31.2 Acute Inflammation Flashcards
What is acute inflammation?
Short-term, beneficial response in which a heightened immune state is locally induced in response to stimulation of the innate (rarely adaptive) immune system by a pathogen.
What is the time course for acute inflammation?
minutes to days
How does acute inflammation fight pyogenic bacterial infection?
Neutrophil recruitment –> to facilitate phagocytosis
What are the cardinal features of acute inflammation?
*Heat (vasodilation)
*Redness (vasodilation)
*Swelling (oedema)
*Pain (chemical mediators)
What are some of the cells that can begin acute inflammation at the site of infection/damage?
Resident macrophages, mast cells and dendritic cells.
What are the cells that are released into the site of infection as part of the acute inflammatory exudate?
Rapidly recruited:
- Neutrophils
- Platelets
Recruited soon after:
- Monocytes (that become macrophages)
- T-lymphocytes
If the site is of allergic inflammation:
- Eosinophils/Basophils
What are the histological differences between healthy and inflamed tissue?
*Vasodilation - increased vessel size + increased presence of blood cells
*Oedema - cells more spread out/ less concentrated on slide.
*Cellular infiltration by leukocytes
What are the different proteins that are released into the site of infection as part of the acute inflammatory exudate?
- Albumin
- Antibodies
- Complement proteins
- Coagulation factors
What is responsible for much of the swelling at sites of acute inflammation?
Albumin released as part of the exudate leads to osmotic pressure that pulls water in.
What are the main stages of leukocyte recruitment in inflammation?
- Margination
- Leukocyte rolling
- Leukocyte activation
- Leukocyte tight adhesion
- Diapedesis (Leukocyte extravasation)
- Chemotaxis
Describe the concept of how neutrophils and macrophages know where to go during acute inflammation.
- The cells at the site of inflammation send out cytokines and other molecules to partially activate neutrophils and macrophages passing through an adjacent venule
- This triggers them to cross the blood vessel barrier
- Once crossed, the neutrophils and macrophages follow chemokines, pathogen molecules and other molecules to the site of infection
- Here they are fully activated
What is pus?
An accumulation of fluid, living and dead white blood cells, dead tissue, and bacteria or other foreign invaders or materials.
What is an abcess?
- A localized collection of pus in any part of the body, often caused by an infection by pyogenic bacteria.
What are the different possible fates of an abscess, in order of desirability?
- Resolution with no scarring
- Resolution with scarring
- Rupture external
- Rupture internal -> Leading to a septic embolus
- Cyst formation (neutral outcome)
What are the best treatment options for abscesses?
Surgical drainage and antibiotics
What makes abscesses difficult to treat?
Their interiors have little or no vascularization, making it difficult to deliver therapeutic agents effectively.
How does pus form?
During immune response to a pathogen, neutrophils and surrounding tissue die accumulating as pus. This collects in a cavity created by the breakdown of tissue (Abscess).
Describe the appearance of neutrophils.
- Multi-lobed nucleus joined by filaments (sausage-like appearance)
- About 10µm
- Has some granules
What percentage of leukocytes are neutrophils?
40-70%
Where does proliferation and differentiation of neutrophils take place?
In the bone marrow.
Where is the reserve pool of neutrophils?
Bone marrow (50% of the nucleated leukocytes in bone marrow are PMNs)
When are neutrophils mobilised from the reserve pool in bone marrow? What is this called?
- In response to infection
- This is called granulocytosis/neutrophilia
Are neutrophils terminally differentiated?
Yes, so they do not synthesis DNA, although they do synthesis some limited mRNA and proteins.
What could an increased neutrophil count indicate?
Bacterial infections and stress.
What are the actions of neutrophils? (5)
- Increase their numbers during acute bacterial infection
- Adhere to vascular endothelium –> migrate into tissues in acute inflammation
- Release granules - contain antibacterial molecules
- Generate toxic mediators derived from H2O2
- Phagocytosis - ingest, kill and digest bacteria (produce pus)
What is diapedesis?
