neutrophils eosinophils lymphocytes · haemopoietic stem cell red blood cells white blood cells but...
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HaemopoieticStem Cell
Red BloodCells
White BloodCells
BUT THINGS ARE MUCH MORE COMPLICATED THAN THIS!
Lymphocytes
neutrophils eosinophils
monocytes
White BloodCells
THE HAEMOPOIETIC SYSTEM
ALL THESE CELLS MUST BE ABLE TO MOVE!
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
Myeloidprecursor
Lymphoidprecursor
Pluripotentstem cell Monocyte
Dendritic cells
CELLS MOVE TO SITES OF INFLAMMATION
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
Myeloidprecursor
Lymphoidprecursor
Pluripotentstem cell Monocyte
Dendritic cells
CELLS MOVE TO SECONDARY LYMPHOID ORGANS
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
Myeloidprecursor
Lymphoidprecursor
Pluripotentstem cell Monocyte
Dendritic cells
Tissue specific homing
White BloodCells
THE HAEMOPOIETIC SYSTEM
ALL THESE CELLS MUST BE ABLE TO MOVE!
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
Myeloidprecursor
Lymphoidprecursor
Pluripotentstem cell Monocyte
Dendritic cells
CELL MOVEMENT IS IMPORTANT IN MANY OTHER CONTEXTS
I) Development
2) Adult stem cell movement
Bone Marrow
HSC HSC
SO HOW IS ALL THIS REGULATED?
CHEMOKINES
Chemotactic Cytokines
CHEMOKINES: Chemotactic Cytokines
CC C C
CXC C C
C C
CXXXC C C
MIP-1α , MIP-1βMCP1-5, EotaxinRANTES, SLC, ELC
IL8, PF4, IP10SDF, MGSA
Lymphotactin
Fractalkine/neurotactin
The cysteines ensure that the chemokines have the same overall structure!
CHEMOKINES: Chemotactic Cytokines
CC C C
CXC C C
C C
CXXXC C C
MIP-1α , MIP-1βMCP1-5, EotaxinRANTES, SLC, ELC
IL8, PF4, IP10SDF, MGSA
Lymphotactin
Fractalkine/neurotactin
CHEMOKINES: Chemotactic Cytokines
CC C C
CXC C C
C C
CXXXC C C
CCL1-28
CXCL1-16
XCL1
CX3CL1
CHEMOKINES REGULATE CELL MIGRATION/NAVIGATION
Cellular MigrationRegulation of cell movement is the key role of chemokines
Chemokines
Regulation of leukocyte migration is the prominent function for chemokines
Inflammatory/inducible
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
Myeloidprecursor
Lymphoidprecursor
Pluripotentstem cell Monocyte
Dendritic cells
Constitutive/homeostatic
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
Myeloidprecursor
Lymphoidprecursor
Pluripotentstem cell Monocyte
Dendritic cells
a) Inflammatory Chemokines: i) normally expressed at low levelsii) can be induced to very high levels in many cell typesfollowing inflammatory insults or woundingiii) recruit inflammatory leukocytesiv) redundant
b) Constitutive chemokines:i) made constitutively at low levelsii) made in discrete tissues by specific cells iii) regulate homeostatic movement of leukocytes iv) non-redundant
These differences are reflected in relevant null mice!
These differences are reflected in relevant null mice!
1) Inflammatory Chemokines
MIP1α /CCL3 -/-
Mice have no overt phenotype: haemopoietic cell numbers etc are normal
But, mice have altered responses to a range of inflammatory stimuli
2) Constitutive chemokines
SLC/CCL21 -/- miceSDF1/CXCL12 -/- mice
These animals have defects in basal leukocyte trafficking
SLC/CCL21 null mice lack lymph node and other secondaryLymphoid organ T cells
SDF1/CXCL12 null mice lack bone marrow haemopoietic stem cells
Bone Marrow
Non-immune/inflammatory cell migrationa) Haemopoietic stem cells:
SDF/CXCL12
AMD3100
CXCR4 or SDF/CXCL12 null micedie from bone marrow
failure
b) Primordial Germ Cells
Genital Ridge
Zebra Fish exposed to antisenseoligos to CXCR4 are infertile
Same seen with CXCR7!
