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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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Influx of neutrophils into the glomeruli after the administration of mCFH. (A) Glomerular neutrophil numbers in Cfh−/− mice 24 h after injection of PBS, 0.75 μg of LPS or 1 mg of mCFH. Bars denote median values with standard deviation (B) Glomerular neutrophils evident in representative glomerular light microscopic images in mice that had received mCFH but not LPS. Original magnification 40× (C) FACS detection of blood neutrophils using rat IgG2b anti-mouse GR-1 antibody in Cfh−/− mice before, 24 and 48 h after neutrophil depletion. Neutrophil depletion was achieved by a single injection of rat monoclonal IgG2b anti-murine neutrophil Ly.6G (αNE). (D) Glomerular neutrophil numbers in neutrophil-depleted Cfh−/− mice 24 h after injection of mCFH demonstrating absence of glomerular neutrophils in mice pre-treated with the αNE antibody. (E) Glomerular C3 stain using polyclonal anti-mouse C3 antibody in neutrophil-depleted mice treated with mCFH. The absence of neutrophils did not influence the change in glomerular C3 staining seen after the administration of mCFH. Compare this image with that shown in Fig. 2. Original magnification 40×. WT: wild-type.
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fig5: Influx of neutrophils into the glomeruli after the administration of mCFH. (A) Glomerular neutrophil numbers in Cfh−/− mice 24 h after injection of PBS, 0.75 μg of LPS or 1 mg of mCFH. Bars denote median values with standard deviation (B) Glomerular neutrophils evident in representative glomerular light microscopic images in mice that had received mCFH but not LPS. Original magnification 40× (C) FACS detection of blood neutrophils using rat IgG2b anti-mouse GR-1 antibody in Cfh−/− mice before, 24 and 48 h after neutrophil depletion. Neutrophil depletion was achieved by a single injection of rat monoclonal IgG2b anti-murine neutrophil Ly.6G (αNE). (D) Glomerular neutrophil numbers in neutrophil-depleted Cfh−/− mice 24 h after injection of mCFH demonstrating absence of glomerular neutrophils in mice pre-treated with the αNE antibody. (E) Glomerular C3 stain using polyclonal anti-mouse C3 antibody in neutrophil-depleted mice treated with mCFH. The absence of neutrophils did not influence the change in glomerular C3 staining seen after the administration of mCFH. Compare this image with that shown in Fig. 2. Original magnification 40×. WT: wild-type.

Mentions: Neutrophils were observed in the glomeruli of Cfh−/− mice 24 h after the injection of mCFH but not after injection of LPS alone (Fig. 5a and b). Human CFH has been reported to act as an adhesion ligand for neutrophils through CD11b (Mac-1) (DiScipio et al., 1998). To investigate whether the administration of mCFH could be directly involved in neutrophil recruitment we administered mCFH to Cfh−/− mice lacking CD11b (Cfh−/−.CD11b−/−). 24 h after mCFH administration we observed significant glomerular neutrophil influx in these animals demonstrating that the neutrophil influx was independent of CD11b. To test if glomerular neutrophil proteases (Carlo et al., 1981), could influence glomerular C3 staining, we administered mCFH to Cfh−/− mice that had been depleted of neutrophils (Fig. 5c). The change in C3 staining pattern persisted despite neutrophil depletion indicating that neutrophils were not involved in C3 changes in the Cfh−/− mice following mCFH administration (Fig. 5d and e).


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)

Influx of neutrophils into the glomeruli after the administration of mCFH. (A) Glomerular neutrophil numbers in Cfh−/− mice 24 h after injection of PBS, 0.75 μg of LPS or 1 mg of mCFH. Bars denote median values with standard deviation (B) Glomerular neutrophils evident in representative glomerular light microscopic images in mice that had received mCFH but not LPS. Original magnification 40× (C) FACS detection of blood neutrophils using rat IgG2b anti-mouse GR-1 antibody in Cfh−/− mice before, 24 and 48 h after neutrophil depletion. Neutrophil depletion was achieved by a single injection of rat monoclonal IgG2b anti-murine neutrophil Ly.6G (αNE). (D) Glomerular neutrophil numbers in neutrophil-depleted Cfh−/− mice 24 h after injection of mCFH demonstrating absence of glomerular neutrophils in mice pre-treated with the αNE antibody. (E) Glomerular C3 stain using polyclonal anti-mouse C3 antibody in neutrophil-depleted mice treated with mCFH. The absence of neutrophils did not influence the change in glomerular C3 staining seen after the administration of mCFH. Compare this image with that shown in Fig. 2. Original magnification 40×. WT: wild-type.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2697322&req=5

fig5: Influx of neutrophils into the glomeruli after the administration of mCFH. (A) Glomerular neutrophil numbers in Cfh−/− mice 24 h after injection of PBS, 0.75 μg of LPS or 1 mg of mCFH. Bars denote median values with standard deviation (B) Glomerular neutrophils evident in representative glomerular light microscopic images in mice that had received mCFH but not LPS. Original magnification 40× (C) FACS detection of blood neutrophils using rat IgG2b anti-mouse GR-1 antibody in Cfh−/− mice before, 24 and 48 h after neutrophil depletion. Neutrophil depletion was achieved by a single injection of rat monoclonal IgG2b anti-murine neutrophil Ly.6G (αNE). (D) Glomerular neutrophil numbers in neutrophil-depleted Cfh−/− mice 24 h after injection of mCFH demonstrating absence of glomerular neutrophils in mice pre-treated with the αNE antibody. (E) Glomerular C3 stain using polyclonal anti-mouse C3 antibody in neutrophil-depleted mice treated with mCFH. The absence of neutrophils did not influence the change in glomerular C3 staining seen after the administration of mCFH. Compare this image with that shown in Fig. 2. Original magnification 40×. WT: wild-type.
Mentions: Neutrophils were observed in the glomeruli of Cfh−/− mice 24 h after the injection of mCFH but not after injection of LPS alone (Fig. 5a and b). Human CFH has been reported to act as an adhesion ligand for neutrophils through CD11b (Mac-1) (DiScipio et al., 1998). To investigate whether the administration of mCFH could be directly involved in neutrophil recruitment we administered mCFH to Cfh−/− mice lacking CD11b (Cfh−/−.CD11b−/−). 24 h after mCFH administration we observed significant glomerular neutrophil influx in these animals demonstrating that the neutrophil influx was independent of CD11b. To test if glomerular neutrophil proteases (Carlo et al., 1981), could influence glomerular C3 staining, we administered mCFH to Cfh−/− mice that had been depleted of neutrophils (Fig. 5c). The change in C3 staining pattern persisted despite neutrophil depletion indicating that neutrophils were not involved in C3 changes in the Cfh−/− mice following mCFH administration (Fig. 5d and e).

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