Leukocytes Polarize in Gradients of Chemoattractants, then Crawl to the Source

RestingLeukocyte

CHEMOATTRACTANT

(<1%)

Lamellipod

Uropod

Crawling Leukocyte

CX3C: ……………CXXXC…………………… C…………………C……………… CX3CL1 non-ELR CXC: ……………CX__C………………………C…………………C……………… CXCL# ELR CXC: …ELR…CX__C………………………C…………………C……………… CXCL# 4C CC: ……………C___C………………………C…………………C……………… CCL# 6C CC: ……………C___C…………C…………C…………………C……C……… CCL# C: ………………………C……………………………………………C……………… XCL#

Class NamesStructural Signature

Chemokines: Defined by Structure, Classified and Named by Patterns of Conserved Cysteines

…but unequally distributed among the 4 classes

CX3C (n=1)

(n~16)

(n~28)

C

CXC CC

(n=2)

The Chemokine Fold

G.M.Clore & A.M.GronenbornPDB ID: 1IL8

C

N

Chemokine receptors are defined by function, not structure: they bind chemokines and transduce signals.

To date all known chemokine receptors are members of the 7TM GPCR superfamily, coupling to Gi.

Chemokine Receptors

Leukocyte Chemoattractants Act Through a Family of 7-trans-membrane domain G Protein-coupled Receptors

7TM Receptor

G protein

Leukocyte Trafficking

Chemoattractant

Chemokine Binding Protein Classification

CXCR1 CXC CXCR2 CXCR3 CXCR4 CXCR5 CXCR6 CCR1 CC CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CCR9 CCR10 XCR1 C CX3CR1 CX3C D6 CC Duffy CC & CXC CCX/CKR CC

Chemokine Subclass Specificity

Chemokine Binding Proteins (7TM architecture)

7TM G Protein-coupled Receptors

Neurotransmitter Receptors

TSH R

LHCG R

b1 AR b2 AR

HT 2 HT 1c A1 P1

A2 P1 a2a AR a2b AR a2c AR

HT 1a M 1

M 5M 3

M 2M 4

RhodopsinOpsin BlueOpsin GreenOpsin Red

SKR SPR IL8RAIL8RB

MIP-1a/RANTES R

FPRL1 R

FPRC5a RPAF R

Leukocyte Chemoattractant Receptors

Polypeptide Hormone Receptors

Light Receptors

Prokaryotic opsins

PTH, calcitonin, secretin receptors

metabotropic glutamate receptors

?

Neuropeptide Receptors

cyclic AMP chemoattractant receptors of Dictyostelium

The GPCR Superfamily

out

+

NH2

COOH

-

P P

P

PS S CC

S/T}

}

}C C S S

VR

LF

F

C

R

i2GDP

21

2 3 4

56

7

N--

1

2C

Chemokine Receptor Models

Hemmerich et al. Biochemistry, 38:13013, (1999)

Model of MCP-1/CCR2 Interaction

Neutrophil

IL-8

IL8RB

IL8RA

GRO

GRO GRO

NAP-2

ENA-78

CXC CC

Eosinophil

Macrophage

CK

R2b

CKR2a

CKR1

CK

R5

CKR4

MIP-1

MIP-1

MCP-3

M

CKR3

CKR1

Eotaxin

Basophil

CKR4

RANTES MCP-1

Chemokine Receptors Can Bind Many Chemokines, but are Class-restricted

i2GDP

i2GDP

21

2 3 4

56

7

N--

21

2 4

56

7

N --++

IL8

C

21

2 3 4

56

77

N--++

3

i2GTP

1

2

PI-PLC 2

?1

2

1

2

IL8

CIL8

C

Extracellular Milieu

Chemokine Receptor Activation Mechanism

Regulation

• Gene expression (cytokines/chemokines, microbes, microbial products, etc.)

• GAG association• Post-translational processing (CD26, cathepsin

G, TACE)• Endogenous antagonistic chemokines• Decoy receptors (Duffy, D6)• Receptor desensitization (phos/intern.)

Chemokines Act as Tethered Ligands

CKR

CK

Tethering mechanism: Genetic or GAGs

*Expressed on the surface of neurons, activated vascular endothelium, and in atherosclerotic lesions

*Chemoattracts monocytes, NK cells, CD8+ T

*Free and membrane bound forms exist

*Membrane-bound form mediates strong integrin-independent adhesion

*Both forms act at CX3CR1

Fractalkine (CX3CL1): an exceptional chemokine

Adapted from Rossi, et al. Genomics 1998

a b c d e f

Endothelium

WBC

aRbR

cR

Area Code forLeukocyteTrafficking

ChemoattractantsSelectins

Integrins

Stage

Flow

Rolling

Firm Adhesion

Transendothelial Migration

Activator

none

IL-1, TNF, lps

Chemoattractant

Chemoattractant

Adhesins

none

Selectins

Integrins

PECAM

Disease

none

LAD-2

LAD-1

LAD-1, LAD-2

Extravascular Space

Intravascular Space

Degranulation Chemoattractant none SGD

Multistep Paradigm for Leukocyte Trafficking

•Inflammatory

•Innate (inducible L -- constitutive R)•CXCR1 & CXCR2 Neutrophils•CCR2, CCR5 Monocytes•CCR3 Eosinophils

