Modulatory role for B (and T) cells in ischemic reperfusion injury Hamid Rabb MD Physician Director, Kidney Transplant Program The Johns Hopkins Hospital.

Modulatory role for B (and T) cells in ischemic reperfusion injury

Hamid Rabb MD Physician Director, Kidney Transplant Program

The Johns Hopkins Hospital

Acknowledgements

• Lorraine Racusen• Wink Baldwin• Robert Colvin• Dolores Ascon• Shannon Bevans• Roshni Molls• Manchang Liu

• Mark Haas• Melissa Burne-Taney • Naoko Yokota• Vladimir Savransky• Carmelo Mendiola• Gilbert Postler• Niamh Kieran

Outline

• Importance of ischemia reperfusion injury (IRI) in transplantation

• Cellular inflammation during IRI

• Role of T lymphocytes in kidney IRI

• Role of B lymphocytes in kidney IRI

• Role of B lymphocytes in intestinal IRI

High incidence of kidney ischemia reperfusion injury

Delayed graft function occurs in 30% of deceased donor (cadaver) kidney transplants (50% at Hopkins-ECD population), which predisposes to acute rejection and chronic allograft nephropathy (NEJM 1995)

All deceased donors have some degree of significant IRI

No specific therapy

ischemic injury

anti-donor Ab

(preexisting)

complement

platelets

toxins

NK cells

T cells

macrophages

pathogenic models

Endothelium + other cells

ischemia hypoxia

accommodation

acute humoral rejection

antigen

antibody

B cells T cells

Ischemia/hypoxia is a major pathophysiologic process during antibody mediated rejection

NIH antibody rejection workshop AJT 2004

VasculatureVasoconstrictionVasodilationThrombosisCoagulationInflammationPluggingNo Reflow

TubulesLoss of PolarityApoptosisNecrosisDetachmentBack-leakObstruction

Pathophysiologic processes in ATN

Wound

Bacteria

MacrophagesNeutrophils

Blood vessel

Skin barrierNK cells

Activation ofserum complement

Cytokines and Chemokines

Phagocytosis and destructionof bacteria by macrophages

and neutrophils

Dendritic cells

Kidney Int 2002

Leukocyte adhesion in renal IRI

CD11/CD18 - ICAM-1PNAS 94, Am J Physiol 94, BBRC 95, JCI 96, KI 97...

SelectinJASN 94, Am J Physiol 96, JCI 97, FASEB 2000,

KI 2001 (selectin ligand), J Immunol 2002(selectin ligand fucose groups)

Assumption: working solely via neutrophils

Role of CD11/CD18 and neutropenia on ischemic ARF.

Mendiola & Rabb, J Am Soc Nephrol 4:741 1993

Neutropenia induced to >98% depletion

No benefit on course of ARF with 60 min unilateral ischemia

CD11/CD18-ICAM-1 pathway blockade protective in same experiment

Human ischemic acute renal failure

• 58 year old one week post cadaveric renal transplant and long ischemia time with no evidence of rejection or calcineurin-inhibitor toxicity. Small arrow: erythrocytes rouloux and sludging in peri-tubular capillaries. Large arrow:marginating leukocytes, mostly mononuclear cells.

Kidney Int 2004

T cell staining in human ischemicacute renal failure

• Immunoperoxidase stain from the same kidney showing that some, but not all of the leukocytes stain positive for CD3.

Kidney Int 2004

Is there data to support the hypothesis that lymphocytes play an important role

in renal IRI?

Sparse lymphocytes are found in the postischemic kidney (Solez)

CTLA-4 Ig (against T cell costimulation) decreases cold ischemic injury (Takada J Clin Invest 1997, Chandraker Kidney Int 1997, B7-1 pathway important in rat model of ARF (DeGreef, Ysebart, De Broe Kidney Int 2001)

Lymphocyte related cytokines are upregulated in the postischemic kidney (Goes/Halloran et al: Transplantation 1995, Lemay et al: Transplantation 2000)

T lymphocytes mediate murine liver IRI (Zwacka et al: J Clin Invest 1997)

CD4 VLA-4CD3

T CELLRECEPTOR

HLAVCAM-1

ICAM-1

T Lymphocyte

Antigen Presenting CellSelectinLigand

SelectinLFA-1

Peptide

ICAM-1 and CD11/CD18 (LFA-1) also mediate T cell adhesion and function

Hours Postischemia

0 24 48

Se

rum

Cre

ati

nin

e (

mg

/dL

)

0

1

2

3

*

control C57 CD4/CD8 (-/-)

CD4/CD8 (-/-) have an improved course of ischemic ARF

Am J Physiol Renal 2000

Nu/nu mice are protected from renal IRI

Hours Postischemia

24 48 72

Se

rum

Cre

ati

nin

e (

mg

/dL

)

