Introduction to Medical Immunology - part 5 ppt

Page 318 Table 17.1 Comparison of the Characteristics of PMN Leukocytes and Monocytes/Macrophages Characteristic PMN leukocytes Monocyte/macrophage Numbers in peripheral blood 3 - 6×10 3 /µL 285–500/µL Resident forms in tissues - + (macrophage) Nonimmunological phagocytosis ++ + Fc receptors FcγRII,III FcγRI,II,III C3b receptors ++ ++ Enzymatic granules ++ ++ Bactericidal enzymes ++ ++ Ability to generate superoxide and H 2 O 2 +++ ++ Synthesis and release of leukotrienes + (B4) ++ (B4, C4, D4) Synthesis and release of prostaglandins - ++ Release of PAF ++ + Release of interleukins + ++ Response to nonimmunological chemotactic factors + - Response to C5a/C3a + - Response to lymphokines + (IL-8) ++ (IFN- γ ) Antigen processing - ++ Expression of HLA class II antigens - ++ Phagocytosis-independent enzyme release ++ - a. Among bacterial products, formyl-methionyl peptides, such as f-methionine-leucine-phenylalanine (f-met- leu-phe), are extremely potent chemotactic agents. b. Tissue damage may result in the activation of the plasmin system that may, in turn, initiate complement activation with generation of C5a, another extremely potent chemotactic agent. c. Many microorganisms can probably generate C5a by activation of the complement system through the alternative pathway. d. After an inflammatory process has been established, proteases released by activated neutrophils and macrophages can also split C5, and the same cells may release leukotriene B4, another potent chemotactic factor, attracting more neutrophils to the site. e. Chemokines such as IL-8, monocyte chemotactic protein-1, and RANTES are also chemotactic for neutrophils and monocytes. 3. After receiving a chemotactic stimulus, leukocytes undergo changes in the cell membrane, which is smooth in the resting cell, and becomes “ruffled” after the cell receives the chemotactic signal. The activated PMN has a marked increase in cell adhesiveness, associated with increased expression of adherence molecules, namely, integrins of the CD11/CD18 complex, which includes: a. CD11a [the α chain of LFA (leukocyte function antigen)-1] b. CD11b (the C3bi receptor or CR3, also known as Mac-1) molecule c. CD11c (also known as protein p150,95) d. CD18 [the β chain of LFA (leukocyte function antigen)-1] 4. These cell adhesion molecules (CAM) are common to the majority of Page 319 leukocytes, but their individual density and frequency may vary in the two main groups of phagocytic cells. a. CD11a and CD18 are expressed virtually by all monocytes and granulocytes b. CD11b is more prevalent among granulocytes c. CD11c is more frequent among monocytes 5. The expression of these CAM mediates a variety of cell-cell interactions such as those that lead to neutrophil aggregation, and, most importantly, those that mediate firm adhesion of leukocytes to endothelial cells. For example, CD11a (LFA-1) and CD11b interact with molecules of the immunoglobulin gene family, such as ICAM- 1, ICAM-2 and VCAM-1, expressed on the endothelial cell membrane. 6. Cytokines released by activated monocytes and lymphocytes, such as IL-1 and TNF- α can up-regulate the expression of VCAM-1 and, to a lesser degree, of ICAM-1 and -2. This further enhances the adhesion of leukocytes to endothelial cells. 7. After adhering to endothelial cells, leukocytes migrate to the extravascular compartment. The transmigration involves interaction with a fourth member of the immunoglobulin gene family—platelet endothelial cell adhesion molecule 1 (PECAM-1)—which is expressed at the intercellular junctions between endothelial cells. The interaction of leukocytes with PECAM- 1 mediates the process of diapedesis, by which leukocytes squeeze through the endothelial cell junctions into the extravascular compartment. 8. The diapedesis process involves the locomotor apparatus of the leukocyte, a contractile actin-myosin system stabilized by polymerized microtubules. Its activation is essential for the leukocyte to move to the extravascular space and an intact CD11b protein seems essential for the proper modulation of microtubule assembly, which will not take place in CD11b-deficient patients. B. Phagocytosis and Intracellular Killing. At the area of infection, PMN leukocytes recognize the infections agents, which are ingested and killed intracellularly. The sequence of events leading to opsonization and intracellular killing is summarized in Figure 17.1. 