THE ACUTE INFLAMMATION AND ACUTE-PHASE RESPONSE

Innate immune mechanisms establish a state of inflammation at sites of infection Illustrated here are the events following an abrasion of the skin. Bacteria invade the underlying connective tissue and stimulate the innate immune response.

Acute inflammation A rapid response to an injurious agent that serves to deliver leukocytes, plasma proteins and fluids to the site of injury The major way by which the innate immune system deals with infections and tissue injury is to stimulate acute inflammation, which is the accumulation of leukocytes, plasma proteins, and fluid derived from the blood at an extravascular tissue site of infection or injury.

Triggers of acute inflammation • Infections (bacterial, viral, fungal, parasitic) & microbial toxins • Tissue necrosis: ischemia, trauma, physical or chemical injury (e.g., thermal injury; irradiation; some environmental chemicals) • Foreign bodies (splinters, dirt, sutures) • Immune reactions (hypersensitivity or autoimmune reactions)

Major components of inflammation – Vascular changes • Vasodilation • Vascular permeability • Increased adhesion of white blood cells – Cellular events • Recruitment and activation of neutrophils (polymorphonuclear leukocytes) and monocytes

Classical signs of acute inflammation Redness (rubor) Swelling (tumor) Heat (calor) Pain (dolor) Loss of function (functio laesa)

Maturation of mononuclear phagocytes and dendritic cells Both dendritic cells and monocytes arise from a common precursor cell of the myeloid lineage in the bone marrow, and differentiation into monocytes or dendritic cells is driven by the cytokines monocyte colony-stimulating factor and Flt3 ligand, respectively (not shown). Dendritic cells further differentiate into subsets, the two major being conventional dendritic cells and plasmacytoid dendritic cells. Some dendritic cells may arise from monocytes in inflamed tissues. When blood monocytes are recruited into tissues, they become macrophages. Long-lived resident macrophages are present in all tissues of the body. At least two populations of blood monocytes exist (not shown), which are precursors, respectively, of macrophages that accumulate in response to infections and macrophages that are constitutively present in normal tissues. Macrophages in tissues become activated to perform antimicrobial and tissue repair functions in response to infections and tissue injury. Macrophages differentiate into specialized forms in particular tissues. CNS, central nervous system; DC, dendritic cell. Cellular and Molecular Immunology, 7th ed., 2014 Elservier

Macrophages respond to pathogens by using different receptors to stimulate phagocytosis and cytokine secretion The left panel shows receptor-mediated phagocytosis of bacteria by a macrophage. The bacterium (red) binds to cell-surface receptors (blue) on the macrophage, inducing engulfment of the bacterium into an internal vesicle called a phagosome within the macrophage cytoplasm. Fusion of the phagosome with lysosomes forms an acidic vesicle called a phagolysosome, which contains toxic small molecules and hydrolytic enzymes that kill and degrade the bacterium. The right panel shows how a bacterial component binding to a different type of cell-surface receptor sends a signal to the macrophage’s nucleus that initiates the transcription of genes for inflammatory cytokines. The cytokines are synthesized in the cytoplasm and secreted into the extracellular fluid.

Effector functions of macrophages Macrophages are activated by microbial products such as LPS and by NK cell–derived IFN-γ. The process of macrophage activation leads to the activation of transcription factors, the transcription of various genes, and the synthesis of proteins that mediate the functions of these cells. In adaptive cell-mediated immunity, macrophages are activated by stimuli from T lymphocytes (CD40 ligand and IFN-γ) and respond in essentially the same way.

Macrophages respond to infection by secreting inflammatory cytokines IL-1β, TNF-α, IL-6, IL-8 (CXCL8), and IL-12 are the five key cytokines that macrophages secrete. The cytokines recruit effector cells and plasma proteins to the infected tissue, where they work together to create a state of inflammation.

Systemic actions of cytokines in inflammation TNF, IL-1, and IL-6 mediate protective systemic effects of inflammation, including induction of fever, acute-phase protein synthesis by the liver, and increased production of leukocytes by the bone marrow. Systemic TNF can cause the pathologic abnormalities that lead to septic shock, including decreased cardiac function, thrombosis, capillary leak, and metabolic abnormalities due to insulin resistance.

