-highly sophisticated innate defense mechanism

 >vertebrate comp. sys. has over 30 different humoral proteins... can be activated by CHO PAMPs, Ab's bound to microbial surfaces and by numerous elements of coagulation/fibrinolytic systems

-comprised of three different activation pathways; alternative, lectin-binding and classical

-all these pathways lead to a common formation of MAC (membrane attack complex)

-oldest

-C3 (complement component 3) and Bf (factor B) are the oldest complement genes

-gene duplications of C3 led to C4 and C5... Bf led to C2

**draw out this pathway!

-MASP gene duplications lead to C1r and C1s

**draw out this pathway!!

-due to complement gene duplication events

**draw out this pathway!!

Birds and reptiles appear to lack C4, C9, properdin and teleost.

T or F

FALSE

they lack C2, C9, properdin and teleost

What complement proteins are produced by MACROPHAGES?

1. anaphylatoxins

2. vasoactive compounds

3. mast cell degranulation

4. platelet activation

In animals that are sick and unhealthy, C3 spontaneously breaks down into C3a which is degraded by chymase and C3b that binds to nearby surfaces.

T or F

FALSE

this happens in healthy normal animals...

C3 spontaneously breaks down into C3a that is degraded by chymase and C3b that binds to nearby surfaces

when C3b binds to microbial surface (which lacks sialic acid) Factor H cannot bind, this means that Factor I remains inactive and C3b therefore does not degrade.

T or F

C8 can also bind to C3b on microbial surfaces and this allow the construction of an anchor molecule for the MAC.

T or F

FALSE

C5 can also bind to the C3b on microbial surfaces and this ENABLES the construction of an anchor molecule for the MAC

it is cleaved by a C5 convertase

>C3bBb - alt. pathway

>C4bC2b - lectin-binding pathway

>and classical pathway

....the cleavage product C5b remains attached to C3b, whereas bioactive C5a is released into the humoral fluid

C3 convertase

>amplifying the production of C3b, which attaces to the microbial surface allowing C5 attachment and cleavage to C5b and subsequent assembly of MAC

C1q and it attaches to the Fc portion of a single IgM molecule or paired to IgG molecules 

IgM is more efficient than IgG for activating the complement system because its complement binding sites are closer together.

T or F

C1-INH

>controls the assembly of C4b2b by blocking the activities if C1r and C1s

CD55 on RBCs, neutrophils, macrophages, lymphocytes, platelets and endothelial cells binds to C3 and C5 convertase to accelerate their decay.

T or F

TRUE!

>Factors H and I, C4-binding protein and CD35 (CR1) also accelerate the decay of these convertases

>block the insertion of C5b678 

>block polymerization of C9 in target cell membrane

C3b, C4b and iC3b

>in primates over 90% of CR1 is found on RBCs and the rest on phagocytic cells

The ligand for CR2 (CD21) is a degraded fragment of C3 referred to as C3d.

T or F

iC3b

>it is expressed on macrophages, neutrophils and NK cells and is involved in mediating phagocytosis and cytotoxicity

>a genetic deficiency in bovine CD18 leads to leukocyte adherence deficiency in cattle

CR4 (CD11c/CD18) is also an integrin that can bind to breakdown fragments of C3.

T or F

Trueeee

>it is expressed on macrophages, neutrophils, T cells, NK cells, platelets, mediates phagocytosis, cytotoxicity and possibly clotting

>pathogenesis of allergic rxns such as asthma 

>they trigger the release of vasoactive compounds such as histamine

>apoptotic bodies

...C1qR-C1q ligation also appears to influence monocyte-to-macrophage differentiation and DC migration

>C3b and iC3b

>found on tissue macrophages such as Kupffer cels, and is importantfor the phagocytic removal of blood-borne pathogens

>opsonins

>phagocytic

...C1q can act as an opsonin for apoptotic bodies

C5a and C5b67 (C1q attracts DCs)

>they can attract various cell types such as neutrophils, eosinophils, basophils and macrophages

>they cause mast cells and eosinophil degranulation and cause platelets to release vasoactive compounds (histamine and serotonin)

>they also increase vascular permeability and cause neutrophils to release lysosomal enzymes and macrophages to release thromboxane

>C3a also disrupts bacterial cell membrane integrity and is therefore also considered to be bacteriolytic

a SNP in CD18 can increase B(beta)2-integrin expression.

