Adhesion Molecules And Lymphocyte Homing · 2019. 10. 25. · J. Exp. Med. 191:61, 2000 CCL21 is Expressed by Lymph Node HEV in Wild Type But Not plt/plt Mice WT plt/plt HEV express

Adhesion Molecules And Lymphocyte Homing

Sharon S. Evans, Ph.D. Dept. Immunology, RPCI Jan. 29 & Feb 3, 2015

Outline

General introduction to lymphocyte trafficking – homeostatic

recirculation vs active recruitment.

Homeostatic trafficking of lymphocytes across specialized high

endothelial venules (HEVs)

Structure/function of adhesion molecules involved in initial tethering/rolling

(L-selectin/PNAd)

Structure/function of chemokine/chemokine receptors that mediate activation

(CCR7/CCL21)

Structure/function of adhesion molecules required for firm

arrest/transendothelial migration (LFA-1/ICAM-1 & α4β7 integrin/MAdCAM-1)

Leukocyte recruitment in acute and chronic inflammation.

Lymphocyte Extravasation in LN HEV

L-Selectin/PNAd-1

Chemokines (CCL21)

LFA-1/ICAM-1/2 1

2

3

Initial

Attachment Rolling Firm

Adhesion Transmigration

CCL21

L-selectin

PNAd

CCR7 LFA-1

ICAM

LN HEV

The CC Chemokine SLC/6CKine/CCL21

Mediates Tissue-Specific Homing

1

2 3&4

C

CXXXC

Chemokines

CXC C CC

C

C N

C

C

N CC

C

CXC

C

C

CXXXC N

CCL (ligand)

• CCL21 (SLC)

CCR (chemokine receptor)

• CCR7

CXCR (chemokine receptor)

• CXCR4

CXCL (ligand)

• CXCL12 (SDF-1)

N

COOH +++ •••• +++ •••• COOH

Structure of CC Chemokines

CCL21

C

C

NH2

COOH +++ ••••

CC

α-helixβ

Anti-parallel

β-pleated sheet

3-dimensional structure at N-

terminus required for binding to

chemokine receptor

Basic (positively charged)

amino acids in C-terminus

required for binding to

glycosaminoglycans (heparin

sulfate) that make up

glycocalyx on endothelial cells

(CXCL12)

(CCL19, CCL21)

(CXCL13)

Chemokine

(CCL21)

Chemokines Stimulate Chemotaxis and Migration

5 mm pore

Transwell

Migration

Assays

Chemokine

(CCL21)

Transwell

Migration

Assays

Chemokines Stimulate Chemotaxis and Migration

5 mm pore

ICAM

CCL21

L-selectin

PNAd

CCR7 LFA-1

HEV

Tissue specific localization

of CCL21 in HEV

Evidence that CCL21 is a Tissue Specific Homing Chemokine

CCL21 mRNA

Capsule

B cell area

(follicle)

T cell area

(paracortex)

HEV

Lymph Node

CCL21 (SLC) is Expressed by Lymph Node HEV

PNAd CCL21

WT+/+

plt/plt

J. Exp. Med. 191:61, 2000 Science 286:2098, 1999

BCL/CXCL13 CCL21/SLC

BCL/CXCL13 – binds CXCR5 (involved in B cell migration)

SLC/CCL21 – binds CCR7 (mediates B & T cell homing across HEV

In Situ Analysis (mRNA) IHC Analysis (protein)

In vivo Imaging of CCL21 Localization on

PLN HEV by Intravital Microscopy (IVM)

VCR

PC

Adapted from von Andrian et al, Curr. Op. Immunol. 2004

Femoral Artery

Injection of FITC-labeled anti-CCL21 Ab)

Inguinal LN

J. Exp. Med. 191:61, 2000

CCL21 is Expressed by Lymph Node HEV

in Wild Type But Not plt/plt Mice

WT plt/plt

HEV express CCL21 [SLC] mRNA and protein

HEV of plt/plt mice lack CC21 [SLC] and CCL19

[ELC/MIP3b); can’t support firm adhesion or homing

Reconstitution of CCL21 in HEV of plt/plt mice restores

firm adhesion

T cells from CCR7-/- suppressed in ability to enter LN, PP

Chemokine desensitization of CCR7 (using high

concentrations of CCL21) blocks arrest of rolling cells

Evidence that CCL21 and CCR7 receptor critical for T

lymphocyte extravasation across HEV

GDP GTP Gai

Gb

Gg

GTP Gai

Signaling

Ca++

Proliferation

Differentiation

Migration Adhesion to

HEV, EC, or ECM

Gene

Transcription

Cytoskeletal

Reorganization

Chemokine (CCL21)

Chemokine Receptor

(CCR7)

PTX

Gb

Gg

Chemokine (CCL21)

