immunology chapter 9

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1

Chapter 9

Immunity Mediated by

B Cells and Antibodies

2

Focus of Chapter 9 How Antibodies Clear Infection

Antibodies recruit “destructive, nonspecific” immune system components to the infecting pathogen How?

Antibodies bind and link the pathogen to effector molecules or cells that will destroy the pathogen

3

RECALL

Pathogens

extracellular

B cell PM cell

Antibodiessecreted in

2ND LT & Bone

Marrow

B cell function

extracellular spaces

virus~~~~~~ ~~~ Next cell

bacteria

YYY

intracellular

virusOther Pathogens

fluids

4

Ab toxic destructive to pathogens

How do Ab reduce infection?

What happens?

Molecular adaptor(opsonize)

PHAGOCYTOSIS

2

pathogen

YY

YY

Y phagocyte

?

?

3

Role of AB =

Y

reduces infectionYYY

Neutralize = pathogen surface coveredGrowth/replication

1

YY

Y YYpathogen

Y

Y YY

5

COMPLEMENT activation

Recall: Chapter 1, Figure 1.5 Opsonization is enhanced by the actions of complement Complement Ag-binding function of Ab

3

Complement = set of proteins that do not discriminate between Ags

6

Antibody production by B lymphocytes

7

The Development Of B Cells Can Be Divided Into six Broad Phases

8

Stem cell in bone marrow to the mature naïve B cell

Location of B cells at the different stages

State of the Ig H- and L- chain genes Form of Ig expressed

Peripheral circulation

Bone Marrow

9

B cells need activated T cell help to mature into Ab-secreting Plasma Cells

“Generally” need T cell help This delays onset of Ab production until a week after infection begins

In addition, B cells take time to switch isotype and undergo and affinity maturation…

10

Last Two Main Phases of B-cell Development

Plasma cells can differentiate directly from: activated B cells isotype switched, somatically hypermutated centrocytes memory B cells

11

T cell help, Isotype Switching & Affinity Maturation

Why? Production of high-affinity antibodies that are MOST effective at

dealing with pathogens During the course of an infection the effectiveness of the Abs

steadily increases Experience retained in the form of memory B cells and high affinity

Abs to provide long-term immunity to reinfection

What’s the alternative to waiting?

12

B cell Activation without T-cell Help

Faster primary response to activate B cells without the need for T-cell help Provides early defense Abs IgM isotype and of low affinity Keeps infection at relatively low level until better antibody response

can develop

How do B cells become activated?

13

7-1 B-cell activation requires cross-linking of surface immunoglobulin

14

Protein or carbohydrate

epitopes

Bacterial cell

Naïve Mature B cell

IgM’s X-linked by repetitive Ag epitopes

Ig

Ig

IgM Ig Ig

Note that BCR signal transduction resembles that of TCR

Signal transductionextracellular intracellular

15

How does BCR signal transduction resemble TCR signal transduction

BCR Associated with cytoplasmic

protein tyrosine kinases PTK’s activated by receptor

clustering

Ig & Ig associate with IgM to form functional BCR Cytoplasmic tails with 2 ITAMs Activates intracellular signaling

pathways

TCR Associated with cytoplasmic

protein tyrosine kinases PTK’s activated by receptor

clustering

CD3 associate with TCR to form functional TCR Cytoplasmic tails with 2 ITAMs Activates intracellular signaling

pathways

Similar intracellular signaling pathways!

16

B cell Signal Cascade

1. Receptor clustering

2. Receptor-associated tyrosine kinases phosphorylate the ITAMs on the Ig & Ig cytoplasmic tails

3. Syk binds to the phosphorylated ITAMs of the Ig cytoplasmic tail

Tyrosine kinases

Ig cytoplasmic tail(mature naïve B cell)

ITAMs

phosphate

Lyn

17

mature naïve

Ig

Lyn

What does Syk bind to? Phosphorylated ITAMS of

chain!!! Recall: clustering of BCRs

minimum of two receptor complexes Syk are close together

What is the result or function of this “closeness”?

Transphosphorylation

Transphosphorylation

18

What is the function of the previous intracellular pathway?

Extracellular Intracellular signals! What is the purpose/function of the B cell signal cascade?

