W3- Pathology of inflammatory responses

what is acute inflammation?

early and immediate response to tissue injury (short term process)

what is produced in an acute inflammatory response?

inflammatory exudate (fluid rich in protein and cells)

what causes oedema in the acute inflammatory response?

microvascular changes cause increased blood vessel permeability

leads to oedema

what is oedema?

excess accumulation of fluid in extravascular space and body cavities

(caused by increased vascular permeability, increased hydrostatic pressure or decreased colloid oncotic pressure)

what stimulates the process of destruction of the pathogen and other cells in the local environment?

-if the fluid in the interstitial spaces contains inflammatory cells (eg neutrophils)

-toxic metabolites are released to cause destruction

what is pus?

purulent exudate made of an accumulation of damaged tissue and dead microbes, granulocytes, and macrophages

why is it useful that exudates contain fibrinogen?

fibrinogen is converted to fibrin, which forms a mesh for healing and repair in the coagulation cascade

what are the 5 clinical symptoms of acute inflammation?

-redness

-heat

-swelling

-pain

-loss of function

what pathway of the complement cascade does the acute inflammatory response involve?

alternative pathway (based on pathogen surface binding)

what are the 4 processes which occur after binding of a pathogen to complement C3 and activating the complement cascade?

-release of histamine and chemoattractants

-altered permeability of local vasculature that allows fluid exudation (oedema)

-altered adhesiveness of endothelium allows cellular migration (extravasation by diapedesis)

-phagocyte recognition of C3b-opsonised pathogen allows it to be engulfed and destroyed

how is the complement cascade activated?

pathogen binds complement C3

what are the events of the alternative pathway?

-pathogen attaches to immature C3

-causes thioester hydrolysis to activate C3 convertase

-activates to common pathway

what occurs in the common pathway of the complement cascade?

C3 convertase --> C3 --> C3a and C3b

C3b --> C5 convertase --> C5

C5 --> C5a and C5b

what is the function of C3b from the complement cascade?

opsonisation- coats bacteria for recognition and engulfing by phagocytic cells

what is the function of C3a and C5a from the complement cascade?

activation of mast cells to release vascular permeability factors and chemoattractants

(allows inflammatory exudate cells to respond to signal at site of infection)

what is the function of C5b-C9 from the complement cascade?

activates membrane attack complex where pathogens are destroyed in cell lysis

what are the 3 functions of cellular and plasma mediators of acute inflammation?

-amplification

-elimination

-regulation

what are some examples of cellular mediators of inflammation?

-vasoactive amines

-cytokines and growth factors

-arachidonic acid derivatives (thromboxane A2, prostaglandins)

-platelet activating factor

-lysosomal enzymes

-oxygen radicals

-nitric oxide

what are some examples of plasma-derived mediators of acute inflammation?

-kinin system

-coagulation and fibrinolytic system

-complement cascade

how is platelet activating factor produced? (cellular mediator of acute inflammation)

phospholipid molecule derived from the cell membrane of basophils, macrophages, mast cells or endothelial cells

what is the potent pathological effect of platelet activating factor?

contributes to:

-inflammation

-endotoxin shock

-allergic reactions (through vasoconstriction and bronchoconstriction)

-leukocyte adhesion

-platelet aggregation

what is different about the systemic effects of low conc platelet activating factor v high conc platelet activating factor?

low conc= vasodilation

high conc= vasoconstriction

what are the 3 main functions of platelet activating factor as a cellular mediator of acute inflammation?

-enhances microbicidal activity in neutrophils through oxidative burst

-enhances phagocytosis

-activates arachidonic acid pathway to produce leukotrienes, prostaglandins and thromboxane

what are the 3 major pathways for regulating acute inflammation?

1. complement cascade

2. platelet activating factor

3. arachidonic acid metabolic pathway

what 3 substances activate the arachidonic acid pathway?

-C3a

-C5a

-platelet activating factor

what are the two pathways involved in the arachidonic acid pathway?

what do they produce?

-cyclo-oxygenase pathway = prostaglandin and thromboxane A2 production

lipo-oxygenase pathway = leukotriene production

what is the function of thromboxane A2 produced in the arachidonic acid pathway?

vasoconstriction and platelet aggregation

what is the function of prostacyclin produced in the arachidonic acid pathway?

platelet disaggregation

what is the function of prostaglandins produced in the arachidonic acid pathway?

vasodilation

what is the function of leukotrienes produced in the arachidonic acid pathway?

