Phagocytes Flashcards
what are phagocytes ?
these are cells that can engulf forgein particles into their cell and break them down
describe neutrophils ?
These are produced by the bone marrow , they have a short half life around 72 days and they die by apoptosis
what can neutrophils also be called ?
polymorphonuclear (PMN) leukocytes
how do neutrophils become activated ?
they can becomes activated by TNF alpha , and IL-8 , 1 , 10 , 12 , 15 , IL-6
do neutrophils activate an inflammatory or anti-inflammatory response ?
pro inflammatory by release of IL-8 , IL-6 , and TNF alpha
anti inflammatory response ?
Stimulate anti-inflammatory reactions – resolvins and lipoxins
IL-10 is an anti inflammatory cytokine
how are the phagocytic qualitites activated by a pathogenic antigen ?
Phagocytoses targets by PRR ( pathogen recognition receptors) which bind to PAMPS on the pathogen
describe neutrophil recruitment ?
Roll along walls of post-capillary tubules in blood vessels
- Respond to pathogen induced chemotactic signals e.g. IL-8, granulocyte chemotactic protein 2 and leukotriene B4
- Neutrophils move into tissues and out of vascular blood system – stop their rolling movement and get firm adherence to endothelial wall
- Process of passing through the endothelial cell layer is called extravasation or diapedesis – as cell moves into tissue
capture and rolling ?
Tethering ( stop rolling) and rolling along the blood vessel wall is due to the reversible binding of transmembrane glycoprotein adhesion molecules called selectins.
- Leucocyte selectin
L-selectin (mel-14, LAM-1, CD62L) believed to bind to a
fucosylated variant of CD34. - Platelet-selectin
P-selectin released onto outer surface of endothelial cells binds to PSGL-1 P-selectin glycoprotein ligand on neutrophil - Endothelial cell selectin
E-selectin (ELAM1; CD62) binds to a variety of sialic acid and fucose containing glycoproteins on neutrophil including
Sialyl Lewis X.
Firm adhesion ?
compeltely stopped
Integrins:
groups of heterodimeric ( 2 different polypeptide chains) transmembrane glycoproteins found on neutrophil mediate cell-cell, cell-extracellular matrix adhesion.
Major integrins on neutrophils include:
Macrophage antigen 1 = Mac1 (CD11b/CD18)
Lymphocyte associated function antigen 1 = LFA1 (CD11a/
CD18)
2 Intercellular adhesion molecules (ICAMs)
Mac1 binds ICAM-1 preferentially on endothelial cells
LFA1 binds ICAM-2 preferentially on endothelial cells
neutrophil homing to transmigration sites on endothelium ?
This happens at tricellular junctions
- PECAM1 (platelet-endothelial cell adhesion molecule-1:CD31) found on neutrophils and endothelial cells
- can serve as its own ligand to form homodimers. Has a junctional location on endothelial cells so may serve as a homing device
- Leads to extravasation (or diapedesis) out of blood into tissue
describe transmigration ?
Little is known how neutrophils migrate through the sub-endothelial matrix.
- May be helped by release of proteases as they break down barriers.
- CD18 also VLA (very late antigens) which bind fibronectin
- laminin and other matrix proteins may be important
- VLA-4 also binds VCAM-1 (vascular cell adhesion molecule) which is expressed by fibroblasts and parenchymal tissues
soluble mediators of migration ?
TNF-α and IL-1 can upregulate adhesion molecules but are not chemotactic themselves. Neutrophils have 5 receptors for chemoattractants.
5 receptors for chemoattractants ?
- PAF (Platelet Activating Factor)
- C5a receptor
- Leukotriene B4
- Formyl-methionyl-leucyl-phenylalanine
- Chemokines – IL-8 plus 6 others identified. All chemokines possess four cysteine residues.
In CXC chemokines what are cysteines seperated by ?
amino acid X ( neutriophils)
in CC chemokines what are the cysteines ?
adjacent ( monocytes)
In neutrophils for rodents what are MIPI alpha nad beta important for ?
chemokines
explain macrophages ?
Monocytes circulate for approx. 8 hours and then enter tissues and differentiate into macrophages.