Neutrophils are directed to sites of infection through interactions between adhesion molecules The upper panel shows the rolling interaction of a neutrophil with vascular endothelium as a result of transient interactions between selectin on the endothelium and sialyl-Lewisx (s-Lex) on the leukocyte. The lower panel shows the conversion of rolling adhesion into tight binding and subsequent migration of the leukocyte into the infected tissue. The four stages of extravasation are shown. Rolling adhesion is converted into tight binding by interactions between integrins on the leukocyte (LFA-1 /Lymphocyte-function associated antigen-1/ is shown here) and adhesion moleules on the endothelium (ICAM-1). Expression of these adhesion molecules is also induced by cytokines. A strong interaction is induced by the presence of chemoattractant cytokines (the chemokine CXCL8 is shown here) that have their source at the site of infection. They are held on proteoglycans of the extracellular matrix and cell surface to form a gradient along which the leukocyte can travel. Under the guidance of these chemokines, the neutrophil squeezes between the endothelial cells and penetrates the connective tissue (diapedesis). It then migrates to the center of infection along the IL-8 (CXCL8) gradient. The electron micrograph at the right shows a neutrophil that has just started to migrate between adjacent endothelial cells but has yet to break through the basement membrane, which is at the bottom of the photograph. The blue arrow points to the pseudopod that the neutrophil is inserting between the endothelial cells. The dark mass in the bottom right-hand corner is an erythrocyte that has become trapped under the neutrophil. Photograph (× 5500) courtesy of I. Bird and J. Spragg.

Neutrophil chemotaxis Activated macrophages release matrix metalloproteinases, which cleave the collagen matrix. A collegen fragment, N-acetyl proline-glycine-proline induces CD11b/CD18-dependent neutrophil adhesion. acPGP: N-acetyl Proline-Glycine-Proline – neutrophil chemoattractant MMP: matrix metalloproteinase

Neutrophil granulocytes 68% of circulating leukocytes, 99% of circulating granulocytes Phagocytic cells Are not present in healthy tissues Migration  elimination of pathogens (enzymes, reactive oxygen intermediates) Main participants of acute inflammatory processes Upper panel: the neutrophil has several different receptors for microbial products. Lower panel: the mechanism of phagocytosis for two such receptors, CD14 and CR4, which are specific for bacterial lipopolysaccharide (LPS). A bacterium binding to these receptors stimulates its own phagocytosis and degradation.

Killing of bacteria by neutrophils involves the fusion of two types of granule and lysosomes with the phagosome After phagocytosis (first panel), the bacterium is held in a phagosome inside the neutrophil. The neutrophil’s azurophilic granules and specific granules fuse with the phagosome, releasing their contents of antimicrobial proteins and peptides (second panel). NAPDH oxidase components contributed by the specific granules enable the respiratory burst to occur, which raises the pH of the phagosome (third panel). Antimicrobial proteins and peptides are activated, and the bacterium is damaged and killed. A subsequent decrease in pH and the fusion of the phagosome with lysosomes containing acid hydrolases results in complete degradation of the bacterium (fourth panel). The neutrophil dies and is phagocytosed by a macrophage (fifth panel).

Killing of bacteria by neutrophils is dependent on a respiratory burst In the absence of infection, the antimicrobial proteins and peptides in neutrophil granules are kept inactive at low pH. After the granules fuse with the phagosome, the pH within the phagosome is raised through the first two reactions, involving the enzymes NADPH oxidase and superoxide dismutase. Each round of these reactions eliminates a hydrogen ion, thereby reducing the acidity of the phagosome. A product of the two reactions is hydrogen peroxide, which has the potential to damage human cells. (In hair salons and in the manufacture of paper, it is used as a powerful bleach.) The third reaction, involving catalase, the most efficient of all enzymes, promptly gets rid of the hydrogen peroxide produced during the neutrophil’s respiratory burst, raising the pH of the phagosome and enabling activation of the antimicrobial peptides and proteins.

Neutrophils are stored in the bone marrow and move in large numbers to sites of infection, where they act and then die. After one round of ingestion and killing of bacteria, a neutrophil dies. The dead neutrophils are eventually mopped up by long-lived tissue macrophages, which break them down. The creamy material known as pus is composed of dead neutrophils.

PUS Pus is a whitish-yellow, yellow, or yellow-brown exudate produced Pus = transudate (liquor puris) + dead pathogens + dead neutrophils + dead tissue cells Pus is a whitish-yellow, yellow, or yellow-brown exudate produced by vertebrates during inflammatory pyogenic bacterial infections. Pus consists of creamy, protein-rich fluid, known as liquor puris, and dead cells.

Acute injury of myocardium 1. Exudate leaves the vessels. 2. Recruitment of large numbers of neutrophils, followed by monocytes, from blood into tissues typically occurs as part of the acute inflammatory response to infections and tissue injury.

Fever production in response to TNF, IL-1, and IL-6 proinflammatory cytokines hypothalamic control of body temperature increased ‚set-point’ value fever TNF, IL-1, and IL-6 all act on the hypothalamus to induce an increase in body temperature, and these cytokines are therefore called endogenous pyrogens.

ACUTE-PHASE REACTION Liver IL-6 The panel shows representative examples, and their functions, of the different types of plasma protein that increase during the acute-phase response.

