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Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase.

Paixão-Cavalcante D, Hanson S, Botto M, Cook HT, Pickering MC - Mol. Immunol. (2009)

Bottom Line: Dense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway.Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma.The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Rheumatology Section, Faculty of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK.

ABSTRACT
Dense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway. Factor H (CFH)-deficient (Cfh(-/-)) mice spontaneously develop C3 deposition along the glomerular basement membrane (GBM) with subsequent development of glomerulonephritis with features of DDD, a lesion dependent on C3 activation. In order to understand the role of CFH in preventing renal damage associated with the dysregulation of the alternative pathway we administered purified mouse CFH (mCFH) to Cfh(-/-) mice. 24h following the administration of mCFH we observed an increase in plasma C3 levels with presence of intact C3 in circulation showing that mCFH restored control of C3 activation in fluid phase. mCFH resulted in the reduction of iC3b deposition along the GBM. The exogenous mCFH was readily detectable in plasma but critically not in association with C3 along the GBM. Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma. Western blot analysis of glomeruli from Cfh(-/-) mice demonstrated the presence of iC3b. Our data show that the C3 along the GBM in Cfh(-/-) mice is the C3 fragment iC3b and that this is derived from plasma C3 activation. The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.

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Detection of CFH-related proteins in Cfh−/− mice and tracking mCFH after administration to Cfh−/− mice. (A) Serum western blot for CFH using cross-reactive polyclonal anti-human CFH antibody. (B) Renal sections from Cfh−/− mice 2 h after the injection of Alexa-488-tagged CFH. At this time point the Alexa-488-tagged CFH was detected within the mesangium and tubulointerstitium but not along the GBM. At this time-point linear C3 staining is evident in Cfh−/− mice similar to unmanipulated Cfh−/− mice (far right panel). (C) Kidney sections immunostained for CFH using anti-human CFH antibody from wild-type, unmanipulated Cfh−/− mice and Cfh−/− mice 24 h after injection of mCFH. Some mesangial reactivity is present in wild-type glomeruli (left panel) whilst marked linear capillary wall staining is evident in the unmanipulated Cfh−/− mice (middle panel). In contrast, a mesangial staining pattern was evident in Cfh−/− mice 24 h after injection of mCFH (right panel) with very little staining along the capillary walls (arrows). Original magnification, 40×.
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fig4: Detection of CFH-related proteins in Cfh−/− mice and tracking mCFH after administration to Cfh−/− mice. (A) Serum western blot for CFH using cross-reactive polyclonal anti-human CFH antibody. (B) Renal sections from Cfh−/− mice 2 h after the injection of Alexa-488-tagged CFH. At this time point the Alexa-488-tagged CFH was detected within the mesangium and tubulointerstitium but not along the GBM. At this time-point linear C3 staining is evident in Cfh−/− mice similar to unmanipulated Cfh−/− mice (far right panel). (C) Kidney sections immunostained for CFH using anti-human CFH antibody from wild-type, unmanipulated Cfh−/− mice and Cfh−/− mice 24 h after injection of mCFH. Some mesangial reactivity is present in wild-type glomeruli (left panel) whilst marked linear capillary wall staining is evident in the unmanipulated Cfh−/− mice (middle panel). In contrast, a mesangial staining pattern was evident in Cfh−/− mice 24 h after injection of mCFH (right panel) with very little staining along the capillary walls (arrows). Original magnification, 40×.

Mentions: 24 h after the injection of mCFH, we could still detect CFH in circulation by western blot but the maximal signal was 2 h following injection (Fig. 4a). To check whether the circulating mCFH could interact with glomerular C3 we tagged purified mCFH with Alexa 488 and assessed its distribution 2 h following the injection. We detected tagged CFH in the mesangium and within the tubulo-interstitium (Fig. 4b). This data showed that the administered mCFH did not directly interact with C3 bound on the GBM at this time point. Immunostaining of glomeruli from unmanipulated Cfh−/− mice showed reactivity with a polyclonal anti-CFH antibody in a linear pattern identical to that seen for C3 reactivity (Fig. 4c). We interpreted this reactivity as a consequence of cross-reactivity the anti-CFH antibody with CFH-related proteins that were associated with the GBM-bound C3 in the Cfh−/− mice. Notably, CFH-related protein staining was not detected along the GBM of wild-type mice. In glomerular sections from Cfh−/− mice reconstituted with mCFH a mesangial staining pattern was seen using the polyclonal anti-CFH antibody at 24 h. This pattern was identical to that seen for C3 using the anti-C3 antibody (Fig. 4c). These data suggested that the presence of CFH-related proteins on the GBM is associated with the presence of C3.


Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase.