•Adaptive (inducible L – inducible R)•CXCR3 •CCR2 •CCR5 •CCR3•CCR9-gut homing•CCR4, CCR10-skin homing

•Homeostatic (constitutive L – constitutive R)

•CXCR4 Bone marrow•CXCR5 B cell homing to LN•CCR7 mDC & T cell homing to LN

Classification of the Chemokine System by Immunologic Function

Homeostatic

CXCL12 Bone Marrow. BCXCL13 LN-BCCL19 LN-TCCL21 LN-TCCL25 gut homingCCL27 skin homingCCL28 mucosal homing

Inflammatory

CXCL1-8 PMNCXCL9-11 Th1CCL2-5, 11 M, Eos, T, NKCX3CL1 M, Th1, NK

Classification of Chemokines by Immune Function

Lumbar Nodes

Axillary nodes

Tonsils

AdenoidsCervical Nodes

Thoracic Duct

L. Subclavian Vein

L. Lymph DuctL. Subclavian Vein

Peyer’sPatches

Appendix

Thymus Bronchial Nodes

Bone Marrow

Intercostal Nodes

Peyer’s Patches

Spleen

Iliac Nodes

Inguinal Nodes

Chemokine Receptors Regulate Effector T Cell Balance

CXCR3CX3CR1CCR1CCR2CCR5

CCR1CCR3CCR4CCR8

Th1

Th2

Host defenseAngiogenesis

chemokines

Autoimmune Disease

Hematopoiesis

InfectionAtherogenesis

Inflammation

Neoplasia

LHD

CHEMOKINES: A DOUBLE-EDGED SWORD

Chemokines/Receptors with Roles in Human Disease

CCR5 HIV/AIDS, WNV Duffy P. vivax malaria CXCR4 WHIM syndrome CXCL4 Heparin-induced thrombocytopenia(CXCR4) HIV/AIDS(CX3CR1) Atherosclerosis

Phenotypes Associated with Chemokine or Chemokine Receptor Deficiency

Abnormal Development: SDF-1/CXCR4

CXCR2CCR7/SLCCXCR5/BLC

Modulation of Inflammation

EotaxinMIP-1CCR1, 2, 3, 4, 5, 8CXCR2, 3CX3CR1

Altered susceptibility to infection

CCR1, 2, 3, 5CXCR2Duffy

Duffy and Malaria• 1950s: serologically defined as a minor blood group antigen

• 1975: pathologically defined as a virulence factor for P. vivax. Duffy is absent from the red cells of most black Africans who are thereby resistant to vivax malaria.

• 1993: molecularly defined as a 7TMD protein, and a promiscuous chemokine binding protein, binding several but not all members of both the CC and CXC groups. The Duffy mutation is in a GATA site in the promoter.

• expressed in Purkinje cells of cerebellum, post-capillary venule endothelial cells and CD45RO+ T cells

• biochemical and physiological roles undefined

Angiogenesis is Regulated by the Balance of Angiogenic and Angiostatic CXC Chemokines

ELR Non-ELR

Angiogenesis

+ -

Wound Healing Chronic Inflammation

Cancer

IFN- +-

Classification of Viral Chemokine Mimics•Structure

-chemokine-7TMD protein-unique

•Function-anti-chemokine-chemoattractant-gene expression-growth factor-membrane fusion factor (cell entry)

Receptor antagonistLigand scavenger

Host ChemokineReceptors (7TM)

Host ChemokineBinding Proteins (7TM)

Viral Chemokine Binding Proteins (non-7TM)

Viral Chemokine Receptors (7TM)

Human & Viral Orphan Receptors

Host Chemokines

(-)

?

(-)

(+)

(+)

Non-chemokineViral Ligands for

Chemokine Receptors

Viral Chemokines

Non-chemokine Host Ligands for

Chemokine Receptors

(+/-)

(+/-)

(+/-)

The Extended Chemokine Family

Host Chemokine Receptors

HIV-1 Infection

HIV-1 Progression

HIV and Chemokine Mimicry

TCL PBMC M

CD4 CD4 CD4

T

HIV-1 Cytotropism and Pathogenesis

AIDSInitial Infection

M

M-tropicT-tropic

HIV-1

EnvHIV-1

Target Cell

gp120 gp120

gp

41 g

p4

1

Target Cell

CD4

Chemokine Receptor

gp

12

0

Genome

gp41

Exploitation of Chemokine Receptors by HIV

Target Cell

CXCR4 CCR5

CD4

CD4

CXCL12

CCL3CCL4CCL5

X4

HIV

R5

12

34

5CCR532

CCR5 & CXCR4 and HIV Pathogenesis

RBDEU HIV+

Fre

qu

ency

of

CC

R5

-/-

(%)

Comparison Groups

5

4

3

2

1

CCR5 is Required for Efficient HIV-1 Transmission: Evidence from Population Studies of the Inactive CCR5 Allele, CCR5 delta 32

(~ZERO)

p<0.001

n= 111 387 614

Chemokines & Cancer Metastasis

Breast Blood Target Organ

Murphy (2001) NEJM; 345:833-835