T or F

FALSE

>a SNP in CD18 can DECREASE beta2-integrin expression

>mammalian neutrophils

>avian heterophils

>fish neutrophils

>phagocytosis

>degranulation

>formation of extracellular traps

...granulocytes also have regulatory function in that they recruit circulating monocytes into the tissues, which mature as tissue macrophages and participate as effector cells and in the resolution of infection

granulocytes are potent effector cells.

T or F

TRUE!!

>they can contribute to extensive host tissue damage, which is minimized by rapid granulocyte recruitment from the circulation to target tissues/short life span/replacement by monocyte-derived macrophages

unlike macrophages, granulocytes are capable of sustaining their phagocytic cell function for very long therefore their effector function is not temporarily limited.

T or F

FALSE

unlike macrophages, granulocytes are INCAPABLE  of sustaining their phagocytic cell function for very long therefore their effector function IS temporarily limited

pluripotent stem cells in bone marrow equivalent organs

>bone marrow stromal cells secrete growth fractors that steer their differentiation

2 days

>they are rapidly produced, in all cases these cells can be rapidly mobilized from the circulation to the target tissues and rapidly replaced by granulopoieses bone marrow equivalent tissues

>based on their nuclear morphology

>granulocytes differentiated based on their granule staining properties 

-EOSINOPHILS take up ACIDIC EOSIN

-BASOPHILS take up BASIC HEMATOXYLIN

-NEUTROPHILS take up very little of either dye

Neutrophills can also be identified based on the presence of myeloperoxidase (MPO) in their granules, chicken heterophils lack MPO.

T or F

TRUE!!

-and some other important oxidative enzymes that are req'd for the production of superoxide radicals 

-may rely more on non-oxidative mechanisms for their effector function

MPO + >primary or azurophil granules (green, packaged very clearly during neut. development, contain elastase/cathepsin G/proteinase 3/defensins/MPO/azurocidin/lysozyme)

MPO - >secondary or specific granules (red, packaged later and contain adhesion molecules/TNF receptors/collagenase/lactoferin/haptoglobin)

-increases vascular permeability

-increases endothelial cell adhesion molecules for monocytes

-attracts monocytes

-destroys peptidoglycans of Gram + bacteria

-innate opsonin

-blue 

-packaged the latest

-contain adhesion molecules, matrix metalloproteins, TNF receptors, lysozyme and arginase

-secretory vesicles (light blue)

-these contain many adhesion molecules, PRMs and many different cytokine receptors that are deposited onto their cell membrane allowing them to be responsive to their ligands

-they also sequentially release their preformed granules; gelatinase granules first during trans-endothelial cell migration, then the azurophil and specific granules within the extravascular space to create a chemokine gradient for the subsequent infiltration of monocytes as well as target the pathogen

-slows down the neutrophils allowing them to interact with endothelial cells

-during infection/tissue trauma, expression of endothelial cell P-selectin is upregulated in response to perceived 'danger signals'

-P-selectin attaches to L-selectin, allowing the neutrophil to transiently attach to endothelial cells as it passes along

-endothelial cells also secrete platelet-activating factor (PAF) triggers neutrophil rolling allowing for closer interaction between the neut and endo cell

-neut adhesion of endo cell adhesion molecules (ICAM-1, ICAM-2) that bind to neutrophil beta2 integrins (CD11a-CD18, LFA-1)

-neuts release sm. amts of elastase this cleaves anti-adhesive leukosialin (CD43) molecules from the endothelial cells allowing for even closer interaction and stronger integrin binding

-occurs over several hours as both cell types become activated

-mediated by various pro-inflammatory cytokines (TNF-alpha, IL-1) and chemokines/chemotactic agents (TNF-alpha, CXCL8, C5a, LTB4, PAF)