SDF/CXCL12-/- mice andCXCR4 -/- would be infertile
SDF/CXCL12
Mesenchymal stem cells also require CXCR4 for migration
The roles for SDF/CXCL12 and CXCR4 in regulatingHSC and PGC Migration reflect the extremely
primitive nature of this ligand/receptor pair
SDF/CXCL12 is probably the primordial chemokine
(HUMAN) KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN
(XENOPUS) KPVSLVYRCPCRYFESNVPKSNIKHLKILSTSNCSLQIVARLKHNGKQICLDPKTKWIQEYLEKALN
|||||*||||||*|||*|***|*||||||*|*||*||||||||*|**|*|||||*||||||||||||
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
Myeloidprecursor
Lymphoidprecursor
Pluripotentstem cell
Monocyte
Chemokines Bind to Seven-TM Receptors
CCRs 1-10
CXCRs 1-7
XCR1
CXXXCR1
Decoy receptors
Viral receptors
MANY RECEPTORS ARE PROMISCUOUSAND MANY LIGANDS UNFAITHFUL!
CCR5
RANTES/CCL5
MCP2/CCL8MIP-1β /CCL4
MIP-1α /CCL3
CCR1
CCR3
D6
Receptors can also be categorised as Inflammatoryor constitutive
Inflammatory: CCRs 1, 2, 3, (4) and 5
CXCRs 1, 2, 3
Constitutive: CCRs 6, 7, 9 and 10
CXCRs 4, 5, 6 and 7
Again, this is reflected in receptor-null mice!
CCR1: CCL2, 3, 5, 7, 14, 15, 16, 23 CCR2: CCL2, 7, 12, 13, CCR3: CCL3, 5, 7, 8, 11, 13, 15, 24, 26CCR4: CCL17, 22CCR5: CCL3, 4, 5, 8CCR6: CCL20CCR7: CCL19, 21CCR8: CCL1CCR9: CCL25CCR10: CCL27, 28
CXCR1: CXCL6, 8CXCR2: CXCL1, 2, 3, 5, 6, 7, 8,CXCR3: CXCL4, 9, 10, 11 CXCR4: CXCL12CXCR5: CXCL13CXCR6: CCL16CXCR7: CXCL12/CXCL11
Promiscuity and unfaithfulness are properties mainly ofinflammatory chemokines and their receptors
This gives great scope, versatility and redundancy to inflammatory responses
Chemokines and their receptors in Disease
1) Immune/Inflammatory disorders
3) HIV pathogenesis
5) Cancer and cancer metastasis
Chemokine receptors and HIV pathogenesis
1) T cells from some individuals who were relatively resistant to HIV infection expressed high levels of MIP-1α/CCL3, MIP-1β/CCL4 and RANTES/CCL5
2) CCR5 was shown to bind these ligands
3) CCR5 and CD4 bearing cells could be infected by M-tropic strains of HIV
4) People with a homozygous null mutation in CCR5 (∆32) are refractory to HIV infection
CD4?
CD4 and a co-factor was neededfor HIV infection of cells
CD4CXCR4 orCCR5
The co-factors turned out to beChemokine receptors
CXCR4 is the main HIV co-receptor for T-tropic HIV strains
CHEMOKINES AND CANCER
1) Angiogenesis:
CXC C C
ELR motif (angiogenic); non-ELR (angiostatic)
2) Metastasis
Breast Tumours
Bone marrow
Lung
Liver
This has been shown to involve CXCR4 and SDF/CXCL12
i) Bone marrow
Lung
Liver
ii)
And is dependent on hypoxia
This may be a much more widespread phenomenon (CCR7, CCR9, CXCR5, CCR10)!
(Plus attraction of EPCs! Plus CXCR7?)
