Week 1, Lecture 10 - Phagocytosis
How does opsonization occur in neutrophils?
The neutrophil lacks the inherent ability to distinguish foreign from self surfaces, so some type of marker is required. This function is performed by serum substances called opsonins which, by virtue of their ability to bind to a particle and to a receptor on a neutrophil, can initiate the phagocytic events.
How potent are the oxidants generated by the neutrophils?
The oxidants are very toxic, and kill virtually any organism ingested by the neutrophil.
What is the oxidative process used by neutrophils?
The oxidative process is the most important part of neutrophil's actions. 1. Superoxide (.O₂-) and 2. Hydrogen Peroxide H₂O₂ are products of NADPH oxidase and give rise to 3. Hydroxyl radical (.OH) and 4. Singlet (excited state) oxygen (¹O₂). In addition, H₂O₂ is the substrate for the granule enzyme, Myeloperoxidase, which uses H₂O₂ and a halide cofactor (Cl-, I-) to generate additional oxidants.
What is phagocytosis?
The process by which particulate matter is engulfed and degraded by a cell.
What happens to the old/damaged/non-functional cells?
The products are degraded by lysosomal enzymes and reutilized by the body.
What causes defective opsonization in neutrophils? What are the consequences?
The recognition function in phagocytosis is performed by humoral factors, and defects in these have been reported to result in abnormal opsonization. Defective opsonization have been observed in patients with agammaglobulinemia, C3 deficiency, and opsonin receptor (CR3 and CR4) deficiency (termed *leukocyte adhesion deficiency - LAD), due to the absence of the CD18 molecule, which is the common β chain of the β2 [leukocyte] integrins). These defects are all due to a genetic deficiency of an opsonin or its receptor. Patients with these defects are at risk for recurrent and frequently life threatening bacterial infections.
What causes defective degranulation in neutrophils? What are the consequences?
The release of lysosomal enzymes in the phagosome has consistently been reported to be abnormal in only one disease, *Chediak-Higashi syndrome.* Other phagocytic functions are also defective in these patients, so that the clinical findings of recurrent bacterial infections are probably not due to faulty degranulation alone. PMNs of these patients characteristically have giant lysosomal granules which can be seen in routine peripheral blood smears. It is a genetically transmitted, autosomal recessive disease, in which lysosomal enzyme content is nearly normal, but maldistributed, and the rate of degranulation is markedly depressed. Defects in the granules of NK cells result in markedly reduced NK function, and defects in the granules of melanocytes results in partial albinism. It is the only disease where NK cells don't work. Chediak-Higashi also comes with partial albinism, so it is "easy" to diagnose.
Since neutrophils take about 14 days to mature, which is too long to respond to an infection in a timely manner, how does the body react with neutrophils?
There are stores of mature neutrophils ready to be released.
How does the granulocyte and monocyte reserve release neutrophils?
There is a large store of mature monocytes, neutrophils, and band cells stored in the bone marrow. These cells are released into the circulation at a rate sufficient to keep the PMN count roughly constant, since PMNs are constantly migrating out of the circulatory system and into the tissues. Upon proper stimulation the rate of cell release from the marrow reserve can increase, causing leukocytosis. Bacterial endotoxin is very effective at stimulating such release, and can be used as a measure of granulocyte reserve.
What causes defective mobilization and chemotaxis in neutrophils? What are the consequences?
These defects occur together often, since both require that cell movement and deformability to be normal. Intrinsic movement defects are known to occur in "lazy leukocyte" syndrom (leukocyte adhesion deficiency) and Chediak-Higashi Syndrome. There are also transient movement defects seen in newborn infants and in patients recovering from some viral infections, such as influenza.
What are the 3 categories of PMNs in human blood?
They are divided based on their staining properties: - Neutrophils - Eosinophils - Basophils.
Why does MPO deficiency not result in as severe infection as CGD?
This is due to the action of the oxygen intermediates generated spontaneously from superoxide, which is produced in elevated amounts, since NADPH oxidase is intact.
T/F: Monocytes and neutrophils have the same precursor
True
How are PMNs different from RES/MPS cells?
