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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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Nature of C3 bound to the glomeruli 24 h after the administration of mCFH. (A) Kidney sections were stained for C3 using polyclonal anti-mouse C3d (red stain) or polyclonal anti-mouse C3 (green stain) antibodies. In the mice injected with PBS linear capillary wall staining was evident using both antibodies. However, in the Cfh−/− mice injected with mCFH a mesangial staining pattern was evident using anti-mouse C3 antibody whilst the linear capillary wall staining pattern remained unchanged with the anti-C3d antibody. The merged images showed that the areas of mesangial reactivity did not co-stain with the anti-C3d. Original magnification 40×. (B) Western blot analysis of C3 under reducing conditions using solubilised laser dissected glomerular tissue from Cfi−/−, Cfh−/− or Cfh−/− mice injected with mCFH. To demonstrate the positions of the intact C3 α-chain, the α′ chain of C3b and the β-chain of C3 plasma from both wild-type (intact C3) and Cfi−/− mice (in which all C3 is circulating as C3b) was also run on the gel. C3 α-chain fragments and the C3 β-chain were evident in Cfh−/− mice with or without the administration of mCFH. In contrast no α-chain fragments were evident in glomeruli from Cfi−/− animals. (C) Glomerular C3 staining in mice with combined deficiency of CFH and CFI (Cfh−/−.Cfi−/−) that have been given CFI. Following adminstration of sera containing CFI, linear capillary wall C3 staining develops that is reactive with both the anti-C3 and anti-C3d antibody. Original magnification 40×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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fig3: Nature of C3 bound to the glomeruli 24 h after the administration of mCFH. (A) Kidney sections were stained for C3 using polyclonal anti-mouse C3d (red stain) or polyclonal anti-mouse C3 (green stain) antibodies. In the mice injected with PBS linear capillary wall staining was evident using both antibodies. However, in the Cfh−/− mice injected with mCFH a mesangial staining pattern was evident using anti-mouse C3 antibody whilst the linear capillary wall staining pattern remained unchanged with the anti-C3d antibody. The merged images showed that the areas of mesangial reactivity did not co-stain with the anti-C3d. Original magnification 40×. (B) Western blot analysis of C3 under reducing conditions using solubilised laser dissected glomerular tissue from Cfi−/−, Cfh−/− or Cfh−/− mice injected with mCFH. To demonstrate the positions of the intact C3 α-chain, the α′ chain of C3b and the β-chain of C3 plasma from both wild-type (intact C3) and Cfi−/− mice (in which all C3 is circulating as C3b) was also run on the gel. C3 α-chain fragments and the C3 β-chain were evident in Cfh−/− mice with or without the administration of mCFH. In contrast no α-chain fragments were evident in glomeruli from Cfi−/− animals. (C) Glomerular C3 staining in mice with combined deficiency of CFH and CFI (Cfh−/−.Cfi−/−) that have been given CFI. Following adminstration of sera containing CFI, linear capillary wall C3 staining develops that is reactive with both the anti-C3 and anti-C3d antibody. Original magnification 40×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Mentions: In order to further define the nature of C3 that was detected in the mesangium after administration of mCFH we immunostained kidney sections using a polyclonal anti-mouse C3d antibody. Our data has shown that this antibody does not recognise intact C3 or C3b (Leung at al paper submitted). Using this antibody, GBM C3 staining was detectable to an equivalent intensity in Cfh−/− mice injected with either PBS or mCFH (Fig. 3a). However, in the Cfh−/− mice injected with mCFH, this antibody did not recognize C3 within the mesangium that was evident using the polyclonal anti-C3 antibody (Fig. 3a). This was clearly seen when the two staining images were merged. This showed complete overlap of anti-C3 and anti-C3d glomerular staining pattern in Cfh−/− mice injected with PBS. In contrast, there was no overlap of mesangial and GBM staining patterns in the sections from Cfh−/− mice injected with mCFH (Fig. 3a). This suggested that the nature of the C3 along the GBM was not the same as that of the C3 within the mesangium.