PI3K

ERK

40

80

Ce

lls

/ U

nit

Are

a

*p < 0.0001

Short-Term Homing Assay (PLN)

CCL21 Is Required for Lymphocyte Trafficking Across HEV

HEV

PTX

*

PTX : - +

*

-

-

- - + - CCL21 mAb :

CCL21 mAb

CCR7

*

-

CCR7 Desens: - + -

Chen et al., Nature Immunology, 2006

Outline






(L-selectin/PNAd)


(CCR7/CCL21)




1

2

3

Tethering/Rolling • L-selectin/PNAd

• α4β7/MAdCAM-1

Activation • CCL21/CCR7

Sticking • LFA-1/ICAM-1/2

Transmigration 4

Adapted from Immunol. Rev. 2002

Dynamic Regulation of Leukocyte-Endothelial Adhesion

HOMING

HEV

LAD – Leukocyte Adhesion Deficiency

LAD patient leukocytes do not express LFA-1 (leukocyte-function

associated antigen-1) – cannot make b2 integrin chain

TIME MAGAZINE 1992

Nature VOL 346 – 2 August 1990

Integrin Family

a4b1 a4b7 aLb2

Butcher and von Andrian Cell 1995

LFA-1 TK1 cells (low mag)

LFA-1 is Localized on Planar Membranes of Lymphocytes

Nature VOL 346 – 2 August 1990

Ig Superfamily

LFA-1 is Required for Lymphocyte Homing

Across HEV of Lymphoid Organs

PLN MLN PP SPL Pancreas

150

250

350

Isotype Control LFA-1 Blocking Ab

* * * 50

Cells /

Un

it A

rea

Quantify red-

labeled cells in

organs

1 h

TRITC-

labeled

cells

Treat +

LFA-1 blocking

Ab

Chen et al., Nature Immunol. 2006

LFA-1 Binds to Domain 1 (D1) of ICAM-1 Dimers

Y Yang, Molecular Cell 14:269-276, April 23, 2004

D4-D4 dimer D4-D4 dimer

D1-D1 dimer

ICAM-1

LFA-1

D1

D2

D3

D4

D5

ICAM-1

D1

D2

D3

D4

D5

ICAM-1

Extended

High affinity Folded

(

1

2

3

Tethering/Rolling • L-selectin/PNAd

• a4b7/MAdCAM-1

Activation • CCL21/CCR7

Sticking • LFA-1/ICAM-1/2

Transmigration • LFA-1/ICAM-1/2

4

Adapted from Immunol. Rev. 2002

Dynamic Regulation of Leukocyte-Endothelial Adhesion

HOMING

HEV

Carman and Springer, J. Cell Biol. 2004

Shaw et al., J. Exp. Med. 2004

Yang et al., Blood 2005

LFA-1

ICAM-1

ICAM-1

LFA-1

Endothelial Cell

Adherent

Leukocyte Cup-like

Structure (forms in min.)

ICAM-1-Enriched Microvilli-Like Projections Form Cup-Like Structure that Guides Transendothelial Migration

Lymphocyte-Endothelial Adhesion in Peyer’s Patches

CCL21

L-selectin CCR7 LFA-1

ICAM-1/2

MAdCAM-1

HEV

MAdCAM-1

a4b7 integrin

a4b7 integrin

1 2 3&4

Briskin, Butcher et al. Nature 363:461, 1993

Mucosal Addressin Cell Adhesion Molecule-1

(MAdCAM-1)

MAdCAM-1 staining

of PP HEV with

MECA-367 mAb

MAdCAM-1 structure

DE antenna Ig

Domain

2

Ig

Domain

1

Ig

Domain

3

Mucin

Domain

GLDTSL

MAdCAM-1

Glycocalyx

10 nm

L-selectin Lectin

I domain

Mg++

b 7

a 4 XXXX b propeller

Function blocking mAb

MEL-14, DREG-56

Function blocking mAb

DATK32, HP1/2

Lymphocyte

Microvillous

Membrane HEV

MIDAS

Ca++

Lymphocyte-Endothelial Adhesion in Lymph Nodes

CCL21

L-selectin

PNAd

CCR7 LFA-1

ICAM-1/2

HEV

1 2 3&4

Homing Receptors Direct Lymphocyte Trafficking

L-selectinhi

a4b7+ LFA-1+

CCR7+

Naïve Central Memory

L-selectinneg

a4b7hi LFA-1hi

CCR7neg

CLA+

CCR5/CXCR3+

Effector Effector Memory

Inflamed Tissues

(Autoimmune pathology)

Infection, Inflammation Tumor Tissues

Lymphoid Tissues

(LN, PP)