Pathway that relays signals produced to the B-cell nucleus

What are the results of the B-cell nucleus receiving signals? Gene expression modulation Why does the IS want to modulate the gene expression in a B-cell

“B-cell Activation”

Is X-linking of the BCR by Ag sufficient to activate a mature naïve B cell?

19

NO! Additional signals are required to “activate” a mature naïve B cell

Requirement for the association of BCR with its co-receptor B-cell co-receptor 3 proteins

CR2 = complement receptor 2 CD19 CD81 = TAPA-1

Functions of co-receptor proteins CR2 = binds to complement deposited on pathogen CD19 = receptor signaling chain CD81 = unknown B-cell co-receptor function?????

20

How are signals delivered? Binding of CR1 (on the B-cell) to C3b (on the pathogen)

makes it susceptible to cleavage to C3d CR2 (part of the B-cell co-receptor) can then bind to the C3d

Signal is sent through CD19

21

Synergetic cooperation between B-cell receptor & B-

cell co-receptor

1. CR2 C3d X-links BCR to its co-receptor

2. Results in clustering together

3. CD19 phosphorylation by BCR-associated tyrosine kinases

4. Phosphorylated CD19 binds intracellular signaling molecules

5. BCR + BCR co-receptor signals, synergize to signals by 1,000- 10,000-fold

Is this “combined effect” of the BCR signal with its co-receptor signal (signal 1) and CD19 BCR intracellular signaling molecules (Lyn, etc) (signal 2) sufficient to activate a mature naïve B cell?

Signal 2

Signal 1

Ig

Ig: Lyn

22

Additional signals from helper T cells are required

“Generally” helper CD4 T cell signal requirement Which CD4 T cells help?

The “effector CD4 T cells” produced when naïve CD4 T cells encounter antigen and became activated

Which “effector CD4 T cells”? TH1 or TH2 ?????

Wait a minute…do all mature naïve B cells even require help?

23

9-3: The antibody response to antigens does not (always) require T-cell help

24

The Ab response to certain Ags DOES NOT require T cell Help

Chemical and antigenical “distinctions” in mammalian versus bacteria polysaccharides, lipopolysaccharides and peptidoglycans

One has repetitive epitopes … Is it bacteria or mammalian?

Repetitive epitopes are a major target of Ab response to extracellular pathogens

Some repetitive epitope Ags can activate mature naïve B cells without CD4 T cell help!

25

Whether a B cell needs T-cell help or not depends on the nature of the Ag

Two classifications of Ags Thymus-dependent Ags (TD Ag) Thymus-independent Ags (TI Ag)

Immunodeficient pts without thymus can make Ab against TI Ags

For TI Ags the need for CD4 T cell help can be overcome in 2 different ways

TI-1 Ag Bind to BCR and other receptors on B cells – Ex. (LPS TLR’s)

Combination = B cell induction to proliferate & differentiate

TI-2 Ag Bind to repetitive Ag’s and cause extensive crosslinking of the

BCR’s that no additional signal is needed to activate the B-cell.

26

What is LPS?

Lipopolysaccharide (surface of pathogens) Ex: LPS=Gram-negative bacteria

What is LBP? Soluble LPS binding protein

27

LPS of gram-negative bacteria can activate B cells to become Ab-producing plasma cells

1. BCR is specific for LPS epitope

2. LPS forms complex with soluble LPS binding protein (LBP)

3. Signals - CD14/TLR-4 + BCR + B-cell co-receptor sufficient to activate B cell plasma cells

28

LPS of gram-negative bacteria

can activate B cells to become Ab-

producing plasma cells

1. LPS binding to CD14/TLR-4 = a co-activating signal

2. Co-activating signal for another Ag on the bacterium to bind to its specific BCR

3. This B cell goes on to produce Ab specific for the bacterial antigen, not LPS

29

There is a second type of TI Ag: TI-2

Repetitive carbohydrates or protein epitopes at a high density on a pathogen’s surface Stimulate B cells specific for the Ag Extensive X-linking of BCR to B-cell co-receptor

What’s the result of this “Extensive X-linking” May over-ride need for additional signals