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)

Nature of C3 bound to the glomeruli 24 h after the administration of mCFH. (A) Kidney sections were stained for C3 using polyclonal anti-mouse C3d (red stain) or polyclonal anti-mouse C3 (green stain) antibodies. In the mice injected with PBS linear capillary wall staining was evident using both antibodies. However, in the Cfh−/− mice injected with mCFH a mesangial staining pattern was evident using anti-mouse C3 antibody whilst the linear capillary wall staining pattern remained unchanged with the anti-C3d antibody. The merged images showed that the areas of mesangial reactivity did not co-stain with the anti-C3d. Original magnification 40×. (B) Western blot analysis of C3 under reducing conditions using solubilised laser dissected glomerular tissue from Cfi−/−, Cfh−/− or Cfh−/− mice injected with mCFH. To demonstrate the positions of the intact C3 α-chain, the α′ chain of C3b and the β-chain of C3 plasma from both wild-type (intact C3) and Cfi−/− mice (in which all C3 is circulating as C3b) was also run on the gel. C3 α-chain fragments and the C3 β-chain were evident in Cfh−/− mice with or without the administration of mCFH. In contrast no α-chain fragments were evident in glomeruli from Cfi−/− animals. (C) Glomerular C3 staining in mice with combined deficiency of CFH and CFI (Cfh−/−.Cfi−/−) that have been given CFI. Following adminstration of sera containing CFI, linear capillary wall C3 staining develops that is reactive with both the anti-C3 and anti-C3d antibody. Original magnification 40×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Related In: Results  -  Collection

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fig3: Nature of C3 bound to the glomeruli 24 h after the administration of mCFH. (A) Kidney sections were stained for C3 using polyclonal anti-mouse C3d (red stain) or polyclonal anti-mouse C3 (green stain) antibodies. In the mice injected with PBS linear capillary wall staining was evident using both antibodies. However, in the Cfh−/− mice injected with mCFH a mesangial staining pattern was evident using anti-mouse C3 antibody whilst the linear capillary wall staining pattern remained unchanged with the anti-C3d antibody. The merged images showed that the areas of mesangial reactivity did not co-stain with the anti-C3d. Original magnification 40×. (B) Western blot analysis of C3 under reducing conditions using solubilised laser dissected glomerular tissue from Cfi−/−, Cfh−/− or Cfh−/− mice injected with mCFH. To demonstrate the positions of the intact C3 α-chain, the α′ chain of C3b and the β-chain of C3 plasma from both wild-type (intact C3) and Cfi−/− mice (in which all C3 is circulating as C3b) was also run on the gel. C3 α-chain fragments and the C3 β-chain were evident in Cfh−/− mice with or without the administration of mCFH. In contrast no α-chain fragments were evident in glomeruli from Cfi−/− animals. (C) Glomerular C3 staining in mice with combined deficiency of CFH and CFI (Cfh−/−.Cfi−/−) that have been given CFI. Following adminstration of sera containing CFI, linear capillary wall C3 staining develops that is reactive with both the anti-C3 and anti-C3d antibody. Original magnification 40×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Mentions: In order to further define the nature of C3 that was detected in the mesangium after administration of mCFH we immunostained kidney sections using a polyclonal anti-mouse C3d antibody. Our data has shown that this antibody does not recognise intact C3 or C3b (Leung at al paper submitted). Using this antibody, GBM C3 staining was detectable to an equivalent intensity in Cfh−/− mice injected with either PBS or mCFH (Fig. 3a). However, in the Cfh−/− mice injected with mCFH, this antibody did not recognize C3 within the mesangium that was evident using the polyclonal anti-C3 antibody (Fig. 3a). This was clearly seen when the two staining images were merged. This showed complete overlap of anti-C3 and anti-C3d glomerular staining pattern in Cfh−/− mice injected with PBS. In contrast, there was no overlap of mesangial and GBM staining patterns in the sections from Cfh−/− mice injected with mCFH (Fig. 3a). This suggested that the nature of the C3 along the GBM was not the same as that of the C3 within the mesangium.

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