3) Inflammation
TNF null mice are refractory to induction of skin tumours
Many tumours are characterised by extensive leukocyte infiltratesand expression of chemokines
The leukocyte infiltrate may play complex and contradictory roles
There is a correlation between chemokine levels and prognosis insome cancers
Tumour associated macrophages
Oncogene induction of chemokines
Chemokines and their receptors are therefore prominentdrug targets
There have been some recent successes (CCR5, CXCR4, CCR9)
1) Immune/inflammatory disorders
3) HIV pathogenesis
3) Cancer
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
Myeloidprecursor
Lymphoidprecursor
Pluripotentstem cell
Monocyte
LEUKOCYTES MUST LEAVE THE VASCULATURE AT APPROPRIATE SITES.
THIS MUST BE REGULATED!
Problems
1) Cells must adhere to the venular wall under shear flow(Cells travel at 4000um/sec)
2) Cells must cross the EC layer without altering vessel properties
3) Cells must know where and when to do all this!(Local address codes)
The Classic Model of Transendothelial Migration
Diapedesis
Step 1: Rolling and tethering
1) Induction of rapid bond formation at leading edge of leukocytes
3) Formation of high affinity binding contacts
5) Rapid dissociation constant
4000µm/second to 4-40µm/second
This allows sampling of the vascular endothelial surface.
This process is regulated by Selectins and their ligands
All are Selectins are C-type lectins and there are P, E and L-selectins
The ligands are specific carbohydrate moieties on cell surface proteins
This interaction slows the leukocytes down by 100-1000 times
In inflammation, P.E and L-selectin ligands include PSGL1 on ECs and leukocytes
On HEVs, the L-selectin ligand is referred to as PNaD
PNaD: Peripheral Node Addressin
(Glycam, CD34, sgp200, podocalyxin and endomucin)
In intestine it is MadCAM
2) Activation and arrest
1) Increased binding and adhesion to the ECs
3) Must be rapid and shear-flow resistant
5) Must be at appropriate sites
Activation and arrest take place in a sub-second time frame
This is essential for initiating diapedesis
This process is regulated by Integrins and their ligands
Integrins are heterodimeric adhesion molecules made up from α and β subchains.
There are 18 α and 8 β subunits and 24 known heterodimers!
The 5 main leukocyte integrins are: LFA1 (αLβ2) CD11a/CD18Mac1 (αmβ2) CD11b/CD18CR4 (αxβ2) CD11cVLA4 (α4β1) CD49d/CD29LPAM1 (α4β7) CD49d/CD
INTEGRINS ARE EXPRESSED ON SPECIFIC LEUKOCYTE SUBSETS
CD11a: All leukocytes
CD11b: Monocytes/macrophages and neutrophils
CD11c: DCs
CD18: Broad leukocyte expression
CD29: Lymphocytes
CD49d: Lymphocytes and monocytes
100 101 102 103 104FL2H
Data.005
M1
100 101 102 103 104FL2H
Data.001
M1
So, what are the ligands for the integrins?
Integrins bind extracellular matrix molecules and other cell surface molecules
LFA1 ICAM1, iC3b, FibrinogenMac1: ICAM1, iC3b, FibrinogenCR4: ICAM1VLA4: Fibrinogen, VCAM1, CS1LPAM1: MadCam, Fibrinogen
beta-1: binds extracellular components e.g. collagens, laminin, fibronectin,but also VCAM-1 (alpha-4);
beta-2: binds ICAMs, clotting factors, and C3b (alphaM) - expressed exclusivelyon leukocytes.
beta-3: binds clotting factors, and more; beta-4: binds laminin beta-5: binds vitronectin beta-6: binds fibronectin beta-7: binds fibronectin, VCAM-1 beta 8: ?
BUT: THE INTEGRINS NEED ACTIVATED FOR FIRM ADHESION
Weak Adhesion Firm Adhesion
THIS IS TRIGGERED BY CHEMOKINES VIA ‘INSIDE OUT SIGNALLING’SO….. SPECIFICITY NOW???
1) Conformational Change (affinity)2) Increased lateral motility and clustering (avidity)
CHEMOKINE RECEPTOR SIGNALLING AND FIRM ADHESION
YY
Chemokine Interaction
YY
Firm Adhesion
Y
LFA1
CCL21
CCR7
ICAM1
This takes place extremely rapidly and is dependent onIMMOBILISED chemokines
Now we can understand site-specific recruitment of leukocytes!!!