1. Several recognition systems appear to be involved in the phagocytosis step: a. The best defined of which is the reaction with the Fc fragment of opsonizing antibodies. Neutrophils express two types of Fc γ receptors, Fc γ RII and Fc γ RIII, both of which are involved in phagocytosis. b. The CR1 (C3b) receptor is also expressed by neutrophils, and the binding and ingestion of microorganisms through this receptor has been well established. c. Opsonization with both IgG antibodies and C3b seems associated with maximal efficiency in ingestion. d. As pointed out in Chapter 13, opsonization is not an absolute requirement for ingestion by neutrophils. Nonimmune recognition systems leading to phagocytosis are believed to be responsible for the ingestion of microorganisms with polysaccharide-rich outer layers. The neutrophil is also able to ingest a variety of particulate matter, such as latex beads, silicone, asbestos fibers, etc., in the absence of opsonizing antibodies or complement. Page 320 Figure 17.1 Diagrammatic representation of the sequence of events that takes place during PMN leukocyte phagocytosis. (Reproduced with permission from Wolach, B., Baehner, R.L., and Boxer, L.A., Isr. J. Med. Sci., 18:897, 1982.) Page 321 2. Ingestion is achieved through formation of pseudopodia that surround the particle or bacteria, with pseudopodia eventually fusing at the distal pole to form a phagosome. The cytoplasmic granules of the neutrophil (lysosomes) then fuse with the phagosomes, and their contents empty inside the phagosomes (degranulation). This degranulation process is very rapid and delivers a variety of antimicrobial substances to the phagosome. a. The azurophilic or primary granules contain, among other substances, myeloperoxidase, lysozyme, acid hydrolases (such as β -glucuronidase), cationic proteins, and neutral proteases (including collagenase, elastase, and cathepsin C2). b. The secondary granules or lysosomes contain lysozyme and lactoferrin. 3. Killing of ingested organisms involves the effects of cationic proteins from the primary granules and lysosomal enzymes, such as lysozyme and lactoferrin, as well as of the by-products of the respiratory burst, activated as a consequence of phagocytosis. a. Cationic proteins can bind to negatively charged cell surfaces (such as the bacterial outer membrane) and interfere with growth. b. Lactoferrin has antimicrobial activity by chelating iron and preventing its use by bacteria that need it as an essential nutrient. c. Lysozyme splits the β -1,4 linkage between the N-acetylmuramic acid peptide and N-acetylglucosamine on the bacterial peptidoglycan. However, the importance of this enzyme as a primary killing mechanism has been questioned due to the relative inaccessibility of the peptidoglycan layer in many microorganisms, which may be surrounded by capsules or by the lipopolysaccharide-rich outer membrane (Gram-negative bacteria). d. From the bactericidal point of view, however, the activation of the superoxide-generating system (respiratory burst) appears considerably more significant. This system is activated primarily by opsonization but also by a variety of PMN-activating stimuli, ranging from bacterial peptides, such as f-met- leu-phe, to C5a. i. A key enzymatic activity (NADPH oxidase) is activated and results in the transfer of a single electron from NADPH to oxygen, generating superoxide (O 2 - ). ii. NADPH oxidase is a molecular complex located on the cell membrane, constituted by: • cytochrome B, which is a heterodimer formed by two polypeptide chains (91 kD and 22 kD, respectively), believed to play the key role in the reduction of oxygen to superoxide, possibly by being the terminal electron donor. • two cytosolic proteins—p47 and p67—one of which (p47) is a substrate for protein kinase C. In a resting cell the complex is inactive and its components are not associated. iii. After the cell is activated, p47 is phosphorylated, and it becomes associated with p67 (and possibly with a third protein, p21 rac . The phosphorylated complex binds to cytochrome B in the phagocytic cell membrane, which is considered to be the active oxidase. iv. Cytochrome b transfers one electron from NADPH to oxygen through at least three steps: Page 322 • reduction of a flavin adenine dinucleotide (FAD) bound to the high-molecular-weight subunit of cytochrome b • transfer of an electron from FADH2 to ferric iron in a heme molecule associated with the low- molecular-weight subunit of cytochrome B • transfer of an electron from reduced iron to oxygen, generating superoxide v. Since at the time this oxidase is fully activated the cell membrane is invaginating around the particle that stimulated the phagocytic process, the brunt of the active oxygen radicals generated by this system is delivered to the phagosome (Fig. 17.2). vi. The respiratory burst generates two toxic compounds essential for intracellular killing of bacteria: superoxide and H 2 O 2 . Through myeloperoxidase, H 2 O 2 can be peroxidated and led to form hypochlorite and other halide ion derivatives, which are also potent bactericidal agents. vii. These compounds are also toxic to the cell, particularly superoxide, which can diffuse into the cytoplasm. The cell has several detoxifying systems, including superoxide dismutase, which converts superoxide into H 2 O 2 and, in turn, H 2 O 2 is detoxified by catalase and by the oxidation of reduced glutathione, which requires activation of the hexose monophosphate shunt. Figure 17.2 Diagrammatic representation of the major events involved in the respiratory burst