How long does this take? 48 hrs after Ag encounter Ex: bacterial cell wall polysaccharides with B-1 cell as the IS

responder

30

Ab responses induced by TI-2 Ags

Induce early Ab response to contain an infection – typically B-1 cells Limitations

Little isotype switching >> IgM (some IgG)

No hypermutation What is the result of no

hypermutation?

no affinity for Ag No long-term

immunological memory no long lasting immunity for 2nd encounter

31

9-4: Activation of naïve B cells by most antigens requires help from CD4 T

cells

32

B cells needing T-cell help and Thymus-dependent Ags (TD Ags)

Bulk of pathogen-specific Ab are produced by TD Ags What does TD antigen do?

TD Ags activate B cells in 2nd LT Does this make sense?

Well…..the 2nd LT is where B cells, specific Ag and helper CD4 T cells are brought together

33

B-cell Meets it’s Antigen

If a B-cell meets it’s antigen signals (BCR + co-receptor) are sent to the nucleus and induce changes in

the expression of adhesion molecules and chemokine receptors at the surface

These changes trap the B-cell in the T-cell area close to the B-cell zone. This allows for effector T-cells to test their TCR’s against the Ag-MHC II on

the B-cell cognate interactions and conjugate pairs

34

Ag delivery dendritic cells

Who delivers the Ag from the afferent lymphatic vessel?

P-APC, MHCII: dendritic cell

Mature naïve B cells need T-cell help with Thymus-dependent Ags (TD Ags)

1. B cell migrate in blood or afferent lymph to LN

2. B cells leave blood HEV LN cortex meets Ag

3. B-cells are drawn to T-cell areas by CCL21 and CCL 19 just like T-cells

4. B-cell doesn’t meet it’s antigen drawn to follicle by CCL13

5. Ags are trapped in the T-cell areas of LN

6. “Ag specific CD4 T cell helpers” activated in presence of IL-4 Th2 help activate the B-cells

Infected tissue

CCL19 and 21

CCL13

35

Recirculating naïve B cells enter the LN T-cell zone from the blood HEV

B cells encounter helper TH2 cells specific for the same Ag

B cells interact with TH2 to form a “1° focus” of proliferating activated B cells and TH2 cells in the medullary cords

Mostly IgM Under influence of IL-

5 & 6

Mature naïve B cells become trapped in the T-cell zone of 2nd LT if they encounter their

“cognate” helper T cell

36

Does the BCR have a role in B cell activation?

Does the BCR have a role in B cell activation? Two distinct BCR roles in B cell activation:

Binding antigen sends a signal to the B cells’ nucleus to change gene expression

Internalizing Ag by receptor mediated endocytosis processing and presenting it to helper T-cells

37

Two signals B cell proliferation & differentiation into plasma cell

B-cell activation in response to TD-Agrequires T cell help

38

What happens when TH2 cell’s TCR binds peptide Ag:MHC class II molecules on the B cell surface?

B cell CD40 :CD40 T cell ligand Why ?

a signal for the B cell to activate the transcription factor, NFB which then up regulates intercellular adhesion molecule 1 (ICAM-1) expression which can bind to LFA-1 on T-cell What’s the functional result of the up-regulation of ICAM-1on the

B cell’s surface? Strengthen the cognate interactions between the B and T-cell Reorganization of cytoskeleton and golgi allow focused secretion of

cytokines onto the B-cell IL-4 being one of the most significant cytokines to drive B-cell

proliferation and differentiation

Signal sent to T-cell nucleus to make of IL-4? Essential for B cell proliferation and development to plasma cells Characteristic of Th2 cells

39

40

What happens to these dividing B cells?

Recall: B cells activated by interaction with cognate helper T cells in the LN T-cell areas form a 1° focus of dividing B and T cells in the medullary cords of the LN

Result is dividing B lymphoblasts and some will secrete IgM How long does a 1° focus of dividing B cells last?

A few days

41

How long does it take for a GC to appear?

What is the physiological symptom of GC formation?

Division rate 1/6hr Large metabolically active cells

Centroblast Morphology of follicle changes

secondary follicle Domination by the germinal center (lots of

new B cells)

B cells Medullary cords PM cells

IL5 & IL6

TH2

YY

Y

IgM

1° focus 1° follicles

Still attached to TH2

1

2Can isotype switching happen in a primary follicle?

