- Email: [email protected]
Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis
Abstracts / Molecular Immunology 47 (2010) 2198–2294
defence and maintenance of immune tolerance. M-ficolin was shown recently to interact with the short pentraxin C-reactive protein (CRP) through a site located close to the junction between the collagen-like fibres and the fibrinogen (FBG) domains. To characterize the interaction of M-ficolin with the long-chain pentraxin-3 (PTX3) and to locate the binding site on PTX3, recombinant Mficolin and PTX3 were expressed in eukaryotic cells and their interaction was characterized using SPR spectroscopy. M-ficolin was shown to bind to immobilized PTX3 with high affinity (KD in the sub-nanomolar range) in the presence of calcium ions. The interaction was abolished in the presence of EDTA and inhibited by GlcNAc, indicating involvement of the FBG domain. Similar results were obtained in the reverse configuration, i.e. using soluble PTX3 and immobilized M-ficolin. Removal of sialic acid from the single N-linked carbohydrate at position 220 of the C-terminal domain of PTX3 abolished interaction with M-ficolin. Likewise, an M-ficolin mutant with impaired sialic acid binding ability did not interact with PTX3. The isolated recognition domain of M-ficolin did not bind PTX3, and conversely the monomeric C-terminal domain of PTX3 did not significantly interact with M-ficolin, suggesting a requirement for oligomerization of both proteins. These data indicate that interaction of M-ficolin with PTX3 involves multivalent binding of the ficolin FBG domains to the terminal sialic acid residues of the N-linked carbohydrate of the PTX3 pentraxin domain. The interaction of M-ficolin with PTX3 arises from its known ability to bind sialylated ligands and thus clearly differs from the binding to CRP. The biological implications of the interaction between M-ficolin and PTX3 are currently under investigation. doi:10.1016/j.molimm.2010.05.109 188 Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis Mei Chen a , Elizabeth Muckersie b , John V. Forrester b , Heping Xu a a
Centre for Vision and Vascular Science, Queen University Belfast, Grosvenor Road, BT12 6BA, UK b Immunology and Infection, Division of Applied Medicine, University of Aberdeen School of Medicine, Foresterhill, AB25 2ZD, Aberdeen, UK
Purpose: We have shown previously that complement factor H (CFH) and factor B (CFB) are constitutively expressed by retinal pigment epithelial (RPE) cells and their production is regulated by inflammatory cytokines, suggesting that alternative pathway (AP) of complement activation might play a role in retinal inflammation. In this study we further investigated the role of AP in retinal inflammation using experimental autoimmune uveoretinitis (EAU) as a model. Methods: EAU was induced in C57BL/6 mice with interphotoreceptor retinoid-binding protein (IRBP) peptide 1–20 immunization. Complement C3d deposition and CFB expression were examined by immunohistochemistry, and CFB gene expression was evaluated by real-time PCR. An AP selective blocker CRIg fusion protein (CRIgFc) was administered to IRBP-immunized mice at different times. The severity of retinal inflammation was evaluated clinically by the topical endoscopic fundus imaging (TEFI) system and pathologically by light microscopy. In addition, the effect of CRIg-Fc on T cell proliferation and cytokine production was investigated in IRBP peptide-activated spleen cells. Results: Mice with EAU show increased levels of C3d deposition and CFB expression in the retina. CFB gene expression increased 60-fold in the retina and 5-fold in RPE/choroid/sclera of EAU mice as compared to control non-EAU mice. Retinal inflammation was
2233
suppressed clinically and histologically by blocking the AP complement activation with CRIg-Fc. In line with reduced inflammation, C3d deposition and CFB expression were also markedly decreased by CRIg-Fc treatment. CRIg-Fc also suppressed T cell proliferation and cytokine IFN-g, TNF-a, IL-17, and IL-6 production in mice with EAU. Conclusions: AP complement activation contributed significantly to retinal inflammation in EAU. Inhibition of EAU by blocking AP complement activation appeared to be mediated by reduction in both the Th1 and Th17 pathways for EAU induction. doi:10.1016/j.molimm.2010.05.110 189 Recombinant form of human wild type mannan-binding lectin (MBL/A) but not its structural variant (MBL/C) promotes phagocytosis of zymosan by activating complement Rema Rajagopalan, Takazvida Nyaundi, Veena P. Salvi, Nenoo Rawal Department of Biochemistry, University of Texas Health Science Center, 11937, US Highway 271, Tyler, TX 75708-3154, United States Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometry analysis revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5–30 g/ml) resulted in a 2.1- and 2.7-fold enhancement in their uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Similar results were obtained when THP-1 phagocytosis of zymosan was analyzed by light microscopy. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and studies conducted in the absence of MBL/C1q Dpl serum exhibited a modest increase in THP-1 uptake of MBL opsonized zymosan. Complement opsonins, C3b and C4b, were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis due to dysfunctional complement activation may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections. doi:10.1016/j.molimm.2010.05.111
