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. 2007 Apr;44(10):2697-706.
doi: 10.1016/j.molimm.2006.12.001. Epub 2007 Jan 17.

The C-terminus of Complement Factor H Is Essential for Host Cell Protection

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The C-terminus of Complement Factor H Is Essential for Host Cell Protection

Mihály Józsi et al. Mol Immunol. .
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Complement is a powerful self-amplifying system of innate immune defense with the capacity to eliminate microbes directly. Factor H is a central regulator in plasma which protects host tissue from complement mediated damage. Here we characterize the relevance of surface attached factor H, and study the regulatory activity of factor H on endothelial cells. Although these cells expressed membrane bound regulators, cell bound factor H contributed substantially to complement regulatory activity at the cell surface. Blockade of the C-terminus of factor H with monoclonal antibodies inhibited cell binding of this soluble regulator and resulted in enhanced complement activation on the cells. In the absence of factor H, increased deposition and slower inactivation of C3b resulted in higher amount of membrane attack complexes on the cell surface. When the membrane regulators CD55 and CD59 were removed by enzymatic treatment, complement mediated cell lysis was enhanced in the absence of factor H. Importantly, inhibition of the C-terminus did not compromise the regulatory function of factor H in fluid phase. Altogether these data point to a highly relevant, yet so far underestimated role of factor H for complement control at cellular surfaces, and reveal a decisive role of the factor H C-terminus in host cell recognition and protection.


Figure 1
Figure 1. Expression of complement regulators on HUVEC
The presence of CD35 (CR1), CD46 (MCP), CD55 (DAF) and CD59 was measured using specific mAbs. The x- and y-axes correspond to relative fluorescence intensity and relative cell number, respectively.
Figure 2
Figure 2. FH binding to HUVEC
(A) Comparison of binding of purified FH to HUVEC in 0.5×PBS and in PBS, as determined by flow cytometry. Mean ± SD of median fluorescence intensities (MFI) from three experiments are shown. (B) Dose-dependent binding of FH from human serum to HUVEC in low ionic strength buffer (0.5×PBS) and under physiological conditions (PBS), as measured by flow cytometry. (C) C3 deposition enhances FH binding to HUVEC. The cells were pre-incubated with anti-HUVEC antiserum, then exposed to 10% human serum in PBS. C3 deposition and FH binding was analyzed by flow cytometry.
Figure 3
Figure 3. Characterization of FH-binding mAbs
(A) Mapping of the binding domain of mAb MH10. Human serum was separated by SDS-PAGE, transferred to a membrane and the blot was developed with mAb MH10 (lane 1) or a polyclonal FH antiserum (lane 2). (B) Recombinant FH fragments, i.e. CCPs 1-7, 8-11, 11-15, 15-18, 15-19, 15-20 and 19-20 were separated by SDS-PAGE and analyzed by Western blotting using mAb MH10 (lanes 1-7). The upper bands in lane 6 are due to oligomerization of the FH fragment CCPs 15-20. (C) Binding sites of the mAbs used in this study. mAb E22 binds CCP3, and mAb C21 binds within CCPs 15-18 of FH. The mAbs C18, E14 and MH10 bind to the C-terminal CCP20 domain of FH.
Figure 4
Figure 4. Effect of mAbs on FH functions
(A) Binding of FH to HUVEC is reduced by the C-terminally binding mAbs C18, E14 and MH10, but not by mAb C21, as determined by flow cytometry. Human serum preincubated with the indicated mAbs was added to the cells. FH binding to the cells in the absence of mAb was set to 100% and the inhibitory effect of the various antibodies is shown (mean ± SD of five experiments). (B) FH binding to immobilized C3b is inhibited by C-terminally binding mAbs. FH preincubated with the indicated antibodies was assayed for binding to C3b by ELISA. Mean ± SD of data from four experiments is shown. (C) The FH-binding mAbs do not inhibit FH-mediated cofactor activity in fluid phase. C3b was incubated with factor I in the presence of FH pretreated with the indicated mAbs. After 10 min, the samples were separated by SDS-PAGE under reducing conditions, and C3-fragments were identified by Western blotting.
Figure 5
Figure 5. Surface deposition and fragmentation of C3b on HUVEC
(A) HUVEC were incubated for the indicated time with human serum, stained with C3c- and C3d-specific antibodies and analyzed by flow cytometry. The C3c-antibody detects C3b and iC3b, but not the final C3d fragment. The C3d-antibody detects the C3d domain, which is present in all types of surface-bound C3b-fragments. Data of a representative experiment are shown as median fluorescence intensities (MFI). (B) HUVEC were incubated with human serum in the absence (open circles) or presence (filled circles) of mAb C18, which blocks FH binding. C3-fragments were analyzed by flow cytometry. The ratio of C3c- and C3d-specific fluorescence values was calculated at the indicated time points. (C) Effect of the various FH binding mAbs on the fate of C3. The C3c/C3d ratio was determined after incubating the cells for 5 min as above. Means ± SD of data from four experiments are shown. (D) Blocking FH attachment affects C3 degradation on the cell surface. HUVEC were incubated for 5 min in human serum which has been pretreated with mAb C18. Cell lysate was separated by SDS-PAGE and analyzed by Western blotting using a C3-specific antiserum. Lane 1, purified C3b is shown to indicate mobility of the α′- and β-chains; lane 2, lysate prepared from HUVEC incubated in PBS; lane 3, cells incubated in human serum; lane 4, cells incubated in serum in the presence of mAb C18. The high molecular weight bands in lanes 3 and 4 represent α′-chain fragments covalently bound to membrane molecules.
Figure 6
Figure 6. Role of FH in C3b and MAC deposition on HUVEC
(A) HUVEC were incubated in 10% FH-depleted human plasma (empty bars) and FH binding, C3b deposition and MAC formation were analyzed by flow cytometry. Reconstitution of the FH-depleted plasma with purified FH resulted in reduced C3b and MAC deposition (filled bars). (B) HUVEC were incubated in 20% normal human serum (NHS) in the absence or presence of the indicated mAbs or in EDTA. MAC formation was detected on the cell surface by flow cytometry using a C5b-9-specific mAb. The mean of specific median fluorescence intensities (MFI) ± SD are shown from four independent experiments.
Figure 7
Figure 7. Role of FH in complement mediated cell lysis
(A) HUVEC were incubated with FH-depleted human plasma in the absence (empty bars) or presence (filled bars) of Mg2+ for 60 min. Cell lysis as assayed by calcein release is shown in arbitrary fluorescence units (AFU). (B) FH protects HUVEC from complement mediated lysis. Incubation of the cells in FH-depleted plasma resulted in low lysis as indicated by the release of calcein (empty bar). Following enzymatic removal of the membrane regulators CD55 and CD59, complement mediated lysis was increased (left panel, filled bar). The protective role of FH is demonstrated when FH-depleted plasma was reconstituted with purified FH (right panel, filled bar). The values of FH-depleted plasma without Mg2+ added was subtracted as negative control. n.d.: not determined.

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