-extravasation, diapedesis

-neutrophils move through endo cell layer

-basement membrane is mediated by the attachment of neut to adhesion molecules within the endothelial cell tight junctions

Neutrophil phagocytosis

stage 1: activation

-require dual signal to become activated

1. ICAM/integrin interaction

2. TNF-alpha, CXCL8 or C5a

-signal causes them to release some granules

-granular MMPs loosen up tissue matrix allowing for cell migration

-cleaves chemokines

-cleaves membrane TNF-alpha from surrounding cells which attracts even more neuts

Neutrophil phagocytosis

stage 2: chemotaxis

-neuts are drawn through tissues via chemokine gradient which is a mileau of chemoattractants that include CXCL8, C5a, LTB4, PAF and certain bacterial peptides

-these chemotactic agents are detected by receptors that are concentrated on neut membrane projections called lamellipodia (direct neutrophil movement)

Neutrophil phagocytosis

stage 3: adherence and opsonization

-host cells/microbes have -ve charge associated with their cell membranes = repel each other

-opsonins +vely charged molecules that attach to microbial surfaces, facilitate host cell-microbe attachment and subsequent phagocytosis

Neutrophil phagocytosis

stage 4: ingestion

-after opsonin-receptor ligation, net lamellipodia surrounds the particle forming a phagosome

-phagosome combines with a primary granule called a lysosome forming phagolysosome

-during ingestion, it is possible for neut to release some of its granules into surrounding tissues and this can be damaging to host tissues

-MBL, lysozyme, serum amyloid A and C-reactive protein, C3 breakdown products

-neonatal mammals rely heavily on these bc of immaturity of their acquired immunity

-immunoglobulins, IgM+C1

-neonatal mammals will also recieve these that are passively transferred from the mother to the offspring via colostrum but also milk

-can also occur in some instances

ex: mannose receptors and certain integrins

oxygen-dependent pathway >uses oxygen to generate oxygen-reactive species

oxygen-ind pathway >involves the release of lytic enzymes and cationic peptides that bind to bacterial and fungal surfaces and disrupt their membrane integrity

the NOX complex converts NADPH into NADP+, liberating 2 e-s

>these e-s are transported into the phagosomal membrane

>bind to two oxygen molecules = formt'n of two superoxide anions (which dismutate into H2O2 w. help of superoxide dismutase [SOD])

-cytotoxic

-membrane-permeable

-relatively stable so it can diffuse away from the site of production 

-can react with Cl- and other halides in the presence of phagosomal MPO to produce more toxic compounds such as hypochloride

-produced by MPO

-kill microbes by oxidizing their membrane proteins and lipids and enhancing lysosomal enzyme activity

1. hydrogen peroxidase - splits H2O2 into water and oxygen

2. large amounts of glutathione (GSH) - reduces oxidants

3. iron-binding proteins - such as lactoferrin and haptoglobin that reduce iron bioavailability

as microbial oxidation continues in the phagolysosome, the pH is increased and this decreases the activity of lytic enzymes.

T or F

FALSE

as microbial oxidation continues in the phagolysosome, the pH is decreased and this increases the activity of the lytic enzymes

-defensins (beta-sheet peptides)

-cathelicidins (alpha-helical peptides), whose main function includes interacting electrostatically with negatively charged mem components such as LPS and LTA (affects cell mem. and osmoregulation)

Defensins have a diverse array of bioactivity, which of the following are true?

a. defensins are chemotactic for neutrophils, macrophages, DC, Mo and fibroblast cells

b. defensins can induce infected host cell necrosis

c. defensins can inhibit activity of certain enzymes

d. defensins are anti-inflammatory

e. defensins can induce mast cell degranulation

a., c., d. and e.

b. defensins can induce infected host cell APOPTOSIS

...side note: defensins are anti-inflammatory bc they can sequester bioactive LPS and LTA

-neutrophil extracellular traps

-dying neutrophils

-nuclear chromatin, histones and the antimicrobial granular cargo (mix after nuclear and granular membranes disintegrate)

-it's prompted by NADPH-oxidase mediated superoxide production

-caspases are not involved with NETosis (but are in apoptosis) and DNA does not become fragmented

what would trigger NETosis? 