Passage of leukocytes through intact capillary walls
How does leukocyte (neutrophil and monocyte/macrophage) activation occur in acute inflammation?
- It occurs via GPCRs on the leukocytes
- The molecules that stimulate these mostly include cytokines, C5a, PAF and others
Note that partial activation occurs during recruitment of the leukocytes, while complete activation occurs at the site of inflammation.
What does (partial) activation of leukocytes during recruitment in acute inflammation do?
- Leads to altered conformations of cell surface integrins on the leukocytes
- The integrins can now form tight interactions with cell adhesion molecules on endothelial cells
- Thus, the leukocytes adhese to the vessel wall
Describe how tight adhesion of leukocytes to the endothelium occurs (during acute inflammation).
ICAM interactions:
- Integrins on the leukocyte surface undergo a conformational change upon activation of the leukocyte
- This allows the integrins to bind to cell-adhesion molecules on endothelial cells
- This ICAM interaction is necessary to halt leukocyte rolling and start diapedesis
(NOTE: The diagram also shows the selectin interactions involved in leukocyte rolling)
What are chemokines?
A family of small cytokines that can induce chemotaxis in nearby responsive immune cells.
What is chemotaxis?
Movement of a cell in a direction corresponding to a gradient of increasing or decreasing concentration of a particular substance.
What receptors on leukocytes detect chemoattractants?
GPCRs
What are some chemoattractants involved in attracting leukocytes to the site of inflammation? What are their receptors?
- Bacterial products, namely N-formylmethionine (fMLP - an amino acid used to initiate protein synthesis in bacterial cells) -> mFLP receptor
- Cytokines and chemokines (e.g. IL-8, CCL5, etc.) -> IL-8 receptor, CCR5, etc.
- C5a (a chemoattractant product of complement) -> C5a receptor
- Platelet-activating factor (PAF) -> PAF receptor
Describe how leukocytes move during chemotaxis and describe their appearance.
- It moves by cytoskeleton remodelling
- The protrusions at the front are called pseudopods, while the tail at the back is called the uropod
Describe the process of phagocytosis
*Opsonisation by Ab and complement
*Lysosomal fusion
*Killing and digestion of micro-organism
What are the different phagocyte killing mechanisms?
*Respiratory burst (NADPH oxidase)
*Non-oxygen-dependent killing mechanisms (antimicrobial peptides, e.g. LL-37)
*Neutrophil Extracellular Traps (NETs)
What is the respiratory burst and why is it required?
- Following uptake of pathogens for phagocytosis, the oxygen demand of phagocytes increases.
- The oxygen burst is used largely to generate NADPH from glucose via the pentose phosphate pathway. The NADPH in turn can be used to generate reactive species that kill pathogens.
What are antimicrobial peptides and what is their function in phagocyte defence mechanisms?
- Peptides (AMPs) of fewer than 50 amino acids that are an evolutionarily conserved part of the innate immune response
- Primarily target the cell membrane and create transmembrane channels, meaning that they have broad specificity within bacteria and other pathogens.
- They are not affected by antibiotic resistance of a bacterium.
- Experimentally, the broad specificity of AMPs can be demonstrated by an in vitro assay.
What are NETs?
- Neutrophil extracellular traps
- Neutrophils have a short lifespan (relative to macrophages), but before and during apoptosis they leave an extracellular net of DNA and histones, with proteins such as MPO bound to them.
- These bound proteins have antimicrobial properties, so the net functions to kill pathogens outside of cells.
Give examples of defects in phagocytic killing mechanisms.
Chronic granulomatous disease, complement deficiencies.
What are some functions of neutrophils?
- Phagocytosis
- Degranulation (of anti-microbial agents)
- Production of NETs
- Mediation of inflammation (via release of cytokines)
What are some functions of macrophages? How do these compare to neutrophils?