defence and maintenance of immune tolerance. M-ficolin was shown recently to interact with the short pentraxin C-reactive protein (CRP) through a site located close to the junction between the collagen-like fibres and the fibrinogen (FBG) domains. To characterize the interaction of M-ficolin with the long-chain pentraxin-3 (PTX3) and to locate the binding site on PTX3, recombinant Mficolin and PTX3 were expressed in eukaryotic cells and their interaction was characterized using SPR spectroscopy. M-ficolin was shown to bind to immobilized PTX3 with high affinity (KD in the sub-nanomolar range) in the presence of calcium ions. The interaction was abolished in the presence of EDTA and inhibited by GlcNAc, indicating involvement of the FBG domain. Similar results were obtained in the reverse configuration, i.e. using soluble PTX3 and immobilized M-ficolin. Removal of sialic acid from the single N-linked carbohydrate at position 220 of the C-terminal domain of PTX3 abolished interaction with M-ficolin. Likewise, an M-ficolin mutant with impaired sialic acid binding ability did not interact with PTX3. The isolated recognition domain of M-ficolin did not bind PTX3, and conversely the monomeric C-terminal domain of PTX3 did not significantly interact with M-ficolin, suggesting a requirement for oligomerization of both proteins. These data indicate that interaction of M-ficolin with PTX3 involves multivalent binding of the ficolin FBG domains to the terminal sialic acid residues of the N-linked carbohydrate of the PTX3 pentraxin domain. The interaction of M-ficolin with PTX3 arises from its known ability to bind sialylated ligands and thus clearly differs from the binding to CRP. The biological implications of the interaction between M-ficolin and PTX3 are currently under investigation. doi:10.1016/j.molimm.2010.05.109 188 Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis Mei Chen a , Elizabeth Muckersie b , John V. Forrester b , Heping Xu a a
Centre for Vision and Vascular Science, Queen University Belfast, Grosvenor Road, BT12 6BA, UK b Immunology and Infection, Division of Applied Medicine, University of Aberdeen School of Medicine, Foresterhill, AB25 2ZD, Aberdeen, UK
Purpose: We have shown previously that complement factor H (CFH) and factor B (CFB) are constitutively expressed by retinal pigment epithelial (RPE) cells and their production is regulated by inflammatory cytokines, suggesting that alternative pathway (AP) of complement activation might play a role in retinal inflammation. In this study we further investigated the role of AP in retinal inflammation using experimental autoimmune uveoretinitis (EAU) as a model. Methods: EAU was induced in C57BL/6 mice with interphotoreceptor retinoid-binding protein (IRBP) peptide 1–20 immunization. Complement C3d deposition and CFB expression were examined by immunohistochemistry, and CFB gene expression was evaluated by real-time PCR. An AP selective blocker CRIg fusion protein (CRIgFc) was administered to IRBP-immunized mice at different times. The severity of retinal inflammation was evaluated clinically by the topical endoscopic fundus imaging (TEFI) system and pathologically by light microscopy. In addition, the effect of CRIg-Fc on T cell proliferation and cytokine production was investigated in IRBP peptide-activated spleen cells. Results: Mice with EAU show increased levels of C3d deposition and CFB expression in the retina. CFB gene expression increased 60-fold in the retina and 5-fold in RPE/choroid/sclera of EAU mice as compared to control non-EAU mice. Retinal inflammation was
2233
suppressed clinically and histologically by blocking the AP complement activation with CRIg-Fc. In line with reduced inflammation, C3d deposition and CFB expression were also markedly decreased by CRIg-Fc treatment. CRIg-Fc also suppressed T cell proliferation and cytokine IFN-g, TNF-a, IL-17, and IL-6 production in mice with EAU. Conclusions: AP complement activation contributed significantly to retinal inflammation in EAU. Inhibition of EAU by blocking AP complement activation appeared to be mediated by reduction in both the Th1 and Th17 pathways for EAU induction. doi:10.1016/j.molimm.2010.05.110 189 Recombinant form of human wild type mannan-binding lectin (MBL/A) but not its structural variant (MBL/C) promotes phagocytosis of zymosan by activating complement Rema Rajagopalan, Takazvida Nyaundi, Veena P. Salvi, Nenoo Rawal Department of Biochemistry, University of Texas Health Science Center, 11937, US Highway 271, Tyler, TX 75708-3154, United States Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometry analysis revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5–30 g/ml) resulted in a 2.1- and 2.7-fold enhancement in their uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Similar results were obtained when THP-1 phagocytosis of zymosan was analyzed by light microscopy. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and studies conducted in the absence of MBL/C1q Dpl serum exhibited a modest increase in THP-1 uptake of MBL opsonized zymosan. Complement opsonins, C3b and C4b, were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis due to dysfunctional complement activation may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections. doi:10.1016/j.molimm.2010.05.111