- Macrophages are 5-10x bigger than monocytes and contain more organelles (e.g. lysosomes) - more effective if activated
- Activity induced by various stimuli e.g. air pollutants, bacteria, viruses, cytokines etc.
how do macrophages recognise pathogen ?
Recognition of foreign material using PAMPS and PRR.
M1 macriphages ?
M1 macrophages are pro inflammatory , microbicidal , pathogen killing and tissue injury. They produce pro inflammatory cytokines such as TNF alpha , Il-12 , Il-6 , Il-1 and MCP-1
M2 macrophages ?
M2 are anti inflammatory , apoptotic cell clearance , high phagocytic capacity and wound healing. They produce TCF beta , IL-10 and MCP-1 , TNF alpha and Il-6.
how are M1 activated ?
M1 are the classically activated macrophages by IFN gamma from LPS which is pro inflammatory
M2 activated ?
While the M2 are alternatively activated by anti-inflammatory cytokines IL-10, IL-4, IL-13, IC.
what does the role macrophage depend on ?
local environment
chronic granulomatous disease ( CGD) ?
recessive X-linked disease
- mutations in gp91, p22, p47 or p67
- a functional oxidase cannot be produced – patients have recurrent fungal or bacterial infections - showing importance of ability to produce an respiratory burst
types of receptors on macrophages >
Macrophage scavenger receptors
- Fc receptors which bind to opsonin’s to allow close contacy
- PRR/Toll receptors e.g. CD14 for LPS
- Interact with pathogen associated molecular patterns PAMP’s on pathogen
agents can affect their activity how ?
Cytokine receptors - e.g activator/deactivator of a cell due to receptor numbers on cell
- Chemokine receptors bring cell to the site where they’re needed by chemotaxis
- Activity can be primed so that they are more responsive to a second signal – experimentally can use LPS and IFN gamma
PAMP dsRNA ?
comes from replicating virus , TLR for PRR and type I interferons produced by infected cells
LPS ?
source is GRAM negative bacteria , PRR is TLR and leads to macrophage activation
Unmethylates CpG ?
source id bacterual DNA , PRR is TLR and leads to macrophage activation
N formylmethionyl peptides >
source is bacterial proteins , PRR is peptide receptors and leads to neutrophil and macrophage activation
Mannose rich glycans ?
source is microbial glycoprotiens and PRR is mannose receptor and response is phagocytosisi , opsonisation and complement activation
phosphotylcholine ?
source is microbial membranes , PRR is plasma C reactive proteins leads to opsonisation and complement activation
why is chemotaxins important for phagocytosis ?
must be next to target so chemotaxis is important
pseudopodia ?
form pseudopodia and enclose target in a phagosome, so target cell with the pathogen must be smaller than immune cell
opsonisation do ?
rate of phagocytosis enhanced (>4000x) if the target opsonised
what can macrophages do ?
macrophages can fuse - form ‘foreign-body giant cells’ or organised structures with other cells – granulomas which block of nutrient supply – formation involves T cells to ‘direct’ cells. This occurs if the target cell is large.
Fcgamma R 2 main types >
Activating human FcgRI, FcgRIIA, FcgRIIIA
Mouse FcgRI, FcgRIIIA, FcgRIV
Deactivating
human FcgRIIB
Mouse FcgRIIB
respiratory burst ?
occurs during phagocytosis
- requires respiratory burst oxidase – NADPH oxidase - membrane bound oxidase that is made up of subunits
- made up of gp91 and p22 subunits which form cytochrome b558, and several cytosolic components e.g. p40, p47, p67 and Rac1 protein
- when phagocyte activated the cytosolic components and cytochrome b558 assemble in the cytoplasmic membrane
- involves phosphorylation and cytoskeletal elements such as microtubules.
NADPH oxidase ?
catalyses reduction of oxygen – production of superoxide 02- , this is a very short-lived element.
- xanthine oxidase (purine metabolism) can also generate 02- important in NADH oxidase deficient mice not Wild Type mice.
- superoxide spontaneously/action of superoxide dismutase – forms hydrogen peroxide
- phagosome fuses with lysosomes – phagolysosome – expose target to hydrolytic enzymes/mediators activates myeloperoxidase in neutrophils
myeloperoxidase >
myeloperoxidase causes formation of hypochlorite (hypochlorous acid- bleach) from hydrogen peroxide and chloride ions - myleoperoxidase deficiency occurs in 1:200-4000 individuals – asymptomatic unless another condition present – suggests other mechanisms can compensate to control a pathogen – only in neutrophils.
hypochlorite ?