Defence:
- Phagocytosis and killing
- Control of inflammation -> Via cytokines and interferons
- Antigen presentation
Tissue maintenance:
- Tissue homeostasis via scavenger receptors
- Tissue remodelling
- Apoptotic cell clearance
- Tissue repair (e.g. in wound repair)
Are all macrophages derived from blood monocytes?
No, some tissue resident macrophages are derived from myeloid precursors in embryonic life and proliferate in situ throughout adult life.
Describe the two origins of macrophages.
- Embryonic yolk sac derived tissue macrophages (long-lived, self-renewing)
- Macrophages that are formed by bone marrow-derived monocytes that infiltrate the tissue
Describe the two functional types of macrophages and what the function of each is.
- Tissue resident (e.g. Kupffer cells) macrophages (mostly arising from embryonic yolk sac) -> Mediate homeostasis, repair and remodelling
- Infiltrating monocytes that become inflammatory macrophages -> Antimicrobial functions
Describe what happens during adhesion?
Integrins expressed by PMNs and macrophages in response to chemokines
Adhere to cell adhesion molecules on vascular endothelium.
Brings leukocytes to a stop.
What happens after adhesion?
Leukocytes spread and squeeze their cell bodies and secrete collagenases to infiltrate the basement membrane (transmigration) = diapedesis
What is needed for PMNs and macrophages to roll along vascular endothelium?
Expression of selectins on vascular endothelium in response to inflammatory mediators (TNF, IL-1)
List the functions of macrophages?
-Defence
-Pathogen sensing
-Cytokine production
-Antigen presentation
-Tissue homeostasis
-Pathogenic role in chronic inflammation
How are macrophages pathogen sensors?
PRRs including TLRs, NLRs (Nod-like receptors) and nucleic acid sensors.
What are NLRs?
NLRs, or nucleotide-binding, leucine-rich, repeat containing (NOD-like) receptors are components of the inflammasome
What do NLRs detect?
Part of the peptidoglycan wall and bacterial toxins. Activation leads to inflammasome activation and recruitment of caspase 1.
What are the cytotoxic mechanisms of Macrophages?
*Oxygen metabolites (produced by NADPH oxidases)
*Nitric oxide (i-NOS)
Which enzyme produces reactive nitrogen species (NO) for phagolysosomes?
i-NOS (inducible nitric oxide synthase). Used to help break down ingested structures.
Which receptors do chemokines act on?
Chemokines acting on the CCR and CXCR receptor classes, as well as others, then provide a similar chemoattractant gradient to direct the infiltrating macrophages to the areas where they will be able to have the greatest effect
What are the main “professional” phagocytes?
Neutrophils and macrophages
(Mast cells and dendritic cells also phagocytose, to a lesser extent)
What is chronic granulomatous disease?
- Individuals have a defect in the phagocyte NADPH oxidase enzyme that catalyses the reaction of NADPH with O2 to produce superoxide.
- The symptoms include a higher susceptibility to and frequency of infection, as well as the development of granulomas (collections of macrophages) in various tissues, both of which are due to the reduced immune capacity of phagocytes.
- Treatment involves antibiotic use to manage infection, and cure is currently only possible via use of hematopoietic stem cell transplant.
Describe how NO is synthesised in phagocytes and how this works as part of the defense mechanism.
- NADPH (from the respiratory burst) here is also used in the generation of nitric oxide (NO), which is a substrate (along with superoxide), for the production of more reactive nitrogen species.
- Nitric oxide synthase 2 is inducible mostly in macrophages (by inflammatory mediators).
- Reactive nitrogen species have broad antimicrobial action by reacting with transition-metals in proteins (e.g. haemoglobin or cytochrome c) or the amino acids in a protein.
What are some of the pro-inflammatory cytokines secreted by macrophages in response to microbial products? What is the function of each?
- TNF-α
- Activates vascular endothelium, increasing cell and metabolite entry
- Fever
- IL-1β
- Activates vascular endothelium and recruitment of lymphocytes, increasing cell and metabolite entry
- Fever, along with IL-6.
