Alloantibody-Mediated Alloantibody-Mediated Rejection:Rejection:

Complement and Complement and non-Complement non-Complement

Dependent MechanismsDependent Mechanisms

B.A. WasowskaB.A. WasowskaJHMIJHMI

Specific Specific recognition of MHCAntibodiesAntibodies

ComplementComplementindependentindependent

FcRcytokines

Endothelial cellsEndothelial cells

MacrophageMacrophage

Specific Specific recognition of MHCAntibodiesAntibodies

ComplementComplementdependentdependentComplementComplement

independentindependent

FcR CRscytokines

cytokines

Endothelial cellsEndothelial cellsC4 C3

MacrophageMacrophageMacrophageMacrophage

Heart donors Recipients Incompatibilities

B10.A C57BL/6 H-2, H-9

IgKOIgKO ( (-/-/-)-)passively transferred with:

mAbsmAbs C activationC activationIgG2b yesIgG1 noIgG2b+IgG1 yes

Experimental DesignExperimental Design

Passive Transfer of Passive Transfer of IgG1IgG1 and and IgG2bIgG2b to to IgKO Recipients of Cardiac AllograftsIgKO Recipients of Cardiac Allografts

0102030405060708090

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Days after Transplantation

% S

urv

ival

IgKOWTIgG2b 50-200IgG2b 25IgG1 100 mAb

Passive Transfer of Passive Transfer of IgG1IgG1 and and IgG2bIgG2b to to IgKO Recipients of Cardiac AllograftsIgKO Recipients of Cardiac Allografts

0102030405060708090

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Days after Transplantation

% S

urv

ival

IgKOWTIgG2b 50-200IgG2b 25IgG1 100IgG2b + IgG1 mAb

Endothelial CellsEndothelial Cells

Pro-Inflammatory Effects of anti-Pro-Inflammatory Effects of anti-MHC Antibodies MHC Antibodies in the Absence in the Absence

of Complementof Complement

Anti-MHC Ab

Contribution of Anti-MHC antibodies to graft Contribution of Anti-MHC antibodies to graft rejection and activation of ECrejection and activation of EC

Ligation of MHC class I molecules with anti-MHC Ligation of MHC class I molecules with anti-MHC antibodies on EC and smooth muscle results in:antibodies on EC and smooth muscle results in:

• Increased phosphorylation of intracellualr proteins and induction of fibroblast growth factor receptor on EC. (Bian, Reed. JI, 1999, 163: 1010).

• Increased phosphorylation of Src, Paxillin and Focal Adhesion Kinase and enhanced proliferation to fibroblast growth factor. (Jin,…Reed. JI, 2002, 168: 5415).

• Activation of P13K/Akt signaling pathway and regulation of apoptotic genes (Bad, Bcl-2 and Bcl-xL). (Jin, Fishbein,...Reed, Human Immunol. 2004, 65: 291).

Experimental Design:Experimental Design: EC Activation EC Activation in vitroin vitro

MethodsMethods:: Addition of different IgG subclasses to mouse endothelial cell line SVEC4-10SVEC4-10

MeasurementMeasurement:: cytokine production by EC in 48 hr cultures

Cytokine Array on Culture SupernatantsCytokine Array on Culture Supernatants

Endothelial CellsStimulated by IgG1 + IgG2bIgG1 + IgG2b

Endothelial Cells

Macrophages

Anti-MHC Ab

MHC

What Effects Do Antibodies Have on What Effects Do Antibodies Have on Endothelial Cells and Endothelial Cells and LeukocytesLeukocytes in the in the

Absence of Complement?Absence of Complement?

?

Cytokine Array on Culture Supernatants

Endothelial CellsStimulated by IgG1 + IgG2b

Peritoneal andEndothelial CellsStimulated by IgG1 + IgG2b

ELISA for MCP-1 and IL-6 in ELISA for MCP-1 and IL-6 in Cultures of Endothelial Cells+ Cultures of Endothelial Cells+

Peritoneal CellsPeritoneal Cells

0

10

20

30

40

Con

cent

rati

on n

g/m

l

Med alone G1+2b alone Med+Mac G1+2b+Mac

0

1

2

3

4

MCP-1 IL-6

ELISA for MCP-1 and IL-6 in Cultures of ELISA for MCP-1 and IL-6 in Cultures of Endothelial Cells + Endothelial Cells + Alloantigen-primedAlloantigen-primed

SPL from Heart RecipientSPL from Heart Recipient

0

10

20

30

40

50

60

Con

cent

ratio

n ng

/ml

Medium alone G1+2b alone Medium+SPL G1+2b+SPL

0

2

4

6

MCP-1 IL-6

Endothelial Cells

Macrophages

FcFcRR

What Effects Do Antibodies What Effects Do Antibodies Have on Have on FcFcR+R+ Cells in the Absence of Cells in the Absence of

Complement ?Complement ?