-pathogens, their components

-activated platelets

>they trap Gram+ and Gram- bacteria, fungi and likely parasites

neutrophils play a role in regulating the shift of inflammation to resolution.

T or F

TRUE!

...they do this by secreting pro-resolution molecules such as lipoxins, resolvins and protectins that terminate the influx of more neutrophils and promote the influx of monocytes

>generated from omega-3 fatty acids

>by an enzyme cascade (includes COX2, cytochrome P450 and LOX)

>resolvin E1

...human resolvin E1 acts as antagonist for neutrophil LTB4 receptors shutting down neutrophil activation and influx

>resolvin E1 can also activate monocytes through resolvin receptor Chem R23

>chemotactic signals

...these signals will also influence whether or not macrophages are in a pro- or anti- inflammatory state

what do neutrophil granules contain?

-azurocidin

-cathepsin G

for ex: that proteolytically activate prochemerin to chemerin, which is an important DC chemokine

neutrophils directly activate DCs via cell-to-cell contact through what? to produce what?

a. neutrophil CD11a ligation with DC-SIGN, IGN-a

b. neutrophil CD12a ligation with DC-SAT, IFN-a

c. neutrophil CD12b ligation with DC-SAT, IGN-y

d. neutrophil CD11b ligation with DC-SIGN, IFN-y

d. is correct!

...neutrophils directly activate DCs via cell-to-cell contact through neutrophil CD11b ligation with DC-SIGN to produce IFN-y. This helps to drive T-cell differentiation and macrophage activation

IL-10

-this may help protect against lung tissue damage during acute microbial infection, but may also contribute to chronic microbial infections

the hallmark of inflammatory tissue resolution is the clearance of necrotic neutrophils by monocytes

T or F

FALSE

the hallmark of inflammatory tissue resolution is the clearance of APOPTOTIC neutrophils by MACROPHAGES

neutrophils decision to undergo apoptosis involves diff pathways coordinated by transcription factors, which two postpone apoptosis?...

a. hypoxia-reducible factor (H1E1) and FAXO2B

b. hypoxia-inducible factor (H1F1) and FOXO3B

c. hypoxia-inducible factor (H1F1) and FOXO3A

c. is correct

neut apoptosis is postponed via hypoxia-inducible factor H1F1 and FOXO3A

the process of neut phagocytosis down-regulates the production of IL23 by the macrophage...this in turn leads to down-regulation of T-cell IL17 secretion

>when you down-regulate IL17 you down-regulate G-CSF which prevents further neutrophil production and release into circulation

IL17 inhibits bone marrow stromal cells to produce G-CSF.

T or F

FALSE!!!

IL17 promotes bone marrow stromal cells to produce G-CSF and this promotes neutrophil proliferation and release

macrophages are both sentinel and effector cells

T or F

TRUE!

macrophages are equally important as effector cells of the innate inflammatory response and cell-mediated immune response 

>also act as regulatory cells and help with tissue repair

during extravasation and chemotaxis, monocytes differentiate into effector cells called macrophages that have sustained phagocytic activity and a much shorter life span than neutrophils, this makes them much less efficient effector cells

T or F

FALSE

during extravasation and chemotaxis, monocytes differentiate into effector cells called macrophages that have sustained phagocytic activity and a much LONGER life span than neuts, this makes them much MORE efficient effector cells

a. endothelial cells >CCL2-CCR2

b. neutrophils >CCL4-CCR1

c. tissue macrophages >CCL7-CCR2

d. chemotactic agents >beta-defensins, chemerin, TNF-alpha (from activated neuts)

CD35 (CR1) and CD11b/CD18 (CR3)

...these enable macrophages to phagocytize particles/microbes opsonized by C3 components and various FcRs (species-specific depending on immunoglobulin isotype) that enable them to carry out Ag-specific phagocytosis