- IL-6
- Fever
- IL-12 (in chronic inflammation)
- Actives macrophages (via IFN-γ production) and NK cells
- CXCL8
- Recruits neutrophils and basophils
Give an example of an anti-inflammatory cytokine that counteracts the pro-inflammatory cytokines produced by macrophages in response to microbial products.
- IL-10
- Downregulates the expression of helper T-cell cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages.
- Enhances B cell survival, proliferation, and antibody production.
- Inhibits pro-inflammatory cytokines TNF-α, IL-1β, IL-12 and IFNγ secretion.
Other than CKs, what else do macrophages secrete?
*Hydrolytic enzymes
*complement components
What is the major role for macriphages?
Defence. Pathogen control in infected regions and clearing viruses from blood.
How do macrophages provide defence against IC facultative pathogens?
Primary response seems to be uptake, linked with activation by IFN-γ (for Mycobacterium tuberculosis and Leishmania especially), and O2-dependent killing in the phagolysosome
What on the pathogen activates macrophages?
Activated by endotoxin
*usually highly conserved, essential components (e.g. Bacterial Lipid A)
How are macrophages able to induce an anti-viral state in surrounding cells?
Through production and secretion of type-1 interferons (alpha/ beta)
What are the functions of macrophages in tissue homeostasis?
They are involved in tissue remodelling and repair.
Give an example of macrophage involvement in cell repair.
In the lung after slowly resolving bacterial pneumonia, macrophages remove fibrin and inflammatory debris.
They also recruit fibroblasts.
How are macrophages involved in remodeling?
*They produce TGF-β1, which activates myofibroblasts that produce ECM.
*Can also produce MMPs that help resolve fibrosis.
What can persistent injury and/or failed resolution of an infection lead to?
Chronic inflammation
How can macrophages contribute to chronic infection?
When inflammation fails to resolve, macrophages can become dysregulated and shift to a pro-inflammatory state: M1 secreting inflammatory CKs and chemokines. M2 (anti-inflammatory) Macrophages can become less active.
How are pro-inflammatory (M1) macrophages activated?
M1 - pro-inflammatory/ pro-immune role.
*activated by IFN-γ during recruitment.
*TNF-α and IL-1/6 are pro-inflammatory CKs.
*endotoxins
What activates anti-inflammatory (M2) macrophages?
IL-4 and IL-13 during recruitment.
How does IL-12 affect macrophage activation?
Promotes secretion of TNF-α and NO when in the Prescence of mycobacteria.
*aids in inflammatory response.
How does IL-10 affect macrophage activation?
Anti-inflammatory role. It limits the production of pro-inflammaotry CKs and Chemokines by macrophages and monocytes.
*prevents overactivation/ potential tissue damage.
What are the different complement pathways?
- Classical pathway: C1 complex binds to antibody on the bacterial surface
- Lectin pathway: mannose-binding lectin (MBL) binds to bacterial mannose
- Alternative pathway: C3b binds to surface hydroxyl groups on bacteria.
How can the complement system be activated in acute inflammation?
- By the Fc portion of antibodies which have combined with specific antigen—the classical pathway of activation
- By the endotoxins of Gram-negative bacteria—the alternative pathway
- By enzymes releases by dying cells in tissue necrosis
- By some products of the kinin and fibrinolytic systems.
What is the role of C3a and C5a in inflammation?
chemotactic for neutrophils, increases vascular permeability, causes release of histamine from mast cells
What is the role of the C567 complex in inflammation?
chemotactic for neutrophils
What is the role of the C56789 complex in inflammation?
the membrane-attack complex which punches holes in cell membranes → cell lysis
What is the role of C4b, C2a, C3b in inflammation?
all opsonize bacteria by binding to them; macrophages have specific receptors for these proteins.
What does factor XII (Hageman factor) activate?
The coagulation, fibrinolytic, and kinin systems.
