Lecture 6 - Complements and cytokines.ppt
- 1. Complements and cytokines in immunity Dr. K. L. Ward Assistant Professor School of Chemistry, Environmental and Life Sciences The College of The Bahamas
- 2. Areas to be covered The Complement System - role of the complement system in immunity - components of the complement system Complement pathways, activation and function - classical pathway - alternate pathway - lectin pathway Regulation of complements Cytokines Interferons
- 3. Complements Complements are the most vital effectors of the humoral response in adaptive immunity. However complements also has a significant role in innate immunity where they recognise pathogen-associated molecular patterns (PAMPs) and then destroy pathogens following activation by acute phase proteins. Complement activity was first noted in the 1800s at the Institut Pasteur in Paris by Jules Bordet. Paul Ehrlich later made contributions to the existence and activity of complements by indicating that complements are responsible for the activity of blood serum that completes the action of antibodies. Later studies revealed that the complement lytic activity was due to interaction and activation by a series of complex proteins.
- 4. Complements The biological activity of complements is readily seen in both innate and adaptive immunity of vertebrate species. Complement activity greatly exceeds the antibody-mediated lysis of bacterial and red blood cells. Complement interaction with cell receptors can subsequently dictate B cell activity thus confirming its role in adaptive immunity. Complement activation results in a regulated cascade that permits these structures to then perform various functions in an attempt to eliminate foreign matter. Basic functions include: cell lysis, opsonization (promote phagocytosis), binding to specific complement receptors to induce specific functions (e.g. inflammation and secretion) and immune clearance to spleen.
- 5. Complements The complement system - defensive system consisting of over 30 proteins - accounts for 5% of serum globulin fraction - proteins are soluble and may be in the form of glycoproteins - these are mainly produced by the liver and also blood monocytes, tissue macrophages and epithelial cells of the gastrointestinal and genitourinary tracts to a lesser extent - circulating in blood serum (mostly) and body tissues in an inactive form (proenzymes or zygomens) - activation results from proteolytic cleavage of an inhibitory fragment to reveal the active site on these proteins - usually designated as C and are inactive until they are split into two fragments (products) - the proteins are numbered C1-C9 - fragments are activated proteins and are indicated by lower case a (smaller fragment) and b (larger fragment)
- 6. Complements e.g. C3 (inactive) splits into 3a and 3b (active forms) - larger fragments bind to target near site of activation - smaller fragment diffuse from the site and initiate inflammatory responses following binding to specific cell receptors - complement fragments interact with each other to form complexes capable of performing various enzymatic activities e.g. C4b2a - together these proteins destroy microbes and foreign matter by: 1. Cytolysis 2. Inflammation 3. Phagocytosis
- 7. Complements - the activated forms carry out the destructive processes - complement proteins act in a cascade - i.e. one reaction triggers another - during the cascade process, more product is formed with each succeeding reaction therefore the effect is amplified many times - the cascade of complement proteins that occurs during an infection is called complement activation and may occur in three pathways Complement activation - complement activation culminates in the formation of C5b and the formation of the membrane-attack complex (MAC) - formation of C5b and MAC are present in all pathways - three complement pathways exist: 1. Classical pathway 2. Alternate pathway 3. Lectin pathway
- 8. Complements 1. Classical pathway - the first to be discovered - initiated by antibodies binding to antigens forming soluble antibody-antigen complexes or via binding of antibody to antigen on a suitable target (e.g. pathogen) - IgG and IgM are the antibody classes that can activate this pathway - complex formation cause changes in Fc region of IgM with resultant exposed site for C1 to bind - antibody-antigen complexes bind and activate C1 (macromolecule in serum; present as C1qr2s2 stabilised by Ca2+) - C1q molecule has 18 polypeptide chains that form six collagen- like triple helical arms - tips of arms bind to exposed C1q binding sites in the CH2 domain of the antibody - C1q globular heads must bind to a minimum of two Fc sites for a stable C1-antibody interaction
- 9. Complements - C1 activates C2 and C4 (not in order because complements were named based on discovery and not order of function) - C2a and C4b combine (now called C3 convertase) and activate C3 - C4a acts as a mediator of inflammation or anaphylatoxin - activation of C3 results in numerous C3b hence much amplification - some of the C3b then binds to the C4b2a to form a trimolecule of C4b2a3b - trimolecule is termed C5 convertase - C3b from trimolecule now binds to C5 and cause conformational change and formation of C5a and C5b - C5b attaches to C6 resulting in activation of C7, C8, C9 and eventual formation of MAC - some C3b diffuses away and acts as opsonins or promotes phagocytosis - hence C3 fragments initiate cytolysis, inflammation and opsonization
- 10. Complements 2. Alternative pathway - discovered after the classical pathway - unlike the classical, this pathway does not involve antibodies - hence this pathway is seen as a component of the innate system - activation is via contact between complement proteins and a pathogen - pathway activation involves the activity of four serum proteins: C3, factor B, factor D and properidin - pathway initiation usually involves the presence of cell surface constituents of foreign matter - the combination initiates the splitting of C3 and the subsequent steps of inflammation, cytolysis and opsonization - activation results in C3 cleavage and later binding of C3b to foreign surface antigens - C3b then binds to factor B to form a complex that is stabilised by Mg2+
- 11. Complements - binding of C3b to factor B results in expose site on factor B that acts as a substrate for factor D (enzymatically active serum protein) - factor D cleaves C3b-factor B complex to generate C3bBb complex that has C3 convertase activity - C3bBb complex is short lived therefore properidin quickly binds - C3 activity is same as that in classical pathway that results in lysis