The combination of Selectins and Integrins and chemokine receptorsand their respective ligands supplies the ADDRESS CODE
PLN homing
LFA1
ICAM1
CCL21
CCR7
Y
L-Selectin
PNaD
Gut homing
α4β7
MadCAM
CCL25
CCR9
Y
L-selectin
MadCAM
Skin homing
LFA1
ICAM1
CCL17/27
CCR4/10
Y
E-Selectin
CLA
WHAT DOES THIS TELL US ABOUT THE SITE OF ACTIONOF CHEMOKINES?
1) Probably much of chemokine function is on the lumenal face of the ECs
6) Gradients are NOT required for this!
3) Transendothelial Migration
1) Cells must move into and through the EC/EC junction
3) This must not disrupt the integrity of the vascular endothelial layer
5) This involves disassembly and reassembly of the leukocyte cytoskeleton
7) Cells must be intact and functional following TEM
ENDOTHELIAL CELLS ARE HELD TOGETHER BY NUMEROUSJUNCTIONAL MOLECULES
PECAM
JAM-A
JAM-B
JAM-C
CD99
EC EC
1) Adherens junctions are maintained by Cadherins
3) Tight junctions are maintained by the JAM proteins
VE-Cadherin VE-Cadherin
This is what the leukocyte has to crawl through!
1) PECAM
The first adhesion molecule met by the transmigrating leukocytes
PECAM forms HOMOPHILIC interactions
As well as ECs, most leukocytes also express PECAM
PECAM
JAM-AJAM-BJAM-C
CD99
EC ECVE-Cadherin VE-Cadherin
Leukocyte
The leukocyte/EC homophilic interaction maintains the integrity of the endothelial layer!
2) CD99
Expressed on most leukocytes and ECs
32kDa glycosylated molecule (not a member of a larger family)
Again, homophilic interactions are important here
PECAM
JAM-AJAM-BJAM-C
CD99
EC ECVE-Cadherin VE-Cadherin
Leukocyte
Antibodies to CD99 block monocytes that have reached into the EC/EC junctionBlocking PECAM and CD99 completely blocks TEM!
THERE ARE PREFERRED SITES FOR TEM!
2) Transcellular migration???
PECAM
JAM-AJAM-BJAM-C
CD99
EC ECVE-Cadherin VE-Cadherin
1) Tri-Cellular Junctions
Paracellular!
THE IMPORTANCE OF SHEAR FLOW FOR TEM
The Classic Model of Transendothelial Migration
Diapedesis
WHAT HAPPENS AFTER THE CELLS LEAVE THE VASCULATURE???
1) GRADIENTS???
4) CHEMOTAXIS?
7) CHEMOKINESIS?
B-cells
T-cells
Neutrophils
Macrophage
Eosinophil
Basophil
Platelets
Erythrocyte
CFU-GM
CommonLymphoidProgenitor
Pluripotentstem cell
Monocyte
CommonMyeloidProgenitor
How are the chemokines presented and immobilised?
1) Local production
8) Transcytosis (DARC)
15) GAG binding
EITHER WAY…. THE EC GREETS THE LEUKOCYTES IN A SIMILAR WAY
Transcellular Paracellular
PECAM
JAM-AJAM-BJAM-C
CD99
EC ECVE-Cadherin VE-Cadherin
Leukocyte
JAMA (on ECs) JAMA (on leukocytes)JAMA (on ECs) LFA1 (on T cells)JAMB (on ECs) JAMC (on leukocytes)JAMB (on ECs) VLA4 (α4β1)JAMC (on ECs) Mac1 and VLA4 (on leukocytes)
OTHER INTERACTIONS IMPORTANT FOR TEM
TEM through the lymphatic vasculature
Activation
VCAM, ICAM, E-Selectin, CCL21(*), CCLs2,5,20
Essential: CCL21, VCAM, ICAM
NOT A PASSIVE PROCESS!