????

Possible Involvement of Possible Involvement of FcFcR IIIR III in in IgG1-Mediated Cell Activation in the IgG1-Mediated Cell Activation in the

Absence of ComplementAbsence of Complement

WT and FcFcR III KOR III KO C57BL/6 splenocytes and lymph node lymphocytes primed in vivo by 10-day exposure to B10.A B10.A skin transplantskin transplant were co-cultured with mouse endothelial cells in the presence of IgG1 Ab-s.

Receptor The affinity of binding

FcFcRIRI 107-108 M-1 for IgG2aIgG2a>>IgG1>IgG2b

FcFcRIIIRIII <107 M-1 for IgG1IgG1>Ig2a>IgG2b

The Affinity ofThe Affinity of Mouse Mouse FcFcRsRs to Bind to Bind IgG SubclassesIgG Subclasses

ELISA for ELISA for IL-6IL-6 in Cultures of Endothelial in Cultures of Endothelial Cells + Cells + IgG1IgG1 + Lymph Node Cells from WT + Lymph Node Cells from WT

and and FcFcRIII RIII KOKO

0

100

200

300

400

IL-6

pg/

ml

Ab WT Med WT Ab KO Med KO

Naive Tx

EC aloneWT FcR KO WT FcR KO

ELISA for ELISA for MCP-1MCP-1 in Cultures of in Cultures of Endothelial Cells + Endothelial Cells + IgG1IgG1 + Lymph Node + Lymph Node

Cells from WT and Cells from WT and FcFcRIII RIII KOKO

0

20

40

60

80

100

120

140

MC

P-1

ng/

ml

Naive Tx

Ab WT Med WT Ab KO Med KO

EC aloneWT FcR KO WT FcR KO

• There is effective interaction between There is effective interaction between mouse endothelial cells, alloantibodies mouse endothelial cells, alloantibodies and alloantigen-primed effector cells.and alloantigen-primed effector cells.

• FcFcR IIIR III participates in IgG1-mediated participates in IgG1-mediated activation of mouse effector cells.activation of mouse effector cells.

SUMMARYSUMMARY

MHC

What Effects Do Antibodies Have on What Effects Do Antibodies Have on HumanHuman Endothelial Cells and Endothelial Cells and

HumanHuman FcFcRI+RI+ Cells?Cells?

?

BMECBMEC

U 937U 937Anti-MHC AbW6/32 (IgG2a)

FcFcRIRI

Receptor The affinity of binding

FcFcRIRI 108-109 M-1 for IgG2aIgG2a=IgG3>>>IgG1, IgG2b

FcFcRIIIRIII 107 M-1 for IgG3>IgG2aIgG2a> IgG2b >> IgG1

The affinity of human The affinity of human FcFcRsRs to bind mouse to bind mouse IgG subclassesIgG subclasses

Expression of CD 64 (Expression of CD 64 (FcFcRIRI) on U-937 ) on U-937 Macrophages after Stimulation with IFN-Macrophages after Stimulation with IFN-

Iso ctr

CD 64

Iso ctr

CD 64

Iso ctr

CD 64

CTR Mac IFN- 24 hr IFN-48 hr

ELISA for MCP-1 and IL-6 in Cultures of ELISA for MCP-1 and IL-6 in Cultures of Human BMEC + anti-HLA Ab (IgG2a) + Human BMEC + anti-HLA Ab (IgG2a) +

IFN-IFN--Stimulated U-937 Macrophages-Stimulated U-937 Macrophages

0

1000

2000

3000

4000

5000

MC

P-1

pg/

ml

MCP-1

0

1000

2000

3000

4000

5000

6000

7000

IL-6

pg/

ml

IL-6

Mac IFN Mac CTR Mac IFN Mac CTR

W6/32 Iso CTR

SUMMARYSUMMARY

• Two lines of evidenceTwo lines of evidence, in mouse and human , in mouse and human models of EC sensitized with anti-MHC models of EC sensitized with anti-MHC antibodies, suggest that:antibodies, suggest that:

• FcFcR R participate in participate in IgGIgG-mediated activation -mediated activation of EC.of EC.