ACUTE-PHASE REACTION Pentraxin family: CRP – opsonization, complement activation SAP – opsonization, complement activation, binding of mannose/galactose Collectin family: MBL – part of the complement system (SP-A/D – collectins of lungs) Complement proteins (C1-C9) Fibrinogen  blood clotting Elevated levels of acute-phase reactants are commonly used clinically as signs of infection or other inflammatory processes. The pentraxins CRP and SAP play protective roles in infections and fibrinogen, the precursor of fibrin, contributes to homeostasis and tissue repair. CRP: C-reactive protein SAP: serum amyloid P MBL: mannose binding lectin SP-A/D: surfactant proteins A and D

The kinetics of acute-phase proteins in the blood The graph shows the change in concentration of five proteins in blood plasma after the initiation of an inflammatory response. C-reactive protein and serum amyloid A are massively increased acute-phase proteins, whereas C3 and fibrinogen are moderately increased. Serum albumin, the most abundant plasma protein, is reduced in concentration during the acute phase. Data courtesy of J.I. Gitlin and H.R. Colten from Lymphokine Reports Volume 1, edited by Edgar Pick and Morris Landy (1980), pp. 123–153.

Chemical mediators Vasodilation Prostaglandins (PG), nitric oxide (NO) Increased vascular permeability vasoactive amines (histamine, serotonin), C3a and C5a (complement system), bradykinin, leukotrienes (LT), PAF Chemotactic leukocyte activation C3a, C5a, LTB4, chemokines (e.g. IL-8) Fever IL-1, IL-6, TNFα, PGE2 Pain Prostaglandins, bradykinin Tissue damage Neutrophil and Macrophage products lysosomal enzymes Reactive oxygen species (ROS) NO NSAIDs and Paracetamol: inhibiting COX-1 and COX-2  preventing the synthesis of prostaglandins

Resolution of acute inflammation Schematic depicting the cellular components of the inflammatory response and the requirements for resolution. Acute inflammation is characterized by the extravascular accumulation of neutrophils (PMN) and edema formation early in the response. Later during the response, mononuclear cells and macrophages accumulate and help prepare the tissue for resolution. Macrophages remove dead cells and then exit the site of inflammation. Stromal cells such as fibroblasts also contribute to the resolution of inflammation by the withdrawal of survival signals and the normalization of chemokine gradients, thereby allowing infiltrating leukocytes to undergo apoptosis or leave the tissue through the draining lymphatics. This sequential set of responses leads to complete resolution and, importantly, the restoration of the inflamed tissue to its prior physiological functioning.

Septic shock Result: Triggering factors : systemic infection (bacteraemia) microbial cell wall products and/or toxins released from the pathogens into blood circulation. Result: Systemic activation of neutrophils and macrophages  High level of cytokine (TNF-alpha) production: „cytokine storm” Excessive inflammatory response A complication of severe bacterial sepsis is a syndrome called septic shock, which may be caused by LPS released from gram-negative bacteria (in which case it is called endotoxin shock) or lipoteichoic acid from gram-positive bacteria. Septic shock is characterized by vascular collapse, disseminated intravascular coagulation, and metabolic disturbances. This syndrome is due to LPS- or lipoteichoic acid–induced TLR signaling leading to the production of TNF and other cytokines, including IL-12, IFN-γ, and IL-1. The concentration of serum TNF may be predictive of the outcome of severe bacterial infections.

The key molecule of the process: TNF-alpha Septic shock The key molecule of the process: TNF-alpha TNF-alpha and other inflammatory cytokines The principal systemic actions of TNF are the following: - TNF inhibits myocardial contractility and vascular smooth muscle tone, resulting in a marked fall in blood pressure, or shock. - TNF causes intravascular thrombosis, mainly as a result of loss of the normal anticoagulant properties of the endothelium. TNF stimulates endothelial cell expression of tissue factor, a potent activator of coagulation, and inhibits expression of thrombomodulin, an inhibitor of coagulation. The endothelial alterations are exacerbated by activation of neutrophils, leading to vascular plugging by these cells. The ability of this cytokine to cause necrosis of tumors, which is the basis of its name, is mainly a result of thrombosis of tumor blood vessels. - Prolonged production of TNF causes wasting of muscle and fat cells, called cachexia. This wasting results from TNF-induced appetite suppression and reduced synthesis of lipoprotein lipase, an enzyme needed to release fatty acids from circulating lipoproteins so that they can be used by the tissues. DIC capillar permeability blood pressure high fever multiorgan failure disseminated intravascular coagulation Therapy: anti-TNF-alpha antibody

DIC Disseminated Intravascular Coagulation pathologic activation of thrombotic process distress of thrombotic process, bleeding other causes: snake bite, septic abortion, acute obstetric complications, malignant tumors, leukemias Proinflammatory cytokines (IL-1, TNFα) and LPS/endotoxin leads to the release of Tissue Factor (TF) from cells which triggers the coagulation cascade. The complement system also has an effect on blood clothing.

DIC: Disseminated Intravascular Coagulation