-chemotaxis and neutrophil adhesion

-increased vascular permeability

-vasoconstriction

what are the 3 processes/substances that lead to systemic effects from localised acute inflammation?

-acute phase reactions

-acute phase proteins

-exogenous/endogenous pyrogens

what are acute phase proteins?

A class of proteins whose plasma concentrations increase (positive acute-phase proteins) or decrease (negative

acute-phase proteins) in response to inflammation

why are acute phase proteins produced in inflammation?

due to altered liver metabolism

what are some examples of acute phase reactions which are caused by production of acute phase proteins in the liver in response to local inflammation?

-increased bone marrow leukocyte production (more neutrophils from bone marrow and other reservoirs are required)

-fever

-rigors

-tachycardia

-drop in BP

-loss of appetite

-vomiting

-skeletal weakness

-aching

what is the substance that causes fever?

pyrogens

how do pyrogens control fever?

reset the temperature control system operated by the hypothalamus

what is an example of an exogenous pyrogen?

lipopolysaccharide from Gr- bacteria (endotoxin)

what are examples of endogenous pyrogens?

cytokines (IL-1, IL-6, TNF) which are involved in acute inflammation

why does fever cause general malaise?

due to the interaction of the hypothalamic-pituitary-adrenal axis

(causes widespread systemic effects)

what are the properties of acute v chronic inflammation?

acute

-short-lived (mins-days)

-neutrophil-rich

chronic

-long-lived (months-years)

-lymphocyte, plasma cells and macrophage-rich

what are the 5 stages of resolution of inflammation? (acute inflammation)

1. injury/infection

2. acute inflammatory response (histamine, 5-HT, complement)

3. lipoxins and resolvins

4. resolution

5. return to normal tissue physiology

what are the 5 stages involved in the failure to resolve inflammation? (chronic inflammation)

1. injury/infection

2. acute inflammatory response (histamine, 5-HT, complement)

3. lipoxins and resolvins

4. 'failed resolution'- ongoing acute inflammation

5. maintains persistent state of acute inflammation = chronic inflammation

what are 3 characteristics of chronic inflammation?

-abscess formation

-excess scarring

-autoimmunity

why is resolution of infection an active (not passive) process?

requires specific molecular and cellular pathways to be activated

(not just passive reduction of stimulus and inflammation)

what are 3 substances involved in resolution of acute inflammation?

lipoxins, resolvins and protectins

(engaging this pathway leads to early resolution of immune system)

why is it important to understand the active processes which trigger resolution?

provides candidates for drug targets

(can modify inflammatory process and enhance resolution when it is not working in chronic inflammation)

what are the 4 types of chronic inflammation?

-non-specific

-chronic suppurative

-granulomatous

-autoimmune

what are the properties of non-specific chronic inflammation?

-persistent viral infection (eg Hep B) continues to stimulate the local immune response

-associated with persistent activation of lymphocytes

what are the properties of chronic suppurative inflammation?

-abscess-continuing stimulus to neutrophil production and recruitment

-often walled off by fibrous tissue and poorly vascularised, so difficult to treat with antibiotics

-may require surgical clearance

what are the properties of granulomatous chronic inflammation?

-usually a response to agents which are difficult to destroy using lysosomal enzymes or lymphocyte-mediated immune responses

-epithelioid or multinucleated giant cells form by fusion

(eg TB, parasitic infection)

what are the properties of autoimmune chronic inflammation?

-involves T, B and plasma cells

-plasma cells may be a prominent feature in inflammatory infiltrate

-can result in the development of tertiary lymphoid structures

what are tertiary lymphoid structures? what do they indicate?

-rudimentary organelles that function similarly to lymph nodes and lymphoid tissues

-found in autoimmune chronic inflammation

-development is a poor prognostic indicator of autoimmune progression

what are some examples of diseases which are underpinned by chronic inflammation?

-atherosclerosis

-arthritis

-chronic neurodegenerative diseases (eg Alzheimer's)

-dementia

-depression

what are the 4 stages of acute inflammation in lobular pneumoniae?

1. congestion

2. consolidation

3. grey hepatisation

4. resolution

what occurs in the congestion phase (phase 1) of acute inflammation in lobular pneumoniae?

-bacteria cause outpouring of protein-rich (fibrin) fluid into alveolus

-first 24 hours after infection

presence of bacteria and neutrophils, but not blood cells

what occurs in the consolidation phase (phase 2) of acute inflammation in lobular pneumoniae?