of phagocytic cells. The occupancy of Fc and/or CR1 receptors triggers the activation sequence, which involves protein kinase activation, enzyme activation, and phosphorylation of at least one cytosolic protein (p47). As a result, a molecular complex, constituted by cytochrome B (Cytb), p47, and p67, is assembled on the cell membrane, which is folding to constitute a phagosome. This complex has NADPH oxidase activity, oxidizes NADPH, and transfers the resulting electron to an oxygen molecule, resulting in the formation of superoxide (O - 2). Page 323 III. Physiology of the Monocyte/Macrophage A. A Comparison of PMN Leukocytes and Monocyte/Macrophages. The two populations of phagocytic cells share many common characteristics, such as 1. Presence of Fc and C3b receptors on their membranes 2. Ability to engulf bacteria and particles 3. Metabolic and enzymatic killing mechanisms and pathways In contrast, other functions and metabolic pathways differ considerably between these two types of cells (see Table 17.1). 1. One important distinguishing feature is the involvement of the monocyte/macrophage series of cells in the inductive stages of the immune response, due to their ability to process antigens and present antigen-derived peptides to the immune system. 2. The monocyte/macrophage is also involved in immunoregulatory signals, providing both activating signals (in the form of IL-1, IL-6, and IL-12) and down-regulating signals (in the form of PGE 2 ) to T lymphocytes. 3. These two types of phagocytic cells have different preferences as far as phagocytosis. For example, PMN leukocytes are able to ingest inert particles such as latex, but have very little ability to engulf antibody-coded homologous erythrocytes, while the reverse is true for the monocyte/macrophage. 4. While neutrophils seem to be constitutively ready to ingest particulate matter, the circulating monocytes and the tissue-fixed (resident) macrophages are usually resting cells that need to be activated by several types of stimuli, including microorganisms or their products and cytokines, before they can fully express their phagocytic and killing properties. B. The Activated Macrophage has unique morphological and functional characteristics. 1. Morphologically, the activated macrophage is larger, and its cytoplasm tends to spread and attach to surfaces. 2. The composition of the plasma membrane is changed, and the rates of pinocytosis and engulfment are increased (phagocytosis through C3b receptors is only seen after activation). 3. Intracellularly, there is a marked increase in enzymatic contents, particularly of plasminogen activator, collagenase, and elastase, and the oxidative metabolism (leading to generation of superoxide and H 2 O 2 ) is greatly enhanced. III. Laboratory Evaluation of Phagocytic Function The evaluation of phagocytic function is usually centered on the study of neutrophils, which are considerably easier to isolate than monocytes or macrophages. Phagocytosis by neutrophils can be depressed as a result of reduction in cell numbers or as a result of a functional defect. Functional defects affecting every single stage of the phagocytic response have been reported and have to be evaluated by different tests. The following is a summary of the most important tests used to evaluate phagocytic function. A. Neutrophil Count. This is the simplest and one of the most important tests to Page 324 perform since phagocytic defects due to neutropenia are, by far, more common than the primary, congenital, defects of phagocytic function. As a rule, it is believed that a neutrophil count below 1000/ µ L represents an increased risk of infection, and when neutrophil counts are lower than 200/ µ L, the patient will invariably be infected. B. Adherence. The increased adherence of activated phagocytic cells to endothelial surfaces is critical for the migration of these cells to infectious foci. Although specialized tests to measure aggregation and adherence of neutrophils in response to stimuli such as C5a desarg (a nonchemotactic derivative of C5a), presently this property is evaluated indirectly, by determining the expression of the different components of CD11/CD18 complex which mediate adhesion by flow cytometry. C. Chemotaxis and Migration. The migration of phagocytes in response to chemotactic stimuli can be studied in vitro, using the Boyden chamber, or in vivo, by means of the Rebuck's skin window technique. 1. Chemotaxis assays using the Boyden chamber a. The basic principle of all versions of the Boyden chamber is to have two compartments separated by a membrane whose pores are too tight to allow PMN leukocytes to passively diffuse from one chamber to the other, but large enough to allow the active movement of these cells from the upper chamber, where they are placed, to the lower chamber. b. The movement of the cells is stimulated by adding to the lower chamber a chemotactic factor such as C5a or the tetrapeptide f-met-leu-phe. c. The results are usually based either on counting the number of cells that reached the bottom side of the membrane, or on the indirect determination of the number of cells reaching the bottom chamber using 51 Crlabeled PMN (as illustrated in Fig. 17.3). d. All versions of this technique are difficult to reproduce and standardize and are not used clinically. 