-M1 inflammatory macrophages

-imp during the acute phase of intracellular bacterial infections

...iNOS2 uses NADPH and O2 to act on L-arginine to produce large amounts of nitric oxide (NO) and citurlline...NO can interact with O2- and produce antimicrobial oxidants >peroxynitrite and nitrogen dioxide radical

excessive or prolonged M1 macrophage activation is damaging to host tissues and has been implicated in many acute inflammatory diseases

T or F

TRUE

diseases such as gastroenteritis, urinary tract infections, neonatal meningitis and sepsis

M2 anti-inflammatory macrophages

...they utilize an enzyme called arginase to convert L-arginine into ornithine and then to citrulline so that NO isn't produced

M2 macrophages play a key role in wound healing by promoting blood vessel formation, tissue remodeling and repair and reducing inflammation

>M2 macrophages are also important effector cells for parasitic infections

IL10-producing M1 macrophages are associated with persistent bacterial infections and to chronic disease

T or F

FALSE!

IL10-producing M2 macrophages are associated with persistent bacterial infections and to chronic disease

macrophages also secrete regulatory cytokines, list the cytokines that stimulate the following:

a. acute-phase response

b. acquired immune response

i. Th1

ii. Th2

iii. Th17

iv. regulatory T cell steering

a. APR >TNF-alpha, IL-1, IL6

b. AIR...

i.Th1 >IL12, IL18

ii. Th2 >IL1, IL6, IL10, IL13

iii. Th17 >TNF-alpha, IL6, IL23

iv. reg. T cell steering >TGF-beta, IL10

TNF-alpha and IL12

...which activates innate NK cells to secrete IFN-y (which activates M1 macro to higher level and induces NOS2 gene transcription, IFN-y can come from neut too)

in the presence of anti-inflammatory cytokines (IL4, IL10, IL13, TGF-beta) and/or glucocorticoids, macrophages differentiate into M2 macrophages

T or F

draining lymph nodes

...where they can interact as Ag presenting cells (APCs) with Tcells

**for species sans lymph nodes: lymph drainage into lymph nodules (birds), circulation and subsequent interaction with Tcells in other secondary tissues spleen, pronephros (fish) or bursa (bird)

Mcells sample particles in the intestinal lumen and pass them on to macrophages that travel to intestinal Peyer's patches and/or mesenteric lymph nodes to interact with T cells

T or F

particles/microbes may gain entry to the circulation, in which case hepatic macrophages or Kupffer cells will trap them

T or F

FALSE

particles/microbes that gain entry to the circulation with be trapped by SPLENIC MACROPHAGES and Kupffer cells

>pigs/ruminants/horses these pathogens removed by macrophages lining capillary of endothelium of lung

>human/rabbit/mouse/fish/bird? use hepatic Kupffer cells and splenic macrophages

tissue/species/developmentally-specific removal of foreign particles is partly attributed to tissue-specific expression of opsonin receptors...

a. Kupffer cells rely on ?

b. splenic macrophages rely on ?

a. Kupffer cells rely heavily on C3 opsonization via CD35 (CR1)

b. splenic macrophages express high affinity Fc receptors CD64 (FcyRI)...so they can effectively remove IgG-opsonized particles

anti-inflam and immunosuppressive IL10 and TGF-beta

TGF-beta is a compound secreted by M2 macrophages that promotes healing, cell division and enhanced deposition of extracellular matrix proteins

T or F

TRUE

fibronectin is an ex: of extracellular matrix proteins that provide a scaffold for tissue reconstruction

SLP1 is serine protease inhibitor, secreted by macrophages

-inhibits elastase activity and the release of oxidants by activated neutrophils

-protects against TGF-beta degradation

IL-1B is an enzyme secreted by macrophages

-promotes fibroblast growth 

-promotes secretion of new collagen that is remodelled over time as part of ECM

*note >M2 macrophages are also stimulated in hypoxic tissues to secrete antiogenic factors that promote new blood vessel development

another 'eat me signal' taht neuts and all other dying cells use to signal phagocytosis by macrophages is the down regulated expression of ___ on dying cells

>comprised of macrophages that consolidate into multinucleated giant cells, fibroblasts, lymphocytes 

>associated with chronic inflammatory response

>restricts the spread of the particle/pathogen