What is the role of C3 in inflammation?
C3 can be activated by the 3 main complement pathways after which it is cleaved into C3a (activates mast cells/ release of vasoactive mediators) and C3b (opsonisation)
Which other cascades interact with the complement system?
Coagulation
Kinin
Fibrolytic
How do serine proteases interact with the complement cascade? Give examples of such enzymes
Act as convertases to cleave C3 and C5 to increase complement activation e.g. plasmin in fibrinolysis cascade and thrombin in coagulation cascade
Which cascade opposes the coagulation cascade?
Fibrinolytic cascade
Outline the alternative pathway of the complement system
Occurs as a result of spontaneous C3 hydrolysis on the surface of the microbe
Can result as cleavage by factor D and B to produce C3b and Bb which can also act as C3 convertase (C3bBb)
(C3 binds to surface hydroxyl groups on bacteria)
Describe the classical pathway
-Complement proteins are bound to an antigen-antibody complex (IgM or IgG)
-C1q (of C1 which is a trimer of C1q, C1r and C1s) binds to the Fc regions of the heavy chains
-C1 is cleaved to form an active protease (C1r and C1s) which cleaves C4 and C2 into C4b,2b complex. C4 –> C4a + C4b C2–> C2a + C2b
- C4b,2b complex is C3 convertase
-C3 convertase cleaves C3 into C3a and C3b
-C3b forms a new complex with C3 convertase (C2b,3b,4b) called C5 convertase
-C5 convertase cleaves C5 into C5a and C5b
Describe what happens in the lectin pathway
-Mannose binding lectin (MBL) on the surface of cells such as macrophages binds to mannan (polymer of mannose)
-Activates proteases which cleave C2 and C4 into their complement components
-Converges with classical pathway
-Also cleaves C3
(e.g. mannose found in bacteria and yeast is bound by mannose binding lectin on macrophages and dendritic cells - acts as an opsonin and activates complement)
How are prostaglandins formed?
Prostaglandins, which are derivatives of arachidonic acid formed mainly through the action of cyclooxygenases (COX-1/2), are lipid molecules which are locally produced and have a further effect to vasodilate as well as causing the aggregation of platelets and clotting (thromboxane activates platelets)
What activates the coagulation cascade?
Extrinisc pathway: Damage to endothelial walls leads to release of tissue factor which cleaves VIII to VIIIa
Intrinsic pathway: Contact of XII to a negatively charged surface such as basement membrane/collagen
What are leukotrienes formed from?
Oxidation of arachidonic acid in mast cells, neutrophils and basophils
What are the effects of cyokines on vascular endothelium? Give specific examples
Proinflammatory cytokines cause increased vascular permeability, increasing plasma leakage into surrounding tissues, improving diapedesis of WBCs
Induce the coagulation cascade
-TNF alpha and IL-1 increases expression of adhesion molecules (E selecting) on endothelial cells
What are the effects of cytokines on PMNs and macrophages?
IFN-gamma and TNF alpha activate PMNS and activates macrophages to secrete more TNF-alpha
TNF -alpha stimulates respiratory burst in PMNS and macrophages
What are the effects of kinins? Give an example
Bradykinin which increases vascular permeability and causes pain (act on B1/2 receptors) to promote the influx of plasma proteins to the site of tissue injury
What are the main families of cytokines?
Haematopoeitin family = interleukins, growth hormone and erythropoietin
TNF family = TNF alpha
Interferon family
What happens during fibrinolysis?
Plasminogen –> Plasmin (catalysed by plasminogen activators)
Plasmin breaks down fibrin into fibrin degradation products (FDPs)
What is the common pathway of the coagulation cascade?
Factor II cleaved to IIa (thrombin)
thrombin cleaves fibrinogen to fibrin to form the fibrin mesh clot
What is the effect of cytokines on mast cells?
Cytokine IL-33 can aid in the activation of mast cells (main driver is the binding of IgE)
What is the effect of cytokines on fibroblasts? Which cytokines are these?
