GF 9

GF 9

1. Roles of Cytokines in Regulating Inflammation

   • Cytokines are low-molecular weight proteins produced by inflammatory (and other) cells during inflammation. They include TNF, interleukins, and interferons. Chemokines are chemoattractants that recruit cells to sites of injury and infection.
   • Tumour necrosis factor up-regulates inflammation. The effects of TNF were first inferred a century ago by a New York physician, William Coley, who inoculated cancer patients with bacterial preparations and obtained tumour regressions.
   • The probable active agent, TNF, was discovered independently in two research projects
     • Bacterial products injected into mice (the tuberculosis vaccine BCG and bacterial lipopolysaccharide LPS) elicited the production of a protein which caused some tumour to become necrotic. This protein was called tumour necrosis factor.
     • People with cancer and other chronic inflammatory conditions may lose fat and muscle protein (undergo wasting, cachexia). The mediator of cachexia (cachectin) was purified in mice infected with the sleeping sickness parasite, Trypanosome. When its sequences was obtained, it was found to be TNF,
   • TNF has been tested as an anticancer agent. It has limited use.
     • Induces unpleasant flu-like symptoms
     • Is not effective against common cancer types.
   • At low concentrations, TNF and IL-1 induce protein synthesis in endothelial cells.
     • Vasodilation
     • Vasopermeability
     • Expression of EC adhesion molecules (selectins, ICAM-1, VCAM-1) that recruit inflammatory cells.
   • At high concentrations
     • Fever
     • Coagulation
     • Development of scar tissue, fibrosis
     • (Over time) cachexia.

2. Recruitment and Antimicrobial Function of Neutrophils

   • Neutrophils comprise the major cell population in acute inflammation. They exist in an armed and dangerous state, roaming the body in a constant search for invading micro-organisms. When trouble is found, neutrophils can turn from angry to downright nasty, releasing a biological cocktail of what might aptly be described as cellular weapons of mass destruction. They posses granules that contain molecules used in defence and signalling.
   • Injury without infection generates sterile inflammation. Cellular responses are initiated by molecules released from dead cells and damaged ECM. Where there is also infection, microbes release PAMPs.
   • Signalling from TLRs and P2X7Rs activates inflammasomes. Caspase-1 is activated and cleaves pro-IL-1ß. In addition, an IC cytokine, IL-1alpha, is released when cells lyse. These induce ECs to become adhesive for leukocytes.
   • Recruitment of circulating neutrophils requires adherence to the endothelium (margination).
     • Blood flow-rate decreases with loss of plasma and increased blood viscosity. Neutrophils in venules leave the axial (central) stream and concentrate in the plasmatic zone (the boundary layer adjacent to the endothelium).
     • Neutrophils loosely contact the endothelium by rolling along it, mediated by P- and E- selectins on stimulated ECs (maximal within a few minutes, and by 2-4 hour respectively) and glycoproteins with sugar residues (sialyl-Lewis-X) on neutrophils.
     • Firm adhesion with flattening occurs when inflammatory mediators up-regulate intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 on ECs and the leukocyte function-associated antigen-1 on neutrophils.
     • Transmigration: adherent neutrophils push their way between the ECs. They release elastase to digest basement membrane so they can enter the extravascular tissue.
     • Leukocytes move directionally in response to chemical signals. Chemotactic signals guide cells in the circulation.
       • Proteins released from broken cells, such as mitochondrial proteins with an N- formylated methionine. These are recognised, like bacterial NFPs, by formulated peptide receptor -1, overriding the effects of cheekiness in the circulation.
       • Chemokines, leukotriene B4 and complement products C3a and C5a
       • Bacterial products
   • Phagocytes such as neutrophils eliminate damaged tissue and microbes by the coordinated processes of phagocytosis, degranulation and the respiratory burst.
     • Opsonisation: Damaged cells or bacteria are phagocytosed more readily when coated with opsonins. These include immunoglobulins and C3b.
     • Adhesion: Phagocytes attach to opsonised particles using receptors for immunoglobulins and for C3b (the Mac-1 integrin). Receptors for LPS bind bacteria directly in non-opsonic phagocytosis.
     • Phagocytosis of bacteria and debris. Pseudopodia extend around particles, and internalize them in membrane-bound vesicles (phagosomes). The fluid in the phagosome is a sample of the extracellular medium, and is not toxic.
     • Phagosomes fuse with lysosomes (containing hydrolytic enzymes) to form phagocytic vacuoles or phagolysosomes. The lysosomal granules disappear from the cytoplasm (hence, degranulation). The fluid in phagocytic vacuoles is acidified (pH 4.5-5.0).
     • An electron transport complex, NADPH oxidase (NOX2), is assembled on phagocytic vacuole membranes. In a respiratory burst, oxygen is reduced to superoxide.
     • Superoxide
       • Depolarises the vacuole, inducing a K+ influx that releases cationic proteases (cathepsin G, elastase) from anionic sulphated proteoglycans.
       • Is reduced to hydrogen peroxide by superoxide dismutase. Myeloperoxidase catalyses the reaction of H2O2 with chloride to generate hypochlorous acid.
       • Reactive proteases and oxidants degrade cell debris and kill micro-organisms.

3. Phenomena Associated with Acute Inflammation

   • Systemic (body-wide) Responses
     • Fever is caused by pyrogens such as TNF, IL-1, and PGE2 (produced peripherally by macrophages, and in the brain), which affect hypothalamic thermoregulation.
     • The acute phase response is induced by IL-6. The liver produces plasma proteins such as coagulation factors and opsonins.
     • Leukocytosis is an increase in the number of circulating leukocytes, caused by the release of leukocytes from the bone marrow.
     • Processing of microbial molecules activates adaptive immunity.
   • Liquefactive necrosis (suppuration). Infection with pyogenic (pus-forming) agents in solid tissues leads to the accumulation of dead neutrophils and tissue cells (pus). Lesions may be localized (as abscesses such as boils) or spreading (cellulitis).
   • Inflammation may run out of control. Systemic inflammatory response syndrome (SIRS, with sterile injury) and sepsis (with infection).
     • The release into the circulation of DAMPs (NFPs, mitochondrial DNA), pro-inflammatory cytokines (TNF, IL-1), and bacterial toxins.
     • An increase in the ratio of pro- to anti-coagulants.
     • Activation of complement
     • Generation of ROS.
   • The Result
     • Widespread vascular dilatation and permeability with loss of blood volume; o formation of thrombi in small blood vessels (disseminated intravascular coagulation, DIC).
     • Depletion of clotting factors (leading to uncontrolled bleeding).
     • Inadequate organ perfusion, ischaemia.
     • Multi-organ dysfunction or failure (with mortality >30%).
   • Regeneration or Repair with Resolution. Inflammation is turned off by...
     • Eliminating the stimulatory effects of dead cell and bacterial products.
     • Negative feedback loops involving anti-inflammatory cytokines, lipid mediators, and protease inhibitors.
   • During inflammation, the life-time of neutrophils is extended by cytokines, growth factors and activated endothelium. During phagocytosis, the C3b receptor Mac-1 induces the respiratory burst, generating ROS, caspase activation and neutrophil apoptosis. Macrophages phagocytose dead neutrophils.
   • Chronic inflammation arises when acute inflammation fails to resolve.