Paixão-Cavalcante D, Hanson S, Botto M, Cook HT, Pickering MC - Mol. Immunol. (2009)

Detection of CFH-related proteins in Cfh−/− mice and tracking mCFH after administration to Cfh−/− mice. (A) Serum western blot for CFH using cross-reactive polyclonal anti-human CFH antibody. (B) Renal sections from Cfh−/− mice 2 h after the injection of Alexa-488-tagged CFH. At this time point the Alexa-488-tagged CFH was detected within the mesangium and tubulointerstitium but not along the GBM. At this time-point linear C3 staining is evident in Cfh−/− mice similar to unmanipulated Cfh−/− mice (far right panel). (C) Kidney sections immunostained for CFH using anti-human CFH antibody from wild-type, unmanipulated Cfh−/− mice and Cfh−/− mice 24 h after injection of mCFH. Some mesangial reactivity is present in wild-type glomeruli (left panel) whilst marked linear capillary wall staining is evident in the unmanipulated Cfh−/− mice (middle panel). In contrast, a mesangial staining pattern was evident in Cfh−/− mice 24 h after injection of mCFH (right panel) with very little staining along the capillary walls (arrows). Original magnification, 40×.
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fig4: Detection of CFH-related proteins in Cfh−/− mice and tracking mCFH after administration to Cfh−/− mice. (A) Serum western blot for CFH using cross-reactive polyclonal anti-human CFH antibody. (B) Renal sections from Cfh−/− mice 2 h after the injection of Alexa-488-tagged CFH. At this time point the Alexa-488-tagged CFH was detected within the mesangium and tubulointerstitium but not along the GBM. At this time-point linear C3 staining is evident in Cfh−/− mice similar to unmanipulated Cfh−/− mice (far right panel). (C) Kidney sections immunostained for CFH using anti-human CFH antibody from wild-type, unmanipulated Cfh−/− mice and Cfh−/− mice 24 h after injection of mCFH. Some mesangial reactivity is present in wild-type glomeruli (left panel) whilst marked linear capillary wall staining is evident in the unmanipulated Cfh−/− mice (middle panel). In contrast, a mesangial staining pattern was evident in Cfh−/− mice 24 h after injection of mCFH (right panel) with very little staining along the capillary walls (arrows). Original magnification, 40×.
Mentions: 24 h after the injection of mCFH, we could still detect CFH in circulation by western blot but the maximal signal was 2 h following injection (Fig. 4a). To check whether the circulating mCFH could interact with glomerular C3 we tagged purified mCFH with Alexa 488 and assessed its distribution 2 h following the injection. We detected tagged CFH in the mesangium and within the tubulo-interstitium (Fig. 4b). This data showed that the administered mCFH did not directly interact with C3 bound on the GBM at this time point. Immunostaining of glomeruli from unmanipulated Cfh−/− mice showed reactivity with a polyclonal anti-CFH antibody in a linear pattern identical to that seen for C3 reactivity (Fig. 4c). We interpreted this reactivity as a consequence of cross-reactivity the anti-CFH antibody with CFH-related proteins that were associated with the GBM-bound C3 in the Cfh−/− mice. Notably, CFH-related protein staining was not detected along the GBM of wild-type mice. In glomerular sections from Cfh−/− mice reconstituted with mCFH a mesangial staining pattern was seen using the polyclonal anti-CFH antibody at 24 h. This pattern was identical to that seen for C3 using the anti-C3 antibody (Fig. 4c). These data suggested that the presence of CFH-related proteins on the GBM is associated with the presence of C3.

Bottom Line: Dense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway.Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma.The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Rheumatology Section, Faculty of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK.

ABSTRACT
Dense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway. Factor H (CFH)-deficient (Cfh(-/-)) mice spontaneously develop C3 deposition along the glomerular basement membrane (GBM) with subsequent development of glomerulonephritis with features of DDD, a lesion dependent on C3 activation. In order to understand the role of CFH in preventing renal damage associated with the dysregulation of the alternative pathway we administered purified mouse CFH (mCFH) to Cfh(-/-) mice. 24h following the administration of mCFH we observed an increase in plasma C3 levels with presence of intact C3 in circulation showing that mCFH restored control of C3 activation in fluid phase. mCFH resulted in the reduction of iC3b deposition along the GBM. The exogenous mCFH was readily detectable in plasma but critically not in association with C3 along the GBM. Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma. Western blot analysis of glomeruli from Cfh(-/-) mice demonstrated the presence of iC3b. Our data show that the C3 along the GBM in Cfh(-/-) mice is the C3 fragment iC3b and that this is derived from plasma C3 activation. The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.

Show MeSH
Related in: MedlinePlus