Unlike the cells of the RES/MPS, which have multiple functions besides phagocytosis, PMNs are aggressively phagocytic, and have few if any other functions.
How does a neutrophil cause collateral damage?
When a neutrophil engulfs a bacteria, granules start releasing enzymes before the vacuole is closed off. So some of the enzymes sneak out and damage the body.
How are "Giant Cells" formed in the RES/MPS system?
When macrophages phagocytizes particles, it undergoes substantial morphologic and metabolic changes and can transform into an "epithelioid" cell and release lysosomal constituents. In lesions where a large number of macrophages are actively involved in phagocytosis, or in chronic inflammation where particles are not effectively eliminated, the epithelioid cells may fuse to form multinuclear "giant cells." This also occurs when a very large particle is introduced into the body. A foreign body giant cell forms around the particle, most likely an attempt to wall it off from the rest of the body tissues.
What does the antigen clearance aspect of the Mononuclear Phagocyte System (MPS) do?
When particulate antigen is injected intravenously it is cleared from the circulation by the mononuclear phagocyte system.
Efficacy of opsonization methods
Works best if they work together
What is the marginal pool?
A large number of PMNs in the blood are not circulating freely, but are attached to blood vessel and capillary walls. These cells are said to be "marginated" and are unavailable when blood is drawn for a cell count. These cells can be released rapidly (less than 5 minutes) into the circulation upon introduction of stimuli such as epinephrine.
What is the other method of microbe killing?
A nonoxidative method uses lysosomal enzymes and defensins. This accounts for about 2% of the killing of most microbes.
What is endocytosis?
A process by which soluble macromolecules are taken into a cell
What is the function of the Mononuclear Phagocyte System (MPS)?
Also known as the Reticuloendothelial System (RES), it participates in: - Antigen clearance - Clearance of damaged cells - "Giant Cell" formation
When do primary (azurophilic) granules come in?
At 3-10 minutes the primary (acurophilic) granules begin to release their contents. These enzymes have an acid pH optimum, about 4.5.
What cells of the RES/MPS remove cells found at wound healing sites, and what are the removed cells?
At sites of wound healing, *tissue macrophages* remove *fibrin deposits*, trapped *red cells*, and* dead or damaged tissue and debris*.
What are the functions of basophils?
Basophils are mainly secretory cells. Like tissue mast cells, basophils have granules which contain heparin and histamine (and other constituents) that are released upon the interaction with complement derived anaphylatoxins or antigen complexed to cytophilic IgE. They are less avidly phagocytic than neutrophiles, although they are probably capable of particle ingestion. Like mast cells, they are important in allergic reactions. Indirectly fights parasites by increasing vascular permeability.
What are the chemotactic factors?
C5a - From complement activation Leukotriene B4 (LTB4) - From neutrophils Interleukin 8 (IL-8, chemokines) - From macrophages, T cells, endothelial cells, etc. F-met peptides - From prokaryotes (bacteria)
What is the important marker that monocytes express?
CD14
What is there no excessive infection with catalase negative bacteria?
Catalase breaks down H₂O₂, therefore catalase negative bacteria (i.e. Stretococci) secrete H₂O₂ as a waste product. CGD patient neutrophils cannot make their own H₂O₂ due to the enzyme defect, but the catalase negative bacteria can supply the missing H₂O₂ to the neutrophil, allowing the MPO to produce the toxic oxidants necesasry for killing.
What cells are most effective at removing damaged or old, non-functional cells, and via what process?
Cells of the MPS (or RES, same thing), mainly splenic macrophages and Kupffer cells, are effective at removing damaged or old, non-functional cells by phagocytosis.
How does chemotaxis work?
Chemotaxis is the directed migration of cells in response to a chemical stimulus, and chemotactic factors are the soluble molecules which mediate this migration. When chemotactic factors are generated at a particular site, they diffuse over a distance, establishing a concentration gradient. PMNs interact with the chemotactic factors through receptors on their cell surfaces and migrate up the concentration gradient, towards the site of chemotactic factor origin or generation. A number of chemotactic factors are known.