2. The Rebuck's skin window technique is used to evaluate the capacity to recruit PMN into an area of inflammation in vivo. Figure 17.3 Schematic representation of the principle of chemotaxis assays using the Boyden chamber and 51 Cr-labeled PMN leukocytes. Page 325 a. A superficial abrasion of the skin is covered with a glass cover slip forming a small diffusion chamber (“skin window”). b. Inflammatory cells reaching it will adhere to the glass and can be stained and counted. c. This technique is also not used routinely. D. Ingestion. Ingestion tests are relatively simple to perform and reproduce. 1. They are usually based on incubating PMN with opsonized particles, and, after an adequate incubation, determining either the number of ingested particles or a phagocytic index: 2. Several types of particles have been used, including latex, zymosan (fragments of fungal capsular polysaccharidic material), killed C. albicans, and IgG-coated beads (immunobeads). All these particles will activate complement by either one of the pathways and become coated with C3, although opsonization with complement is not the major determinant of phagocytosis. 3. The easiest particles to visualize once ingested are fluorescent latex beads; their use considerably simplifies the assay (Fig. 17.4), particularly if performed in a flow cytometer. 4. This test is not routinely used because others are available (e.g., the nitroblue tetrazolium reduction test, see below) which test both for ingestion and for the ability to mount a respiratory burst Figure 17.4 Use of fluorescent latex beads for evaluation of phagocytosis. The panel on the left reproduces a photograph of microscopic field showing the phagocytic cells that have ingested latex beads under visible light. The panel on the right shows the same field under UV light. Page 326 E. Degranulation. When the contents of cytoplasmic granules are released into a phagosome, there is always some leakage of their contents into the extracellular fluid. The tests to study degranulation involve ingestion of particulate matter, as mentioned above, but in this case the supernatants are analyzed for their contents of substances released by the PMN granules such as myeloperoxidase, lysozyme, β -glucuronidase, and lactoferrin. F. Measurement of the Oxidative Burst. Several different techniques have been proposed to measure the oxidative burst. 1. Chemiluminescence. The chemiluminescence assay is based on the fact that the superoxide ion is unstable, and that its dissociation can be measured either directly or indirectly after addition of luminol that is activated during superoxide dissociation. This is perhaps the most sensitive and directly quantitative assay for the oxidative burst, but it has a major drawback in that it requires special and complex instrumentation. 2. Reduction of cytochrome C. The reduction of the cytochrome C can be used to measure superoxide release because this pigment, when reduced by superoxide, will change its light absorbance properties. The change in color of cytochrome C can be measured with a conventional spectrophotometer. The main drawbacks of the assay are its relatively low sensitivity and difficulties in reproducibility. 3. Fluorescence assays. Several techniques for the measurement of the superoxide burst are based on the oxidation of 2',7' -dichlorofluorescein diacetate (nonfluorescent), which results in the formation of 2',7' -dichlorofluorescein (highly fluorescent). a. The respiratory burst is induced with phorbol myristate acetate (or any other soluble PMN activator) b. The numbers of fluorescent cells and fluorescence intensity of activated and nonactivated PMN suspensions from patients and suitable controls can be determined by flow cytometry. c. In patients with primary defects of the enzymes responsible for the respiratory burst, both the mean fluorescence intensity and the numbers of fluorescent cells after stimulation are considerably lower than those determined in normal, healthy volunteers. 4. Nitroblue tetrazolium (NBT) reduction tests. Tests based on NBT reduction are the most commonly used for the evaluation of neutrophil function. a. Principle. Oxidized NBT, colorless to pale yellow in solution, is transformed by reduction into blue formazan. The test usually involves incubation of purified neutrophils, NBT, and a stimulus known to activate the respiratory burst. Two types of stimuli can be used. i. Opsonized particles, which need to be ingested to stimulate the burst. In this way the test examines both the ability to ingest and the ability to produce a respiratory burst. ii. Diffusible activators, such as phorbol esters. Those compounds diffuse into the cell and activate protein kinase C, which in turn activates the NADPH -cytochrome B system and induce the respiratory burst directly, bypassing the ingestion step. b. Microscopic technique. The simplest NBT reduction assays rely on conventional microscopy to count the number of PMN with blue- [...]