Extralymphoid Sites

Girard JP et al., Nat Rev Immunol. 12:762-73, 2012

Current View of Lymphocyte

Trafficking in PLN HEVs

Girard JP et al., Nat Rev Immunol. 12:762, 2012

Moussion C and Girard JP, Nature 13;479, 2011

DCs Maintain Homeostatic Trafficking in HEVs

Outline






(L-selectin/PNAd)


(CCR7/CCL21)




Lymphocyte Recirculation and Recruitment

PLN (HEV)

Peyer’s Patches

(HEV)

Spleen

(no HEV)

Trafficking to

extralymphoid sites

(HEV-like):

• Low basal level

• Active recruitment

induced by infection,

injury, acute & chronic

inflammation

Recruitment Recirculation

Efficient trafficking to

lymphoid organs:

Dynamic Regulation of Endothelial Adhesion at

Sites of Infection/ Injury/ Inflammation

Control

ICA

M-1

Inflammation

Chen, et al. Nature Immunol. 2006

Role of cell adhesion receptors in inflammation.

An inflammatory response to foregin antigens of

bacteria begins with the attraction of leukocytes

that release biologically active mediators (A).

These cytokines or bacterial products stimulate the

expression of adhesion molecules such as ICAM-1,

E-selectin, P-selectin, and VCAM-1 on the surface

of cuboidal endothelial cells (B). White blood cells

then adhere to the activated endothelial cells,

migrate through the endothelium and enter areas of

infection or tissue damage (D). (•) Up-regulated

adhesion receptors on the surfaces of cells; Endo,

endothelium.

Local Cytokines Increase Lymphocyte Recruitment at Inflammatory Sites

TNF-a

IL-1b

IL-6

IFN-g LTab

Sites of Infection, Local Tissue Damage

Transcription/

Expression of

Chemokines &

Vascular Adhesion

Molecules

• ICAM-1

• VCAM-1

• E/P-selectin

• CXCL9/10

• CCL5

- 4.4

- 2.4

- 1.4

- 4.4

- 2.4

- 1.4

1 2 24 48 72 0 Hrs:

VCAM-1

E-selectin

mRNA Expression in Human Umbilical Vein Endothelial

Cells (HUVEC) Following TNF-a Treatment

VCAM-1 Terminal Ig domains

Lectin, EGF Repeat

E-selectin

Nuclear Translocation

RIP

TNF-α

TNFR

TRADD

TRAF2

IKK

IκB

NF-κB

MEKK3 P

P

VCAM-1 ICAM-1 E-selectin

κBBE

Cytoplasm

Nucleus

IκB P

Endothelium

Taming the Flames Within FEBRUARY 17, 2004

Jalkanen, Butcher et al.

Science Vol. 233:556, 1986

RA

Venules Lined by High Endothelium in Synovium of Rheumatoid Arthritis Patients

Chronic Inflammatory Diseases

Associated With HEV-Like Vasculature

Disease Affected organ Plump CAMs

endothelium on endothelium

Crohn’s disease Gut + MAdCAM-1/PNAd/ICAM-1

Ulcerative colitis Gut + MAdCAM-1/PNAd/ICAM-1

Diabetes mellitus Pancreas + MAdCAM-1/PNAd/ICAM-1

Grave’s disease Thyroid + PNAd/ICAM-1

Hashimoto’s thyroiditis Thyroid + PNAd/ICAM-1

Rheumatoid arthritis Synovium + PNAd/ICAM-1

Cutaneous lesions Skin + PNAd/ICAM-1

Sjogren's syndrome Lacrimal/salivary glands + PNAd/MAdCAM-1/ICAM-1

Adapted from J.P. Girard and T.A. Springer, Immunol. Today 16:449 1995;

S.D. Rosen, Annual Rev. Immunol. 22:129, 2004.

IL-8/MIP2 KC

CXCR2

1 2

Mechanisms of Neutrophil Recruitment to Inflamed Tissues

E/P-Selectin

LFA-1

ICAM-1

Mucin-like CAM

PMN

Endothelium

3&4

Intravital Microscopy

Ischemia-Reperfusion Injury

Control: Ischemia: Reperfusion:

Cells move through venules at high velocity (~2400 mm/sec); few adherent cells. Reduced blood flow; distinct cells visible. Increased number of rolling (~40 mm/sec) and adherent cells; adhesion is L-selectin and P-selectin dependent.

R. Hynes, Cell 68:303-322, 1992

Process of Metastasis of Malignant Cells Involves Similar

Adhesive Mechanisms As Lymphocyte Trafficking

Outline






(L-selectin/PNAd)


(CCR7/CCL21)




Documents

Adhesion Molecules And Lymphocyte Homing · 2019. 10. 25. · J. Exp. Med. 191:61, 2000 CCL21 is Expressed by Lymph Node HEV in Wild Type But Not plt/plt Mice WT plt/plt HEV express