- 12. Complements 3. Lectin pathway - most recently discovered - aka as MBLectin pathway because lectin binds to mannose - similar to alternate pathway in that is not dependent on antibody-antigen complex for activation - functions like the classical pathway with activation of C4 and C2 - involves the production of lectin from the liver following stimulation by macrophages ingesting microbes (e.g. Listeria spp., Neiserria spp., Candida albicans) and foreign objects - lectin binds to CHO (mannose) and leads to the activation of C2 and C4 - a cascade involving MBL-associated serine proteases, MASP-1 and MASP-2 binding to MBL results in cleavage of C2 and C4 - activation of C4 and C2 leads to the formation of C5 convertase and subsequent C5 activation and foreign matter elimination
- 13. Complements Membrane attack complex (MAC) - forms from the terminal action of C5b, C6, C7, C8 and C9 - MAC forms a large channel through target cell membrane - channels allow for easy diffusion of ions and small molecules across the membrane - formation is dependent upon the activity of C5b binding to C6 to form C5bC6 complex which later binds to C7 - binding activity induces conformational change of C5bC6C7 complex - change involves exposure of hydrophobic regions that permit membrane phospholipids to bind - complex binding to C8 also undergoes conformational change to expose a hydrophobic region that interacts with plasma membrane - C5bC6C7C8 complex creates a tiny hole in membrane with resultant cell lysis - final step involves binding of C9 completes MAC formation
- 14. Complements - the completed MAC creates a much larger pore that permits the ions and small molecules to diffuse freely through its central channel - constant diffusion results in the cell’s inability to maintain osmotic stability hence cell death is due to water influx and loss of electrolytes - MAC is capable of lysing the walls and capsids of various foreign matter such as bacteria (both Gram –ve and +ve), viruses, erythrocytes (single MAC), parasites and nucleated cells (many MAC) - at times Gram –ve and Gram +ve bacteria may evade complement activity Some bacteria evade the complement system by means of their capsules (Streptococcus spp), which prevent complement activation. e.g. Gram –ve E. coli (LPS) and Salmonella spp. Gram +ve has thick cell wall
- 15. Complements Inactivation of complement and complement clearance - once activated, complement destructive capabilities usually cease very quickly so as to minimize destruction to host cells - inactivation is accomplished by various regulatory proteins in the host blood or passive mechanisms (e.g. short half life and spontaneous hydrolysis of C3 when unbound from C2aC4b) - the proteins break down activated complements and function as inhibitors and destructive enzymes - complements play a vital role in clearing immune complexes thus preventing various immune disorders (e.g. SLE) - uncleared complexes present in large numbers can induce tissue damage
- 16. Complements Complement and disease - can cause a disease as a result of inherited deficiencies e.g. deficiency in C5 and C9 result in increased susceptibility to Neisseria species e.g. deficiency C1, C2, C4 and CR1 predisposes persons to SLE
- 17. Cytokines Cytokines - named according to their role in cell- to-cell communication - low molecular weight proteins that mediate the interactions between lymphoid cells, inflammatory cells and haematopoietic cells - secreted by white blood cells and other cells in the body in response to a number of stimuli - serve as regulatory proteins in the development of immune effector cells - term cytokines collectively describes all proteins that mediate cell-to-cell communication however cytokines are also named according to tissue or cells that secrete them (e.g. lymphokines from lymphocytes, adipokines from adipose tissue, monokines from macrophages and monocytes, interleukins from leucocytes) - cytokines work by binding to specific receptors on target cells and trigger signal transduction that results in altered gene expression in target cells
- 19. Cytokines Cytokine function - quite similar to hormones but activity is over a shorter distance and these are produced and secreted by several tissues and cells - regulate intensity and duration of the immune response by either stimulating or inhibiting activation, control proliferation and/or differentiation of various cells - these also regulate the secretion of antibodies or other cytokines hence these influence the activity of many other cells involved in immunity (e.g. B cells, T cytotoxic cells, macrophages, stem cells) - cytokine activity can be described as pleiotrophy, redundancy, synergy, antagonism and cascade induction
- 20. Complements Because viruses depend on their host cells to provide many functions of viral multiplication, it is difficult to inhibit viral multiplication without affecting the host cell itself. One way the infected host counters viral infections is with interferons. Interferons (form of cytokines) - a class of similar antiviral proteins produced by certain animal cells such as lymphocytes and macrophages after viral stimulation - small proteins (MW 15,000 – 30,000)that are stable at low pH and are fairly resistant to heat - a principle function of these is to interfere with viral replication - these are host-cell specific and not viral specific e.g. interferons produced by human cells protect human cells but produce little antiviral activity for cells from other species (rat, mice, chicken etc)
- 21. Interferons - lymphocytes, macrophages and fibroblasts that become infected with viruses will secrete interferons - inteferons released from infected cells will diffuse to uninfected cells and induce the formation of antiviral proteins (AVPs) - antiviral proteins produced from uninfected cells interfere with viral replication - interferons do not actually stop viral particles from attaching and penetrating but rather stop viral replication thus protecting uninfected cells - these can also bind to receptors on uninfected cells and make them resistant to viral infection - viruses only cause disease if they are able to multiply - human interferons exist in three types: 1. interferon alpha, 2. interferon beta, 3. interferon gamma - each type have a slightly different affect on the body
- 22. Interferons Interferon alpha and interferon beta - induces the production of AVPs by uninfected cells resulting in: 1. oligoadenylate synthetase causing degrades viral mRNA 2. protein kinase (P1 kinase) inhibiting protein synthesis Interferon gamma - produced by lymphocytes/NK cells and stimulates neutrophils and macrophages to kill bacteria via phagocytosis Similar to complements, interferons are only active for short periods but play a major role in viral infections that are acute (intense but short lived). Also possess anticancer properties. However these proteins have no effect on viral infected cells. High concentrations of interferons can be toxic to major organs like the heart, kidneys and red bone marrow.
- 23. Questions?