Specific Specific recognition of MHCAntibodiesAntibodies

ComplementComplementindependentindependent

FcRcytokines

Endothelial cellsEndothelial cells

MacrophageMacrophage

Specific Specific recognition of MHCAntibodiesAntibodies

ComplementComplementdependentdependentComplementComplement

independentindependent

FcR CRs

MacrophagesMacrophages

cytokines

cytokines

Endothelial cellsEndothelial cellsC4 C3

MacrophagesMacrophages

Complement Receptors for C4 and C3Complement Receptors for C4 and C3

ReceptorReceptor LigandsLigands Cell expressionCell expression

CR1CR1 C3b>C4b>iC3bC3b>C4b>iC3b Monocytes, Monocytes, Macrophages, PMN, Macrophages, PMN,

B, T cells, RBC, B, T cells, RBC, FDCFDC

CR2CR2 C3d/C3dg>iC3bC3d/C3dg>iC3b B cells, FDCB cells, FDC

CR3 CR3 (Mac-1)(Mac-1)

iC3b>C3b, iC3b>C3b, ICAMICAM

Monocytes, Monocytes, Macrophages, PMN, Macrophages, PMN,

NK cellsNK cells

Possible Interactions ofPossible Interactions of iC3b iC3b and and C3bC3b with with CR3CR3 in IgG1+IgG2b-Mediated Cell in IgG1+IgG2b-Mediated Cell

Activation in the Activation in the Presence of ComplementPresence of Complement

WT and CR3 KOCR3 KO C57BL/6 peritoneal macrophages were co-cultured with mouse endothelial cells in the presence of IgG1+Ig2b mAb-s +Complement.+Complement.

ELISA for MCP-1 and IL-6 in Cultures of ELISA for MCP-1 and IL-6 in Cultures of Endothelial Cells + IgG1+IgG2b +Endothelial Cells + IgG1+IgG2b +CC+ + Macrophages from WT and Macrophages from WT and CR3 KOCR3 KO

0

10

20

30

40

50

60

MC

P-1

ng/m

l

WT

EC alone

KO

MCP-1

0

1

2

3

4

5

6

IL-6

-1 n

g/m

l

WT

EC alone

KO

IL-6

Ab Med

KOMa alone

WTKO

Ma aloneWT

Other Complement Receptors Other Complement Receptors

ReceptorReceptor LigandsLigands Cell expressionCell expression

CR1CR1 C3b>C4b>iC3bC3b>C4b>iC3b

Monocytes, Monocytes, Macrophages, PMN, Macrophages, PMN,

B, T cells, RBC, FDCB, T cells, RBC, FDC

CR3 CR3 (Mac-1)(Mac-1)

iC3b>C3b, iC3b>C3b, ICAMICAM

Monocytes, Monocytes, Macrophages, PMN, Macrophages, PMN,

NK cellsNK cells

C5aRC5aR C5aC5a

Monocyte, Monocyte, Macrophage. T cells, Macrophage. T cells,

Endothelial CellsEndothelial Cells

C3aR C3aR C3aC3a

Monocyte, Monocyte, Macrophage. T cells, Macrophage. T cells,

Endothelial CellsEndothelial Cells

Complement and Complement and non-Complement non-Complement

Dependent MechanismsDependent MechanismsContribute to Contribute to

Alloantibody-Mediated Alloantibody-Mediated Activation of Endothelial Activation of Endothelial Cells and MacrophagesCells and Macrophages

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Zhiping Qian, M.D.Zhiping Qian, M.D. Wink Baldwin, M.D., Ph.D.Wink Baldwin, M.D., Ph.D.

Salma RahimiSalma Rahimi Millie Samaniego, M.D.Millie Samaniego, M.D.

Mike CoonfieldMike Coonfield

Justin GaryuJustin Garyu

Jinhuan LiuJinhuan Liu

Karen FoxKaren Fox

Miriam ReynoldsMiriam Reynolds