoxide anion. (see attached-slide 17 of pp). From superoxide, hydroxyl radicals and singlet oxygen species are the most toxic ROS.
In addition to toxic oxygen molecules, nitric oxide (NO) is formed when arginine is converted to the citrulline and NO by Inducible Nitric Oxide Synthase (iNOS) in the presence of NADPH. NO in the presence of superoxide anion form a toxic peroxynitrite, which leads to lipid peroxidation, protein oxidation, protein nitration and inactivation of microbial enzymes.
In the following syndromes, state what the defective gene/protein is, the functional effect, and the clinical effect:
chronic granulomatous disease (CGD)
glucose-6-phosphate dehydrogenase (G6PD) deficiency
Note that myeloperoxidase deficiency often actually has no clinical effect bc the other ROS produced are more potent than myeloperoxidase (hydroxy radical, singlet oxygen radical)
attached is slide 19 of PP
What is the major cell type involved in antigen presentation to T-lymphocytes?
Phagocytic cells are also critical for initiation of acquired immune response. T-cell responses would not occur without phagocytes since they present peptide antigens in the context MHC molecules to T-cells receptors.
The major cell type involved in antigen presentation to T-
lymphocytes is a dendritic cell (DC).
Where are dendritic cells (DCs) found? Where and what do they develop from?
Explain how dendritic cells mature. What receptor is key for this process?
How do DCs activate T-cells? Where do they go to activate them?
- DCs typically are found in tissues and develop from myeloid progenitors within the bone marrow. In normal tissues, DCs are in an “immature state”. Tissue-invading microbes during inflammatory responses come into contact with immature DCs.
- The PAMPS on the microbes trigger PRRs on immature DCs. TLR signaling is critical for maturation of DC and their migration to LNs. The TLRs induce both phagocytosis and activation of the DC. The activated DCs are now “mature”.
- The mature DCs begin to migrate from the inflamed tissue via the lymphatic ducts to draining lymphoid tissues (e.g., lymph nodes) where a concentration of antigen specific T-lymphocytes are present.
- Once in the lymphoid tissues the “mature” DC will trigger an antigen-specific T-lymphocyte that posses the specific T-cell antigen receptor.
Explain the difference btwn macrophages and DCs in antigen presentation to T-cells as it pertains to location in which presentation occurs.
Although macrophages are antigen-presenting cells to T-cells they are onlylocalized tissues and never migrate outside of tissues like dendritic cells are capable of doing. Thus, macrophages can only present to T-cells within the tissue where the microbe or its components are present.
Microbes and their components are capable of moving in lymph ducts via the lymphatic vessels between inflamed tissues and the lymph node. Therefore, the microbe and its components entering the lymph node via the lymph ducts could be phagocytized by a “lymph node macrophage” and the macrophage presents the peptide antigen to an antigen specific naïve T-cells with the T-cell rich zone of the lymph node.