Hypochlorite causes chlorination of targets can inactivate proteins – e.g. enzymes, membrane proteins or origin of replication of DNA
how is waste excreted from phagocytes.
by exocytosis
oxisative mechanisms ?
respiratory burst using NAPDH-oxidase complex to produce superoxide
- hydrogen peroxide - broken down by myeloperoxidase to hypochlorous acid - most bactericidal oxidant in neutrophils - chlorinate targets and effect their function
- also get production hypobromous/hypoiodous acid from iodine
- Reactive nitrogen intermediates may play less of a role in neutrophils but neutrophils still produce nitric oxide and peroxynitrite.
reactive nitrogen species ?
Activated macrophages (IFN g + lipopolysaccharide (LPS) – bacteria or muramyl dipeptide e.g. Mycobacteria - causes expression of iNOS - inducible nitric oxide synthase - NOS2
- 2 constitutive forms in cells NOS1 ( nerve cells) and NOS3 ( macrophages) calcium independent - causes oxidation of L-arginine
- balance competitively regulated - Th2 cytokines inhibit NO production e.g. IL4 or IL13 - Th1 cytokines enhance NO production e.g. IFN g
- activate arginase-1 and deplete arginine through a STAT-6-dependent mechanism
- Infection – 2 signals increase production e.g. activated macrophages (IFN g + lipopolysaccharide – bacteria or muramyl dipeptide – Mycobacteria) causes expression of iNOS – NO production
phagosomal enzymes form lysosomes ?
Hydrolytic enzymes – pH 4.5-5.0 as this is optimum temperature
- pH maintained by a membrane bound ATP-dependent pump - H+ ATPase
- nucleases, proteases, glycosidases, lipases, phosphatases, sulfases, phospholipases break down DNA and carbohydrates
- significant contribution to cell killing
- results in the generation of antigenic peptides for class II MHC loading
neutrophil non oxidative mechanisms ?
Important in Granulocytes
- enzymes – polypeptides that disrupt integrity of the pathogen – present in azurophil granules, specific and small storage granules
- block respiratory burst with inhibitor and still get killing of bacteria as enzymes play a larger part.
- neutrophil elastase – 4 µg/106 cells - produced as a preproenzyme – needs to be processed (e.g. cleaved) before it is active. Packaged with proteinase 3 and cathepsin G in primary granules
- Neutrophil elastase powerful protease -one target is OmpA – conserved protein present in gram negative bacteria.
- shown important in protection in KO mice - neutrophil elastase deficient mice were 50% more likely to die than wild-type animals exposed to a bacterium
- Cells can produce TNF alpha which is a cytokine which is directly toxic
diferences between neutriphils nad macrophages?
Neutrophils contain granules – macrophages do not
Neutrophils contain myeloperoxidase whereas macrophages do not
Neutrophils are more numerous
describe neutrophil killing by apoptosis ?
The neutrophil receives a signal , this could be from a pathogen or form an immune cell. This triggers the neutrophil to go into a type of cell death. Apoptosis can occur which causes no inflammation , autophagy can help recycle cellular components.
neutrophil autophagy ?
programmed cell death under cellular starvation and recycles organelles/proteins. There is no inflammation and can recruit phagocytes
pyroptopsis ?
is programmed cell death resulting in cell lysis and inflammation occurs and recruitment of phagocytes. Normally associated with fever
oncosis ?
cell death by lysis – e.g. pore formation due to exposure to bacterial toxins – inflammation and tissue damage
NETosis ?
uses NET - neutrophil extracellular traps and it involves oxidative stress –which could cause damage.
Intrinsic signal apoptosis ?
Intrinsic signal e.g. high amount of ROS
extrinsic signal apoptosis ?
Extrinsic signal e.g. TNF alpha, engagement of Fas with FasL or TRAIL (TNF related apoptosis-inducing ligand) engagement with TRAIL-R2/TRAIL-R3 mediated.
TNF low conc ?
TNF is at low conc (0.1ng/ml) this delays apoptosis but if high conc (10-100ng/ml) this increases incidence.
how can apoptosis be delayed ?
