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!