... an HY-derived peptide-MHC-Db complex were shown to become tolerant to the HY peptide, but only in Db+ animals The tolerance in this model was apparently due to clonal deletion, since no mature cytotoxic T cells reactive with MHC-Db/HY were detected in the tolerant animals Page 343 to mediate a cytotoxic reaction against any cell expressing the HY antigen i Female transgenic mice (HY-) were found to. .. presented to helper T cells The resulting lack of co-stimulation signals will favor the development of tolerance b Exceptions to these rules have been noted Some autoantigen-derived peptides have been used to induce tolerance in laboratory animals It is possible that such peptides are able to bind directly to MHC-II molecules and may deliver tolerogenic signals to T lymphocytes with the corresponding autoreactive... defined for B-cell anergy The CD40 (B cells)-CD40 ligand (T cells) interaction is critically important for B-cell differentiation In the absence of CD40 signaling, B cells are easy to tolerize d It is possible to interpret the differences between high-zone vs low-zone tolerance as a result of differences in the degree of co-stimulation received by T cells i In high-zone tolerance, the co-stimulatory signals... context able to induce an active immune response (i.e., by activated APC able to deliver co-stimulatory signals to the autoreactive T and/or B cells) b On the other hand, the recognition of autoantigens in the absence of costimulatory signals by helper T cells is likely to contribute to the perpetuation of a state of T-cell anergy, which seems to have an important contribution to perpetuate tolerance... are down-regulated ii Very low antigen doses fail to induce the delivery of co-stimulatory signals to T cells, and low-zone T-cell tolerance ensues 5 Conclusions Clonal deletion seems extremely efficient during embryonic differentiation, but a large number of potentially autoreactive clones seem to escape deletion a Whether those autoreactive clones are activated may just depend on whether the autoantigens... erythematosus—variety of autoantibodies to DNA, cytoplasmic antigens, etc.) II MHC class I-associated A HLA-B27-related spondyloarthropathies (ankylosing spondylitis, Reiter's syndrome, etc.) B Psoriasis vulgaris (which is associated with HLA-B13, B16, and B17) Page 348 to production of autoantibodies In humans, bacterial and viral infections (particularly chronic) may lead to the production of anti-immunoglobulin... clonal deletion as mechanisms leading to T-cell tolerance has been obtained in transgenic mouse models 1 Models for T-lymphocyte clonal deletion There is solid evidence supporting clonal deletion as a mechanism involved in T-cell tolerance: a Transgenic mice were transfected with the gene coding for the T-cell receptor (TcR) cloned from a MHC-I restricted CD8+ cytotoxic T-cell clone specific for the male... cells, and recover the ability to mount an immune response B Cross-Immunization Exposure to an antigen that cross-reacts with a tolerogen may induce the activation of T helper lymphocytes specific for the cross-reacting antigen are activated and provide autoreactive B lymphocytes with the necessary co-stimulatory signals necessary to initiate a response against the tolerogen C Co-Stimulation of anergic clones... NBT (to check for spontaneous activation of neutrophils), with opsonized particles (to check for interference of cells and particles with the colorimetric assay), and with opsonized particles and phorbol myristate acetate (PMA) in the presence of NBT (to check for the induction of the respiratory burst) A kinetic colorimeter is used to monitor changes in O.D due to the reduction of NBT over a 2 5- minute... B virus-determined antigens expressed on the surface of myocardial cells may induce CD8+mediated tissue destruction, causing a viral-induced autoimmune myocarditis b Activated CD4+ helper cells appear to be frequently involved in cell-mediated autoimmune reactions Their pathogenic effects are mediated by the release of cytokines (IFN-γ, IL-1, L-2, IL-4, etc.) that can either trigger inflammatory reactions . interleukins + ++ Response to nonimmunological chemotactic factors + - Response to C5a/C3a + - Response to lymphokines + (IL-8) ++ (IFN- γ ) Antigen processing - ++ Expression of HLA class II antigens - ++ Phagocytosis-independent. antigens - ++ Phagocytosis-independent enzyme release ++ - a. Among bacterial products, formyl-methionyl peptides, such as f-methionine-leucine-phenylalanine (f-met- leu-phe), are extremely potent. membrane. 6. Cytokines released by activated monocytes and lymphocytes, such as IL-1 and TNF- α can up-regulate the expression of VCAM-1 and, to a lesser degree, of ICAM-1 and -2 . This further