How do antibody and the classical complement pathway opsonize for neutrophils?
Complement activating antibodies (IgM, IgG₁, IgG₂, and IgG₃) are extremely efficient at depositing C3b on the bacterial surface (via C1, C4, and C4), thus opsonizing the particle for ingestion. Antibody mediated C3b deposition is more efficient than the alternative or lectin pathways, so that this reaction sequence is the major opsonic pathway when some specific antibacterial antibody is present.
What chemicals mediate mobilization from granulocyte and monocyte reserve? How long does it take?
Cytokins (IL-1 and TNF-α), glococorticoids (at physiologic concentrations) and even a complement activation fragment (C3e) also mediate mobilization. This reaction takes 6-12 hours to reach its peak.
What can cause defective neutrophil production?
Defective neutrophil production, also known as *neutropenia*, results in the depletion of neutrophil count to less than 1000/mm³. Neutropenia may be: - drug induced - result from production of anti-neutrophil antibodies - secondary to leukemia
How are neutrophils mobilized?
During an episode of infection or inflammation, neutrophil counts may rise dramatically. This mobilization is due to the release of cells from storage pools in the body, and not necessarily reflective of an increased synthetic rate in the bone marrow.
What are the functions of eosinophils?
Eosinophils are slightly less avidly phagocytic than are neutrophils. They are not so great against bacteria. Their major role is most likely in host defense against parasites, but they also release mediators which play an important role in local inflammatory response. They contain major basic protein, which is what kills the parasites. If eosinophil levels are elevated, there is a parasite.
What causes phagocyte mobilization?
Glucorticoids endotoxins TNFα IL-1 Chemokines C3e
How does a deficiency in glucose-6-phosphate dehydrogenase (G-6-PDH) lead to defective metabolic stimulation and killing in neutrophils?
Glucose-6-phosphate dehydrogenase (G-6-PDH) is an essential enzyme in the HMP. Its absence in the PMN results in a failure to reduce NADP to NADPH. Thus H₂O₂ formation is severely impaired.
What happens if H₂O₂ can come from another source? What are these sources?
H₂O₂ can come from the bacteria itself, such as with catalase negative organisms. In this situation, killing is not defective, but there is no metabolic stimulation.
What is the enzyme used in the killing stage of neutrophil action?
H₂O₂ reacts with the enzyme *myeloperoxidase (MPO)*, which is present in high levels in PMN primary granules, and has an optimum of 4.5. A halide ion, Cl- or I-, is required for enzyme activity, and killing of ingested viable organisms occurs through oxidation of bacterial surface and internal constituents. Chemiluminescent (light) emission accompanies oxidative killing.
What are the main effectors of the MPS?
In a normal animal about 90% of the particles will be taken up by the Kupffer cells and about 10% by the splenic macrophages. The other cells of the MPS take up trace amounts of the particles, but a histological examiniation of all the tissues will show the distribution of the MPS cells.
What are the names of macrophages in the liver, lung, kidney, CNS, lymph system, and the peritoneal?
Liver macrophages = Kupffer cells (largest amount of macrophages) Lung macrophages = alveolar macrophages Kidney macrophages = Mesangial macrophages CNS macrophages = Microglial cells Peritoneal (everywhere else) macrophages = Tissue macrophages (or Histiocytes in histo) Spleen, lymph nodes, tonsils, Peyer's Patch, etc = Lymphoid tissue
What role does degranulation play in neutrophils?
Lysosomal granules fuse with the phagosome, releasing their contents into the vacuole containing the ingested particle. The vacuole is now called a phagolysosome. There are at least two types of lysosomal granules, called primary (azurophilic) and secondary (specific). They are called primary and secondary because of the time their appearance during development of the cell. Extracellular release of lysosomal enzymes is a major cause of tissue damage during inflammation.
How much oxidative killing do macrophages use?
Macrophages use oxidative killing to a lesser degree, thus they can survive the process.
How does the alternative and lectin complement pathways (C3b, iC3b) act as opsonins for neutrophils?