-occurs 2-3 days after insult and lasts 2-4 days

-neutrophils and rbc enter alveolus

-fluid and cells spread to adjacent alveoli, leading to solidification

-marked with cellular exudate (neutrophils with ingested bacteria, extravasated rbc, epithelial cells, fibrin deposits)

-fibrin deposition replaces the fluid from the congestion phase

why is the consolidation phase of acute inflammation in lobular pneumoniae known as 'red hepatisation'?

texture appears to be like liver

what are the properties of the affected lung in lobular pneumoniae in the consolidation phase?

dry, granular, airless

what occurs in the grey hepatisation stage (phase 3) of the acute inflammatory response in lobular pneumoniae?

-lasts up to 8 days

-macrophages recruited to ingest dead neutrophils and begin digestion of the fibrin mesh

what are the properties of the affected lung in lobular pneumoniae in the grey hepatisation stage?

-grey due to fibrin exudate (fibrin being digested by macrophages)

-disintegration of rbc leads to deposits of haemosiderin

-texture= cooked liver

what occurs in the resolution phase (phase 4) of the acute inflammatory response in lobular pneumoniae?

-restoration of normal tissue when architecture is in tact

-begins centrally in lobe and spreads peripherally

-liquifies fibrous content from earlier phases to recover aeration in the alveoli

(occurs after 8 days)

what occurs when resolution is not possible in the acute inflammatory reaction?

repair- doesn't restore normal architecture, but allows continued life

what is repair after the acute inflammatory response associated with?

organisation- scar formation due to loss of structural integrity

what is an abscess?

accumulation of neutrophils walled off by fibrin and often surrounded by chronic inflammatory cells (macrophages and fibroblasts)

why does an abscess suggest a chronic condition of persistent acute inflammation?

acute inflammation= accumulation of neutrophils

chronic condition= fibrin and chronic inflammatory cells (macrophages and fibroblasts) preventing resolution of acute inflammation

what is suppurative inflammation?

condition where purulent exudate is accompanied by significant liquefactive necrosis

(large amounts of pus due to dense accumulation of neutrophils in the abscess)

what is suppurative inflammation difficult to treat with antibiotics?

-poorly vascularised

-fibrin wall surrounding neutrophils prevents penetration

(may require surgical excision)

what is granulomatous inflammation?

histological pattern of chronic inflammation which occurs following cell insult

what are the various causes of granulomas?

fungi

bacteria

mycobacteria

chemical

autoimmune

cancer

metazoa

protozoa

why are granulomas of histological interest?

involved in many different diseases

what stain do we use to observe nuclear and cytoplasmic morphology of granulomas?

H+E stain

why are macrophages found within granulomas?

granulomas are sites of chronic inflammation

what happens to macrophages in granulomas?

take on unusual shapes or characteristics

what are the two most common macrophage shapes/characteristics in granulomas?

-epithelioid cell- macrophages start to resemble (but do not transform into) cuboidal epithelial cells

-macrophages acquire multiple nuclei

what does the presence of epithelioid cells in the granuloma indicate?

chronic immune stimulation of macrophages in the granuloma

what are the two reasons that macrophages acquire multiple nuclei in the granuloma?

-failure of cell division with nuclear division still occurring

-fusion of multiple macrophages within the granuloma to give rise to large cytoplasmic mass with multiple nuclei contained within

what are the 4 types of multi-nuclear macrophages that are formed in the granuloma?

-foreign body cell- random distribution of nuclei

-langhan giant cell- horse-shoe arrangement of nuclei (eg TB)

-Warthin-Finkeldey cell- contain inconspicuous nuclei

-touton giant cell- nuclei arranged in centre of cell and surrounded by lipid bodies

what are the properties of a non-caseating (sarcoid) granuloma?

-non-necrotising (absence of dead cells in the core)

-macrophage-rich core surrounded by lymphocytes (CD4+, CD8+)

-network of fibroblasts surrounded granuloma

(raised structure if appears on skin)

what are the properties of caseating granuloma?

-macrophage-rich core surrounded by lymphocytes in a cuff

-infiltration of neutrophils/polymorphic cells resulting in a caseating centre (dead cells in centre)

-acute inflammation continues in centre (neutrophil-rich) at same time as chronic inflammation

how is formalin fixed paraffin embedded tissue used when looking at granulomas?

useful to understand the dynamics of the inflammatory process in animal models

(how granulomas are formed and maintained)

how are formalin fixed paraffin embedded tissue slides produced?

