P-244 Poster of distinction

A new concept for autoregulation of complement activation on cell surfaces

Y. Teramura¹, K.N. Ekdahl², J. Hong¹, P. Magnusson¹, D. Ricklin³, J.D. Lambris³, H. Iwata⁴, B. Nilsson^1
1 Uppsala University, Uppsala, Sweden; ² Linnaeus University, Kalmar, Sweden; ³ University of Pennsylvania, philadelphia, USA; ⁴ Kyoto University, Kyoto, Japan

Objective: Ischemia-reperfusion injury andxenogenic/allogeneic antibody-mediated rejection cause acute and long-termdamage in whole organ and cell t ransplant ation. In these conditions complement activation is one of the major mediator of cell damage. Here, we present a new concept for autoprotection against complement attack on cell surfaces using a factor H-binding peptide, which is bound to surface using a PEG-lipid as an anchor to the membrane.

Methods: We conjugated the factor H-binding peptide (14 aa-residues¹) to the end of poly(ethylene glycol)-phospholipid (fH-bp-PEG-lipid), which spontaneously inserted into cell membrane. To examine the function of fH-bp-PEG-lipid, we performed three different assays. 1) Generation of C3a, sC5b-9 on material surfaces coated withfH-bp-PEG; 2) a hemolytic assay of the alternative pathway using rabbiterythrocyte treated with fH-bp-PEG-lipid; 3) complement activation (C3a,sC5b-9) elicited by adherent porcine endothelial cells treated withfH-bp-PEG-lipid and in contact with human whole blood using a slide chambermodel. Control cells were treated with PEG-lipid conjugated with scrambledcontrol peptide.

Results: Factor Hcould be detected by ELISA, confocal microscopy or FACS, on material and cellsurfaces, after the surface was exposused to purified factor H, hirudin-plasmaor whole blood. Bindingof fH-bp-PEG to material surfaces protectedthe surfaces against complement attack at specific surface densities of the fH-bp-PEGconstruct. Lysis of rabbiterythrocytes in human serum was attenuated by the surface modification comparedwith the control cells . Porcine endothelial cellsmodified with fH-bp-PEG-lipid and exposed to human whole blood, were alsoprotected against complement activation, as demonstrated by much lower C3a andsC5b-9 generation.

Conclusions: These results indicate that factor H can specifically be recruitedby the peptide from blood and inhibit complement activation on the cellsurface. The use of the fH-bp-PEG-lipid is a promising tool for autoprotectionagainst ischemia reperfusion injury and antibody-mediated graft rejection inclinical transplantation.

Ref 1) Wu YQ et al, J Immunol, in press

P-244 Poster of distinction

A new concept for autoregulation of complement activation on cell surfaces

Y. Teramura¹, K.N. Ekdahl², J. Hong¹, P. Magnusson¹, D. Ricklin³, J.D. Lambris³, H. Iwata⁴, B. Nilsson^1
1 Uppsala University, Uppsala, Sweden; ² Linnaeus University, Kalmar, Sweden; ³ University of Pennsylvania, philadelphia, USA; ⁴ Kyoto University, Kyoto, Japan

Objective: Ischemia-reperfusion injury andxenogenic/allogeneic antibody-mediated rejection cause acute and long-termdamage in whole organ and cell t ransplant ation. In these conditions complement activation is one of the major mediator of cell damage. Here, we present a new concept for autoprotection against complement attack on cell surfaces using a factor H-binding peptide, which is bound to surface using a PEG-lipid as an anchor to the membrane.

Methods: We conjugated the factor H-binding peptide (14 aa-residues¹) to the end of poly(ethylene glycol)-phospholipid (fH-bp-PEG-lipid), which spontaneously inserted into cell membrane. To examine the function of fH-bp-PEG-lipid, we performed three different assays. 1) Generation of C3a, sC5b-9 on material surfaces coated withfH-bp-PEG; 2) a hemolytic assay of the alternative pathway using rabbiterythrocyte treated with fH-bp-PEG-lipid; 3) complement activation (C3a,sC5b-9) elicited by adherent porcine endothelial cells treated withfH-bp-PEG-lipid and in contact with human whole blood using a slide chambermodel. Control cells were treated with PEG-lipid conjugated with scrambledcontrol peptide.

Results: Factor Hcould be detected by ELISA, confocal microscopy or FACS, on material and cellsurfaces, after the surface was exposused to purified factor H, hirudin-plasmaor whole blood. Bindingof fH-bp-PEG to material surfaces protectedthe surfaces against complement attack at specific surface densities of the fH-bp-PEGconstruct. Lysis of rabbiterythrocytes in human serum was attenuated by the surface modification comparedwith the control cells . Porcine endothelial cellsmodified with fH-bp-PEG-lipid and exposed to human whole blood, were alsoprotected against complement activation, as demonstrated by much lower C3a andsC5b-9 generation.

Conclusions: These results indicate that factor H can specifically be recruitedby the peptide from blood and inhibit complement activation on the cellsurface. The use of the fH-bp-PEG-lipid is a promising tool for autoprotectionagainst ischemia reperfusion injury and antibody-mediated graft rejection inclinical transplantation.

Ref 1) Wu YQ et al, J Immunol, in press