Many bacterial surface products can activate the alternative or lectin pathways in the absence of any specific antibody. This activation results in the deposition of C3b and iC3b on the bacterial surface, and these molecules act as opsonins. PMNs and macrophages have receptors for C3b and iC3b on their surfaces. This pathway plays a critical role in defense against bacteria in the early stages of a first infection, before antibody has been snythesized to the bacterial antigens.
What is the difference between monocytes and neutrophils?
Monocytes and neutrophils are both phagocytic. The difference is that monocytes keep their MHC class II molecules, so they can still present antigens, while neutrophils cannot.
What causes defective metabolic stimulation and killing in neutrophils?
Most defects in metabolic stimulation and killing can be traced to a deficiency in a single essential enzyme in the PMN.
When is neutropenia often found?
Neutropenia is often found in other neutrophil dysfunction disease in which the primary defect is considered to be in one of the other stages of phagocytic cell function.
How much of circulating leukocytes are basophils, eosinophils, and neutrophils?
Neutrophils - 65% Eosinophils - 3-5% Eosinophils numbers increase dramatically in atopic (allergic) conditions or during parasitic infections. Basophils - < 1%
Where does production of neutrophils occur?
Neutrophils are made in the bone marrow and are produced quite rapidly, since their half life in circulation is only 6-7 hours.
What are the functions of neutrophils?
Neutrophils are the most aggressive phagocytic, the most rapid to react, and are the body's major cellular defense against extracellular bacterial pathogens. It is the study of neutorphils that has provided most of our knowledge of the phagocytic process.
What is a good indicator of oxidative metabolism in neutrophils?
Nitroblue tetrazolium (NBT) dye reduction test. The dye is an almost colorless dye that is reduced to a dark blue insoluble precipitate in the presence of superoxide anion. The test is negative in CGD.
What is the consequence of neutropenia?
Patients are at risk for bacterial infections.
What happens to patients with chronic granulomatous disease (CGD)?
Patients with CGD get severe infections with catalase positive bacteria and fungi: *Don't need to know this list* 1. Staphylococcus aureus 2. Klebsiella pneumoniae 3. E. Coli 4. Serratia marcescens 5. Pseudomonas species 6. Salmonella species 7. Nocardia species 8. Aspergillus species 9. Candida albicans
What is the process of neutrophil engulfment?
The attachment of opsonized particles to the cell surface opsonin receptors initiates the engulfment process, and as the particle is ingested, the external membrane surrounding it is pinched off, forming a vacuole around the particle. This structure is called a *phagosome*. The uptake process r*equires the assembly of actin and myosin into microfilaments.* Ingestion *requires metabolic energy*, which is obtained mainly through *anaeroblic glycolysis*.
What is the first line of active cellular defense against invading extracellular pathogens?
The first line of active cellular defense against invading extracellular pathogens are the *polymorphonuclear leukocytes (PMNs).*
What is the *key* intermediate in the bactericidal event?
The generation of superoxide anion, .O₂-
What happens with MPO deficiency?
The infections are not nearly as severe as CGD.
How can you measure the function of the MPS?
The most common experimental system used to study MPS function in animals is the injection of gelantin-stabilized carbon particles into the tail vein of a rat or mouse. The rate of elimination of the particles from the blood stream provides a measure of MPS function. The clearance of IV injected dyes was the original experimental procedure used to define the MPS.
*Exam* How does the absence of NADPH Oxidase lead to defective metabolic stimulation and killing in neutrophils?
The absence of NADPH Oxidase results in *chronic granulomatous disease (CGD)*, causing H₂O₂ to be absent.
What are the changes to glucose use during phagocytosis?
The amount of glucose metabolized via the hexose monophosphate (HMP) shunt increases from 2% of the glucose used (resting) to 50% (phagocytizing). This supplies cytoplasmic NADPH which is reoxidized through non-mitochondrial oxygen uptake through the enzyme NADPH oxidase, which produces the superoxide anion molecule, .O₂-
Describe specific granules.