•apoptosis can be delayed if infection present for example exposure to inflammatory mediators e.g. IL1, IL2, IL6, IL8, IL15, LPS from bacteria.
cell cooperation occurs , describe transferred molecules such as lactoferrin ?
Transferred molecules e.g. lactoferrin ( binds iron away from pathogen = antibacterial) released by neutrophils taken up and increases ability to kill Mycobacteria.
intracellular iron and transferrin receptor CD71 ?
- Cells need iron to grow
- extracellular iron loaded on to the transferrin receptor CD71 on cell membrane
- This is internalised during phagocytosis
- regulate iron available by altering the expression of the transferrin receptor or amount of intracellular ferritin. More ferritin = less iron.
- iron a co-factor in generation of ROI
- need to have a balance
CD71 expression is often decreased on macrophages during a bacterial infection
explain pathway of macrophages and transferrin ?
Macrophages use multiple pathways to restrict the essential growth factor iron for intracellular mycobacteria. First, cytokines such as IFN-γ inhibit the transcriptional expression of transferrin receptor (TfR). TfR is a major source of iron for mycobacteria, because the bacteria can utilize its ligand, transferrin iron, following its endosomal transfer. Macrophages produce lipocalin-2 (Lcn2), which binds and neutralizes siderophores produced by M. tuberculosis to scavenge and re-utilize cytoplasmic iron. Furthermore, macrophage-derived cytokines such as TNF-α induce the formation of the iron-binding protein ferritin, which incorporates iron into its core rendering it unavailable for intracellular bacteria as well as the iron binding protein lactoferrin (Lf), which also scavenges this metal. Activated macrophages express the phagolysomal protein Nramp1, which among other effects pumps iron out of macrophages, thereby reducing the availability of the metal in the phagosome for mycobacteria. Finally, upon formation of nitric oxide, the transcription factor Nrf2 is activated, stimulating the expression of the major iron export protein ferroportin (FP1) pumping iron out of the phagolyosome and of the cytoplasm of macrophages. By mechanisms that remain elusive, the stimulation of hepcidin expression in the liver, which is a major mechanism for iron restriction to extracellular pathogens, is circumvented. All these events result in reduction in intracellular iron levels and a limited availability of iron for intra-macrophage bacteria. Based on the negative regulatory effects of iron on IFN-γ activity, the reduction in this metal’s availability results in strengthened innate anti-microbial immune responses. Importantly, some of the pathways shown in this figure have been investigated for other intracellular bacteria such as S. typhimurium, and their importance for M. tuberculosis remains to be shown.
describe the cysteine rich cationic peptide defensins /
Cysteine-rich cationic peptides
- Produced from 29-35 amino acids with 6 invariant cysteine residues produced as a precursor
- 5-10% of the total protein in human and rabbit neutrophils
- effect gram -ve and +ve bacteria, fungi, some enveloped viruses
- interact with organisms’ membrane and insert themselves into the membrane to form voltage regulated channel so cell contents leak out.
- inhibit protein kinase C
why aren’t the phagocytes damaged by the ROS ?
mitochondria have a very active cytochrome oxidase - decreases 02 availability
- mild-uncoupling electron pathway by free fatty acids - keep proton potential below threshold to stimulate superoxide production
- cytochrome C - catylases oxidation of superoxide ion to oxygen – protective
- presence of superoxide dismutase in mitochondria - coverts superoxide into hydrogen peroxide which escapes mitochondrion because more permeable to it
- antioxidants - tocopherol, CQH2, ascorbate and low molecular weight anti-oxidants - quench ROS.
- glutathione peroxidase and catalase - decompose hydrogen peroxide
when will very high concentrations of ROS be produced ?
by necrosis
RoS by necrosis ?
some anion acids become oxidised by ROS - formation of permeability transition pores in inner mitochondrial membrane
- release of cytochrome C into cytosol catylases oxidation of superoxide ion to oxygen
- oxidation of cytochrome b5 on outer mitochondrial membrane will upset electron transport chain - prevents oxygen being converted into superoxide ion.
- inhibition of Krebs - accumulation of citrate - this complex’s with Fe2+ and stops Fe2+ being oxidised to Fe3+ - prevents conversion of hydrogen peroxide to hydroxyl radical.