Several days

1 week Lymph node swelling

some

Primary Focus

42

Germinal Center

Specialized B-cell microenvironment where: proliferation Somatic hypermutation Selection of antigen binding

What are dark zones? Close packed centroblasts

What are light zones? Non-dividing centrocytes that

will interact with FDC’s

43

9-7 Activated B cells undergo somatic hypermutation and isotype switching in the specialized microenvironment of the

B-cell zone

44

Activation proliferation selection

B cell maturation in germinal center

Activation proliferation selection

Common theme of lymphocyte development

45

Proliferation in the GC, OkayWhat about hypermutation and affinity

maturation in the GC?

centroblasts dividing

in germinal center

somatic hypermutation

& isotype switching

T-cellcytokines

Nondividing centrocytes

Mutated surface Ig

Post hypermutationSurface Ig of centrocyte

Affinity for a specific antigen

higher

lowerequa

l

46

What’s the upshot of this hypermutation & affinity maturation

GC centrocytes express Igs with a range of affinities for the specific Ag The B-cells have to compete again for access to antigen on the

FDC’s and then for access to antigen specific T-cells.

What happens to centrocytes that fail to bind Ag:T-cell CD40 ligand? Centrocytes that fail to bind Ag/CD40 ligand on helper T cell

interaction Apoptosis

47

Mutated centrocytes compete!

Mutatedcentrocyte

To engage a helper T cellCentrocyte bind Ag process antigen surface = MHCII + Ag

Mutated centrocytes now compete!

Access of Ag on FDC’s Ag-specific helper T cells

MHCII

1 2

Follicular dendritic cells provide a source of intact Ag

48

B cells recognize Ag as FDC surface “immune complexes”

Localized in GC’s FDC’s bind Ag in

form of immune complexes (Ag:complement or Ag:Ab:complement)

1. Bind to FDC Fc receptors

2. Fc and complement receptors on FDCs

Immune complexes are not internalized

Persist on FDC’s for long periods (years)

49

Iccosomes are immune-complex coated bodies

Bundles of membrane coated with immune complexes also bud off from the surface of FDCs iccosomes FDCs have a prominent cell

body and dendritic processes Immune complexes are bound

on the FDC surface become clustered prominent beads are formed along the dendrites

Beads shed from the cells iccosomes Iccosomes taken

up by Ag specific B cells in the GC bound B cells process & present Ag

50

Newly formed centrocytes move from the dark zone of the gc

to contact FDCs in the light zone

Newly formed centrocytes move from GC dark zone captures Ag from FDC or iccosomes moves to GC light zone outer regions to helper T cells

Engagement of peptide:MHCII by TCR complex & CD40 ligand induces the centrocyte to express Bcl-xL proteins Bcl-xL functions to prevent death by apoptosis

51

Hypermutated B cells interact with FDCs displaying surface immune complexes

B cells do not bind Ag or poor Ag binding receptors due to mutated beyond recognition, therefore they cannot compete for access to the FDCs apoptosis

B cells receptors that bind Ag well receive signals from the T cell are induced to express Bcl-xL Results in preventing apoptosis Therefore, these B cells survive

52

After somatic hypermutation…B cells with high-affinity receptors for Ag are rescued from apoptosis

I.e. highest-affinity Ag receptors are selected for survival differentiation into Ab-producing plasma cells and into long lived memory cells

Affinity of Abs for specific Ag increase during immune response is called Affinity maturation

53

Survivors interact with Ag-specific T cells…Why?

Mutual engagement of ligands and receptors on surviving centrocyte with T helper cell leads to further proliferation of both B and T cells, serves to: increases population of selected high-affinity, isotype-switch B cell Some B cells are directed down the path of plasma or memory cells

Height of adaptive immune response = need large # of Abs to fight infection selected centrocytes leave GC differentiate into Ab-producing plasma cells

54

What happens to centrocytes that fail to obtain, internalize & present Ag?