-chemicals to allow hydration and dehydration of tissue

-machine allowing to embed fixed material into paraffin

-machine allowing to cut thin sections of the block

-animal tissue can be embedded into paraffin blocks to cut the tissue so it can be viewed microscopically

why is using formalin fixed paraffin embedded tissue microscopy useful?

can look at nuclear and cytoplasmic morphology of granulomas over time

(see infection of cells, infiltration of inflammatory cells, macrophages becoming multinucleate, cuff of lymphocytes outside the core, resolution of granuloma and subsequent reduction in immune cells)

what other use do formalin fixed paraffin embedded sections have> (not staining and microscopy)

formalin fixed paraffin embedded sections can yield molecular information through genome techniques

(static, but can capture more info than by H+E staining or immunofluorescent staining)

how are formalin fixed paraffin embedded sections used in application of genome techniques to study molecular pathology?

-FFPE blocks are cut to produce FFPE slides with sections of tissue

-DNA is extracted from tissues

-next generation sequencing technologies can be used to understand genetic makeup of cells in tissues

how is genetic technology applied to the genetic sequences from the formalin fixed paraffin embedded tissues to understand the transcriptome (what genes are being expressed as proteins by the tissue)?

-based on RNA analysis (RNA sequencing of FFPE sections)

-minute amounts of RNA are extracted from FFPE sections

-RNA sequencing analysis is conducted

-this builds up a gene expression profile of different cells within the tissue

what occurs in immunostaining technology when looking and granulomas?

-used to expand on info from H+E staining

-involves identifying cell types based on molecules rather than morphology

-take antibody molecules which recognise different characteristics of cell types and label them with specific dye under UV light

-allows for greater understanding of granuloma (types of cell, position of cells relative to other cells, number of cells)

what is the process involved in immunostaining granulomas?

-monoclonal antibodies are stained an placed on a section

-they are visualised under a camera equipped with UV light source which captures images digitally

what is involved in intravital fluorescence multiphoton microscopy for observing granulomas?

intravital = in live animals (visualising cell dynamics)

multiphoton microscopy = allows good depth penetration into tissues at high depth resolution to be able to see individual cells and how they behave

why is intravital fluorescence multiphoton microscopy useful for identifying granulomas?

-allows dynamic impressions of inflammatory response associated with granuloma

-specific cells in granuloma can be labelled individually in distinct ways (capture differences in shape, size, motility of inflammatory cells)

-can view different types of cells associating with granuloma moving in and out over time

what are the properties of intravital and non-invasive probes which are being developed for looking at granulomas?

small microscopic objectives placed at the end of a small catheter into live animals for representation of tissue structures and dynamics during the course of infection

(may be able to be done in humans in the future)

what is an example of non-invasive imaging that can be used to look at granulomas?

functional MRI- imaging technique to look at details of immune response in humans in real time

how does cytometry work to look at cells involved in granulomas?

identifies individual cell populations based on cell surface characteristics

(flow cytometry= 10-20 molecules)

(mass cytometry= 100 molecules)

what is the process of cytometry for looking at cells involved in granulomas?

-small pieces of tissue are disaggregated and passed through cytometer

-allows interrogation of surface molecule expression of each cell in population

why is cytometry not always used for looking at cells involved in granulomas even though it is the best method in cell phenotyping?

causes loss of spatial info about cells and structures surrounding the m

(disaggregation means you can no longer understand where cells are in relation to each other/which part of the tissue they came from)

what is digital space profiling when looking at granulomas?

-combines spatial analysis with genomics platforms

-uses highly multiplexed antibody and RNA probes

what is the process of digital spatial profiling?

-stain slide tissue section with RNA probe

-image slide and select regions of interest

-UV cleave RNA off antibodies in the region of interest

-aspirate RNA with microcapillary

-dispense RNA in a plate

-repeat for each region of interest

-hybridise and count RNA

why is digital spatial profiling the best method to use for looking at granulomas?

-defines gene expression profile of individual cells (specifically selected) while still understanding where cells are placed within a tissue section

-allows us to produce more detailed maps about how cells behave in relation to one another or to pathogens at site of infection

what is in silico modelling of granulomatous inflammation?

all data acquired by various techniques can be integrated into mathematical models that allow us to build up the architecture of a tissue

3D models allow us to try and computationally understand how granulomas behave