Alloantibody- Alloantibody- Mediated Rejection: Mediated Rejection: Complement and Complement and non-Complement non-Complement Dependent Mechanisms Dependent Mechanisms B.A. Wasowska B.A. Wasowska JHMI JHMI
Dec 21, 2015
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