What happens when centrocyte fails does not obtain, internalize and present Ag Apoptosis Macrophage engulfment in the gc

Tingible body macrophages are macrophages that have recently engulfed the apoptotic centrocytes are a characteristic feature of gc, because of their contents they are called “tingible body

macrophages”

What happens when somatic hypermutation produces centrocytes bearing Ig reactive to self-Ag? Contact with helper T cells in the GC render these centrocytes

anergic

55

Chapter 9 – Lecture Notes

Immunity Mediated by

B Cells and Antibodies

56

Chapter 9

continued

57

9-9: Interactions with T cells are required for isotype switching in B cells

58

Interactions with T cells are required for isotype switching in B cells, Why?

Cytokine effects on the switching of Ig synthesis to a particular isotype

Induce augment inhibit

1st Igs = IgM and IgD B cells in GC activated by Ag switch H-chain isotype IgG, IgA or

IgE

The isotype to which an individual B cells switches is determined by?

59

…Cognate interactions with helper T cell

What does “cognate” mean? Cognate interactions are cell-cell interactions between B

and T lymphocytes specific for the same antigen How does the helper T cell control the particular isotype to

which a switch is made? controlled by the cytokines secreted by the helper T cell

Roles of specific cytokines in switching is area of hot research in immunology

60

Interactions with T cells are required for isotype switching in B cells

“Based on mouse B cell experiments” Differences in humans (from mouse) are not worked out

Example, switching to IgA in humans involve TGF- and IL-10, not IL-5

61

Which cytokines are most involved?

Prominent players include cytokines secreted by TH2 cells

IL-4, IL-5 and TGF

What do these cytokines do? Activate naïve B cells to differentiate into plasma cells

Secreting IgM Induce the production of other antibody isotypes

IgG2, IgG4 (weak opsonizing Abs) IgA, IgE

IFN- is a characteristic TH1 cytokine Switches B cells to make IgG1 Strong opsonizing Ab in humans

62

How do T-cell cytokines induce isotype switching?

Stimulate transcription from the switch regions that lie 5’ to each H-chain C gene

Example, activated B cells exposed to IL-4 Transcription from a site upstream of the switch regions of C1 and

C is detected prior to switching

What’s the molecular mechanism for isotype switching? Transcript opens up the chromatin Makes the switch regions accessible to somatic recombination

machinery Somatic recombination machinery places a new C gene in

juxtaposition to the V-region

63

What else does isotype switching by cognate helper T cells require?

B cell surface CD40 binding to T-cell CD40 ligand This is very important…

How do we know this is important? Example, pts who lack CD40 ligand (Hyper-IgM

syndrome) Immunodeficient Abnormally high levels of IgM in blood serum Almost no IgG and IgA B cells are unable to switch isotype Cannot make Ab responses to TD Ags

Males gene for CD40 ligand is on the X-Chr X-linked

64

Hyper-IgM syndrome

No GC in Lymph Nodes GC in Lymph nodes

65

Isotype switched, affinity matured B-cells can differentiate into plasma cells or memory cells

When the infection is bad, and secreted Ab’s are needed then the centrocytes will be told to become plasma cells – IL-10

As the infection begins to subside, the centrocytes will be told to become memory B-cells – IL-4

66

67

Antibody effector functions

What does isotype switching do? Isotype switching diversifies Ab Fc region functional

properties Two distinct functions of Fc regions

Deliver Ab to anatomical sites otherwise inaccessible Link bound Ag to molecules or cells that will effect it’s destruction

These cells carry Fc receptors (Examples: macrophages and neutrophils).

68

9-11 IgM, IgG and IgA Abs protect the blood and extracellular fluids

69

IgM, IgG and IgA Ab functions

Protect the blood and extracellular fluids IgM = 1st Ab, pentamer, enters the blood carried to site of

infection Pentameric nature = strong binding to microorganisms or

particulate Ags Large size limits penetration to infected tissue

Reduced ability to passively leave the blood to penetrate infected tissue

No IgM Fc receptors on phagocytic cells or leukocytes IgM cannot directly recruit these cells IgM Fc regions does, however, bind complement and can

activate the complement system

70

Which blood-borne Ab is dominant later in an immune response?

IgG Smaller Together IgM and IgG function to prevent blood-borne infection

septicemia

71

What about IgA?