Fibroblasts are required to lay down a new collagen matrix following damage, which develops either granulation tissue or scarring (type III followed by type I collagen)
The elevated levels of cytokines in these situations promotes two main processes – the differentiation of mesenchymal stem cells into transient fibroblasts, and the increased activity of tissue fibroblasts
Stimulated by PDGF, FGF and TGF beta
What is the function of leukotrienes?
Act on GPCRs to cause increased vascular permeability and bronchoconstriction
What are the effects of CKs and other mediators on the vascular endothelium?
*Proinflammatory cytokines + positive mediators –> decrease in the tightness of the endothelium, promoting leakage of plasma contents into the surrounding tissues and improving diapedesis of white blood cells into these environments. Furthermore, these molecules can help to induce the coagulation cascade, and use VEGFs and TNF-α to increase the proliferation of endothelial cells, as well as guiding these cells along fibronectin structures for angiogenesis in new or healing areas
What is the effect of CKs and other mediators on PMNs and macrophages?
Proinflammatory cytokines help to prime most leukocytes, putting them in an active position where their sensing PAMPs by PRRs will lead to an improved response (phagocytosis) of pathogens or cellular debris. They will also release further inflammatory mediators, including prostaglandins, proteases, and leukotrienes. Anti-inflammatory mediators, however, can work to downregulate this response – these include IL-1β, TNF-α, and the glucocorticoids
Name an inhibitor of proteases in the body. What is its function?
alpha1 anti-trypsin which protects bystander cells from the effects of the immune system, with a key role in the lungs – the serpin can decrease the activity of neutrophil elastases and thus reduce the risk of these enzymes breaking down the extracellular lung tissue which could lead to emphysema
Why are there inactivators for active mediators of inflammation?
To prevent excessive damage (esp. to bystander cells)
What is sepsis?
Life threatening organ dysfunction caused by a dysregulated host response to infection
Can develop septic shock as a result
What is septic shock?
Happens to subset of patients with sepsis
Persisten hypotension (MAP under 65mmHg) and elevated serum lactate despite adequate fluids
Purpleish haemorrhagic rash due activation of coagulation cascade
What is the acute phase response?
A rapid, systemic increase in various plasma proteins such as the acute phase proteins which drive a range of neuroendocrinal, physiological and metabolic changes after stimulation of the innate immune system
What is the strongest trigger for sepsis?
Endotoxin (lipid A in LPS) in circulation
What triggers the acute phase response?
Production of proinflammatory cytokines (IL-1, IL-6 and TNF-alpha) which act on hepatocytes to change the profile of proteins they secrete (synthesise and secrete acute phase proteins)
Where are acute phase proteins synthesised?
Hepatocytes in the liver
Describe how sepsis occurs after bacterial infection
Lipid A component of LPS binds to TLR4
TLR4 binding stimulates production of TNF, IL-6 and IL-1
Cytokines cause fever, recruit neutrophils and macrophages and initiate acute phase response and increase vascular permeability leading to plasma leak and a drop in blood volume (also vasodilation contriibutes)
Endotoxin activates the coagulation cascade causing disseminated intravascular coagulation (DIC) due to stimualting production of tissue factor
=thrombi in capillaries hindering blood flow
=drop in blood volume and pressure
=anoxia of vital organs
How do TNF-alpha, IL-1 and IL-6 cause fever?
ENOGENOUS PYROGENS (not exogenous like LPS)
-Induce the synthesis of prostaglandin E2 by COX-2
-Prostaglandin E2 acts on hypothalamus causing increased heat production from catabolism of brown fat and heat retention from vasoconstriction
List some acute phase proteins made by the liver
C-reactive protein
Complement proteins
Mannose binding lectin
Fibrinogen
alpha 1-antitrypsin
Serum amyloid A
Haptoglobin
Coagulation cascade proteins
What causes gram positive sepsis?
Peptidoglycan or teichoic acid