Specific (secondary) granules compose ~75% of the granules and release their enzymes first, 0-3 minutes after ingestion. These enzymes have a pH near 7.0.
What are the most important chemotactic factors?
- C5a and its catabolie, C5a des arg (C5a which lacks the carboxyterminal arginine) are the most important of the plasma derived chemotactic factors. - Bacterial proteins are also chemotactic (F-met peptides such as F-met-leu-phe). - Stimulated T-lymphocytes produce several cytokines that are chemotactic for PMNs and/or macrophages (chemokines). These include IL-8 and a variety of other cytokines. Chemotactic factors are also released by stimulated PMNs. These included leukotriene B₄ (LTB₄) which has very potent chemotactic activity.
What are some defects of neutrophil function?
- Defective neutrophil production - Defective mobilization and chemotaxis - Defective opsonization - Defective degranulation - Defective metabolic stimulation and killing
What are the major sources of the "extra" neutrophils during mobilization?
- Marginal pool - Granulocyte and monocyte reserve
What are the 3 categories of opsonins for neutrophils?
- The alternative & lectin complement pathways (C3b, iC3b) - Antibody and the classical complement pathway. - IgG alone
What metabolic stimulation/changes happen with PMN metabolism that accompanies phagocytosis?
- increased phosphatidyl inositol synthesis - increased calcium uptake - incrased glycogen breakdown - increased anaerobic glucose metabolism which supplies energy for phagocytosis and chemotaxis. The end product is lactate, since PMNs are relatively deficient in pyruvate dehydrogenase. - The lactate buildup results in decreased intraphagolysosomal pH (pH ~4.5). - Lysosomal enzyme release and - Lysosomal enzyme activation then occur
What are the enzymes responsible for H₂O₂ generation that may be missing, resulting in defective metabolic stimulation and killing?
1. Glucose-6-phosphate dehydrogenase (G-6-PDH) 2. NADPH Oxidase <- critical enzyme
What are the stages of macrophage life?
1. Monocytes circulate blood for 6-7 hours, then migrate into tissues to become resident (non-activated) macrophages 2. Cytokines (especially IFNγ) activate macrophages, which are better at killing ingested pathogens. 3. If macrophages fail to kill/remove the particles (too many particles or whatever), macrophages differentiate to epitheloid cells (no granules). They're basically failed macrophages. 4. Epitheloid cells fuse together to form granulomas (often caused by chronic inflammation and failure to remove stimulus). Granulomas are the body's way of protecting the body from something it can't get rid of. Fibroblast forms around the granuloma, eventually calcifying.
What are the stages of phagocytosis?
1. Production - bone marrow for 14 days 2. Mobilization - granulocyte and monocyte reserves get mobilized into the blood (4-12 hours) 3. Chemotaxis (due to C5a, Leukotriene B4 (LTB4), IL-8 (chemokines, small cytokines) 4. Opsonization - Antibody IgG, C3b, iC3b 5. Engulfment - formation of *phagosome* 6. Degranulation - lysosomal enzyme release 7. Metabolic stimulation -> Killing
What is the process of neutrophil phagocytosis?
1. Production of cells 2. Mobilization of cells 3. Chemotaxis (directed migration of cells) 4. Recognition (opsonization) 5. Engulfment of the particle 6. Degranulation - release of lysosomal enzymes 7. Metabolic stimulation 8. Killing
What are the additional oxidants generated by meyloperoxidase (MPO)?
1. Singlet (excited state) oxygen (¹O₂) 2. Hypochlorous acid (HOCl) 3. Iodonium ions (I+) 4. Chloridne (Cl₂) and 5. Chloramines (R-NHCl)
How does IgG alone opsonize bacteria for neutrophils?
Since PMNs have Fc receptors for IgG, high levels of specific IgG antibody can be opsonic even in the absence of complement. Clinical studies show that both IgG antibody and C3b or iC3b are needed for adequate host defense against extracellular bacterial pathogens, since either C3 deficiency (or deficiency of the iC3b receptors, CR3 and CR4) OR IgG deficiency predispose to severe, recurrent infection with encapsulated bacteria.