Synthesized by plasma cells in 2nd LT Two forms

Monomeric Dimeric

Monomeric IgA Secreted from plasma cells derived from B cells that switched their

Ab isotype in the LN or spleen Enters extracellular spaces and helps IgG protect against bacteria

and virus particles

Dimeric IgA Made in 2nd LT underlying mucosal surfaces

72

IgA & IgG are transported across epithelial barriers by specific receptor proteins

Recall: IgM, IgG and monomeric IgA provide Ag-binding within fluids and tissues of the body

Dimeric IgA protects epithelia surfaces that communicate with the external environment (mucosal membranes susceptible to infection) Linings of the GI tract, eyes, nose, throat, mammary glands,

respiratory, urinary and genital tracts Dimeric IgA made in the lamina propria

Connective tissue underlying basement membrane of mucosal epithelium

IgA-secreting plasma cells are on one side and the target pathogens are on the other side

The dimeric IgA molecules are transported across the epithelium by receptors on the basolateral surface of the epithelial cells

73

How are IgA & IgG transported across epithelial barriers?

Dimeric IgA bound by a J chain diffuses across the basement membrane

Dimeric IgA bound by the poly-Ig receptor on the basolateral surface of epithelial cell

Transcytosis – receptor mediated transport from one side of a cell to the other of a macromolecule

Complex transcytosis vesicles released apical surface Poly-Ig receptor cleaved releases IgA from epithelial cell

membrane IgA still bound to a fragment of the receptor called the “secretory

component” (holds IgA to mucus) Residual membrane-bound fragment of the poly-Ig receptor is

nonfunctional degraded

74

How are IgA & IgG transported across epithelial barriers?

Apical surface

Basolateral surface Receptor

mediated endocytosis

Transcytosis

75

Brambell receptor (FcRB)

IgG actively transported from blood into extracellular spaces by an Fc receptor present on endothelial cells Receptor called FcRB Similar structure to MHC class I (1 & 2 domains to bind the Fc

regions of the Ab) Ab:receptor complex

2 molecules of FcRB bind to the Fc region of one IgG IgG delivery to extracellular spaces in connective tissue

protects IgG from degradation pathways of serum proteins IgG relatively long lived half-life

76

Brambell receptor (FcRB)

77

Passive transfer of immunity

Fetus physically protected by the mother from extracellular microorganisms

What about after birth? Lack actively acquired immunity Vulnerable to infections (microbial colonization of epithelia) Receives IgA from its mother - secreted into breast milk

Transferred to baby’s gut Bind to microorganism preventing attachment to the gut epithelium

Pass in the feces

78

What about IgG during pregnancy?

IgG from maternal circulation transported across placenta Directly into the babies bloodstream Therefore babies have a level of IgG protection in their

plasma equal to the mother’s level of IgG protection Transport of the IgG antibodies across the placenta is

also mediated by the FcRB.

79

Antibody production is deficient in very young infants

1st year = vulnerable to infection, deficient in Abs During pregnancy maternal IgG Ab transported across the

placenta What happens to this IgG Ab?

Maternally derived IgG is catabolized Gradually decreases until infant’s immune system produces its own

Ab (6 months) Therefore, IgG levels are lowest in infants aged 3-12 months (time

the infant is most vulnerable to infection) Premature babies are even more vulnerable Take longer to attain immunocompetence after birth than full

term babies

80

High-Affinity IgG and IgA are used to neutralize microbial toxins and animal venoms

Some bacteria secrete toxins that disrupt the normal function of cells. Diphtheria and tetanus toxins have a binding part and a toxic part. Ab’s that bind to the binding part of the toxic molecule will block the toxins

ability to enter the cell thus blocking its toxic effects. For venoms, passive immunization is used. (antibodies created using a

large animal inoculated with the venom) These antibodies are gathered from the animal and given to a snake bite

victim to neutralize the toxins (no long term immunity)

81

High-affinity neutralizing Ab’s prevent viruses and bacteria from infecting cells

82

Ab’s link effector cells to the antigen

Abs = only effector molecules produced by B cells Abs = 1 function = “adaptor molecules”

neutralize the pathogen (doesn’t destroy pathogen) bring “pathogens” and “effector” cells together

Why? So effector cells clear/destroy the pathogen

83

Fc receptors

Abs bind through their Fc regions Bind to what?