0

1

2

3

4

*

Control C57 nu/nu

J Clin Invest 2001

Adoptive transfer of T Cells into nu/nu mice reconstitutes injury phenotype

Hours Postischemia

24 48 72

Seru

m C

reati

nin

e (

mg

/dL

)

0

1

2

3

4

*

Control C57BL/6 nu/nu Transfer nu/nu

J Clin Invest 2001

Nu/nu mice have reduced tubular necrosis following renal IRI

A B

C D

J Clin Invest 2001

0

1

2

3

0 24 48 72

Hours postischemia

Ser

um

cre

ati

nin

e [m

g/d

L]

Sham

Double Abdepletion

Triple Abdepletion

*

Effects of T cell depletion on renal IRI

Transplantation 2002

0

20

40

60

80

100

120

140

-5 0 5 10 15 20 25 30min

Mea

n ar

teria

l blo

od p

ress

ure

(mm

Hg)

Blood pressure during murine cardiac arrest and CPR

Am J Physiol 2003

Hours Postischemia

0 24 48

Se

rum

Cre

atin

ine

(m

g/d

L)

0

1

2

3

4

**

T cell deficient mice have significant protection from acute renal failure after cardiac arrest and CPR

wild type CPR T cell deficient CPR wild type sham arrest T cell deficient sham arrest

Am J Physiol 2003

Evidence for T cell modulation of IRI in lung and liver

• Am J Transp 2002 FTY 720 liver

• Hepatology 2003 Stat 4 & Stat 6 liver

• Am J Path 2003 Fibronectin-VLA-4 liver

• Mol Ther 2004 CD40Ig liver

• J Immunol 2003 rat lung tx model

Hours Postischemia

0 24 48 720

1

2

3

4 C57BL/6 wild typeMu (B cell deficient)

Se

rum

Cre

atin

ine

(m

g/d

L)

B cell deficient mice are protected from renal injury following IRI

J Immunol 2003

Normal wild-type Wild-type B cell-deficient

J Immunol 2003

Hours after ischemia

0 72

Un

ite

s M

PO

/g p

rote

in

0

50

100

150

200

250

300

350

400Wild-type MT

Phagocyte Infiltration

J Immunol 2003

Renal complement (C3d) deposition is similar post ischemia in wild type and B cell deficient mice

WT WT C3d B-/- C3d

J Immunol 2003

Hours Postischemia

0 24 48 72

Ser

um C

reat

inin

e (m

g/dL

)

0.0

0.4

0.8

1.2

1.6

2.0

2.4

MTB cell transferWild-typeSerum transfer

#

* **

Serum transfer, but not B cell transfer, partially restores injury response in B cell deficiency

J Immunol 2003

Hours Postischemia

0 24 48 72

Se

rum

Cre

atin

ine

(m

g/d

L)

0.5

1.0

1.5

2.0

2.5

3.0

* **

Hours Postischemia

0 24 48 72

Se

rum

Cre

atin

ine

(m

g/d

L)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

* *

RAG-1 (T and B cell deficient) mice are not protected postischemia in both C57BL/6 and BALB/c background

strains

RAG-1 deficient mice Wild type mice T cell deficient (nude mice)

Am J Transplant 2005

Am J Transplant 2005

Am J Transplant 2005

Hours Postischemia

0 24 48 72

Seru

m C

reatin

ine (

mg/d

L)

0

1

2

3

4

*

**

B or T cell transferred RAG-1 deficient mice were protected from renal IRI compared to RAG-1 deficient mice that did not receive a

transfer of cells

RAG-1 deficient B cell transferred RAG-1 deficient T cell transferred RAG-1 deficient

wt mice Am J Transplant 2005

-+- RAG-1 + B cell

--+ RAG-1 + T cells

--- RAG-1 knockout

+-+ B cell deficient

++- T cell deficient

-++ Wild type

ProtectionB cellT cell Mouse strains

Am J Transplant 2005

Proc Natl Acad Sc 2004

Specific IgM mediates intestinal IRI in mice

LOCAL DISTANT

Thymus

Lymphnodes

SpleenBone Marrow

Kidney

BLOOD VESSELMacrophages

Neutrophils

Endothelial cells

INTERSTITIUM

EPITHELIUM

Platelets

Collagen

T Cell

T Cell

T Cell

Cytokines and chemokines

Kidney Int 2002

B Cell

Summary

• IRI is a major cause of delayed graft function, increases graft immunogenicity, and decreases long term function

• Ischemic injury is a final mediator of antibody mediated rejection

• T cells are a newly identified mediator of IRI

• B cells are a newly identified mediator of IRI

• T and B cells may interact for full expression of IRI

• Role of T and B cells in IRI has similarities and differences between kidney and intestine

• B and T cells are a novel therapeutic target for IRI

• The mechanisms underlying the role for lymphocytes in IRI is largely unknown.