Fc receptors on effector cells Fc receptors are specific for Ig isotype Fc receptors have 2 main purposes

Deliver Abs to sites where they would not be carried by blood and lymph circulation

Attach Ag:Ab complexes to effector cells allowing effector cells to destroy the pathogens

84

Fc of Ab and Fc receptor of effector cell

85

IgE binds to high-affinity Fc receptors on mast cells, basophils and activated eosinophils.

FcRI has high affinity for IgE. Crosslinking of receptors by antigen causes release of

histamine from mast cells inflammation recruits cells and proteins needed for host defense.

86

High points of 9.11 - 9.16 & 9.21 - 9.25

High-affinity IgG and IgA Abs are used to neutralize microbial toxins and animal venoms

High-affinity neutralizing Abs prevent viruses and bacteria from infecting cells

Fc receptors of hematopoietic cells are signaling receptors that bind the Fc regions of Abs

Phagocyte Fc receptors facilitate Ab-coated pathogen recognition uptake destruction

IgE binds to high-affinity Fc receptors on mast cells basophils activated eosinophils

Fc receptors activate NK cells to destroy Ab-coated human cells Antibody-dependent cell-mediated cytotoxicity (ADCC) (look up)

87

Immune complexes and the complement system

Immune complexes of Ag:Ab are often ingested by phagocytic cells and destroyed intracellularly fate of most foreign materials that have Abs produced

against them But the fate of immune complexes also depends heavily

upon their ability to activate the complement system

88

Immune Complex Clearance

C3b can coat particulate antigen (immune complexes) that can then be bound by erythrocytes and cleared in the liver or spleen.

89

90

Classical Complement Cascade

Need one IgM or at least two IgG. C1 binds to the Fc regions and needs at least two binding

sites to become stable on the pathogens surface. The rest of the cascade proceeds the same way as you

learned previously. Don’t forget that the alternative pathway can amplify the

cascade that was started by the Ab’s (classical).

91

The complement system – A review of what you have already learned.

Slides 91 – 119 are to help you in remembering complement

~ 30 serum proteins Interact in a complex reaction sequences

“complement cascade”

Results in the manifestation of inflammation phagocytosis lysis of foreign cells

Why call it COMPLEMENT? The action of these proteins

“complements” antibody-mediated reactions

92

Complement components circulate in blood and body tissues

What is the complement system & how does it function? Enzymes that work in a cascade one becomes active

cleaves another to activate it etc. Center stage = C3 cleaved (C3b and C3a) becomes

activated by Abs aggregated in immune complexes bacterial compounds that 1st bind and activate other complement

proteins

93

Complement components C3b and C3a

C3b and C3a degradation products have direct opsonizing effects chemotactic effects inflammatory effects

One of the functions is to activate the lytic pathway involving C5-C9

94

95

Several complement pathways Classical pathway Alternative pathway Lectin pathway

Proteins of the complement system (CS) 5% of serum proteins in vertebrates

Classical pathway proteins are designated by number following C for complement

Range C1 C9

Proteins of the alternative pathway C3, C5 C9 proteins factor B factor D

96

Cascade

Complement system proteins act in a sequence (cascade) Each protein activates the next one in the series

cleaves the next protein Resulting components have new functions

What jump starts the classical pathway? Ag:Ab

Is it the same for the alternative pathway? No…initiated by bacteria and fungi cell-wall polysaccharides

97

C3

C3 has major role in initiation of mechanisms that lead to microbial destruction In fact, both classical and alternative pathways lead to the cleavage

of C3 into fragments C3a C3b

C3a and C3b initiate cytolysis inflammation opsonization

98

The complement component C1

99

100

Cleavage of C4 exposes a reactive thioester bond that covalently attaches the C4b fragment to the pathogen surface

101

Activated C1s cleaves C4 and C2 to produce C4b and C2a, which associate to

form the classical C3 Convertase

102

Formation of the alternative C3 convertase

103

The two types of C3 convertase have similar structures and functions

104

C3 activation by the alternative C3 convertase is a process analogous to C3 activation by the classical C3 convertase

105

View from above of complement deposition

Keep in mind that classical activation is also amplified by the alternative pathway C3 convertase.

106

Cytolysis

Most important function of the complement cascade is… destroy foreign cells (microbial/pathogens)

How does the IS do this? damages the cell membrane to the point that the cellular contents

leak out cytolysis

C5 - C9 = membrane attack complex MAC

produces transmembrane channels through the cell membrane

leads to cell lysis or cytolysis

107

Inflammation

C3a and C5a contribute to acute inflammation What exactly do they do?

bind mast cells, basophils and blood platelets release of histamine

Histamine increases blood vessel permeability

C5a acts as a powerful chemotactic factor What’s this mean?

C5a is able to attract phagocytes to areas where complement has been fixed

108

The Classical Pathway-Step 1: C1 component

C1 component is comprised of three parts C1q (hexamer) C1r (dimer) C1s (dimer)

Held together by calcium ions

109

The Classical Pathway-Step 1: Activation of C1

Initiated by the binding of C1q to C1q-specific receptors on the Fc portions of adjacent Abs (minimum 2 IgG Abs or one IgM) Humans, all Abs have these receptors

exceptions of IgG4, IgA and IgE

Abs possessing these receptors bind or “fix” complement Abs without these receptors, cannot fix complement

Once C1q has bound the C1q specific receptors of two adjacent antibodies, it activates C1r, which in turn activates C1s

Activated C1s subcomponent then activates C4

110

The Classical Pathway-Step 2: Activation of C4

C4 is the 2nd complement component of the cascade but was the 4th complement component identified, “C4”

Activated C1s activates C4 by cleaving it into two fragments C4a C4b

C4b binds to the surface of the cell membrane near the site of the Ag:Ab complex

Once bound to the surface, C4b then binds the C2a complement component

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The Classical Pathway-Step 3: Activation of C2

C2 component is cleaved by the combined activities of C4b and C1s C2a C2b

C2a portion remains attached to the C4b component on the cell membrane

C4b-C2a complex is referred to C3 convertase C3 convertase activates the C3 component of complement

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The Classical Pathway-Step 4: Cleaving C3 and amplification

C3 is activated by the activity of C3 convertase This cleaves C3

C3a C3b

A single molecule of C3 convertase is capable of activating hundreds of molecules of C3 amplifying the cascade by providing large amounts of C3a and

particularly C3b

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The Classical Pathway-Step 4: Activation of C3

Both C3a and C3b have biological activity C3a functions as an anaphylatoxin C3b attaches to the cell membrane near the site of activation

where it is capable of acting as an opsonin

Some C3b combines with the C3 convertase to form active classical C5 convertase (C4b2a3b)

Active classical C5 convertase activates complement component C5

Alternative C5 convertase (C3b2Bb)

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Wait a minute…What about MAC?

What about it’s formation? Who is MAC?

The membrane attack complex (MAC) formed as the result of the assembly and activation of

complement components C5 through C9 involved in cytolysis of the target cell

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The Classical Pathway-Step 5: Activation of C5, C6 and C7

C5 is cleaved by C5 convertase C5a C5b

Both of which have biological activity C5a functions as an anaphylatoxin and a chemotaxin C5b binds C6 and C7

forming a complex that attaches to the surface of the target cell membrane

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The Classical Pathway-Step 6: Activation of C8 and C9

C8 component of complement then binds the C5b-C6-C7 complex on the cell membrane

This complex is capable of forming small pores in the membrane of the target cell = compromising its integrity

This is enhanced by the polymerization of the C9 complement component

The polymerization of C9 leads to the formation of transmembrane channels target cell lysis

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MAC

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Complement System = Major defense mechanism against microbial infection

Especially extracellular bacteria Complement components are plasma proteins of several functional

groups Become activated by infections in 3 different ways

classical pathways alternative pathways lectin pathways

The activation of phagocytes and inflammatory reactions by complement provides protection before the antibody response develops after the antibody response develops

The defense mechanism of complement-mediated phagocytosis of pathogens evolved long before the existence of Ab

Molecular Hx = Ab that actually provided the “complementary function” rather than the complement

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