Complement fragments are biomarkers of antibody-mediated endothelial injury

Abstract

Antibody-mediated rejection (AbMR) adversely affects long-term graft survival in kidney transplantation. Currently, the diagnosis of AbMR requires a kidney biopsy, and detection of complement C4d deposition in the allograft is one of the diagnostic criteria. Complement activation also generates several soluble fragments which could potentially provide non-invasive biomarkers of the process. Furthermore, microvesicles released into the plasma from injured cells can serve as biomarkers of vascular injury. To explore whether soluble complement fragments or complement fragments bound to endothelial microvesicles can be used to non-invasively detect AbMR, we developed an in vitro model in which human endothelial cells were exposed to anti-HLA antibodies and complement sufficient serum. We found that complement fragments C4a and sC5b-9 were increased in the supernatants of cells exposed to complement-sufficient serum compared to cells treated complement-deficient serum. Furthermore, complement activation on the cell surface was associated with the release of microvesicles bearing C4 and C3 fragments. We next measured these analytes in plasma from kidney transplant recipients with biopsy-proven acute AbMR (n = 9) and compared the results with those from transplant recipients who also had impaired allograft function but who did not have AbMR (n = 30). Consistent with the in vitro results, complement fragments C4a and Ba were increased in plasma from patients with AbMR compared to control subjects (P < 0.001 and P < 0.01, respectively). Endothelial microvesicle counts were not increased in patients with AbMR, however, and the number of microvesicles with C4 and C3 bound to the surface was actually lower compared to control subjects (both P < 0.05). Our results suggest that plasma complement activation fragments may be useful as non-invasive biomarkers of antibody-mediated complement activation within the allograft. Complement-opsonized endothelial microvesicles are decreased in patients with AbMR, possibly due to enhanced clearance of microvesicles opsonized with C3 and C4 fragments.

Keywords: Antibody mediated rejection; Biomarker; Complement; Donor specific antibody.

FIGURE 1.. C4 and C3 fragments are deposited on HMEC-1 cells after exposure to anti-HLA antibody and normal human serum.

HMEC cells were treated with W6/32 antibody and then exposed to normal human serum. Complement activation on the cells was then analyzed by flow cytometry after various periods of time. Data are plotted as mean fluorescence intensity (MFI) ± SEM. A) C4c was rapidly deposited on the cell surface (**P<0.01, ***P<0.001 vs control) but was significantly decreased by 60 minutes (#P<0.05 vs 60 minutes). B) C4d was also quickly deposited but remained on the cell surface with no significant change from 30 to 60 minutes (***P<0.001 vs control). C) Immunofluorescence microscopy confirmed deposition of C4d on the cell surface. Nuclei are stained with Dapi (blue). Original magnification x600. D) C3b/iC3b was detected on the cell surface at 15 minutes but was not statistically different than control by 60 minutes (*P<0.05, ***P<0.001 vs control). E) C3b/iC3b/C3dg was rapidly deposited and did not change significantly from 15 to 60 minutes (***P<0.001 vs control). C) Immunofluorescence microscopy confirmed deposition of C3d on the cell surface. Nuclei are stained with Dapi (blue). Original magnification x600.

FIGURE 2.. Anti-HLA antibody, C4 fragments, and C3 fragments are cleared from the surface of HMEC-1 cells within 48 hours.

HMEC cells were treated with W6/32 antibody followed and then incubated with 10% normal human serum in culture medium for 2 hours to allow activation complement on the cell surface. They were then washed with PBS and incubated with normal culture medium for another 0 to 48 hours. Complement fragments were then analyzed by flow cytometry. Data are plotted as mean fluorescence intensity (MFI) ± SEM). Anti-HLA antibody (A), C4d (B), C4c (C), C3b/iC3b (D), and C3b/iC3b/C3dg (E) were detected on the cells at time zero, but by 48 hours the levels of these proteins were not significantly different than untreated cells. Endothelial cells also express the complement regulators CD46 (F), CD55 (G), and CD59 (H). Expression of CD55 and CD59 was stable, but levels of CD46 were reduced 24 hours after complement activation on the cells. (*P<0.05, **P<0.01, ***P<0.001 vs control).

FIGURE 3.. C1q or C4 deficiency reduces C4 and C3 deposition on HMEC-1 cells.

HMEC-1 cells were exposed to W6/32 antibody followed by medium with 10% normal human serum, 10% C1q-depleted (C1(−)) serum, or 10% C4-depleted (C4(−)) serum for one hour. Complement fragments were then analyzed by flow cytometry. Data are plotted as mean fluorescence intensity (MFI) ± SEM. A and B) No C4c or C4d was deposited on cells exposed to C1q(−) serum or C4(−) serum (P<0.001 vs control). C) C3b/iC3b was not detected in any condition. D) C3b/iC3b/C3dg was deposited with all serum conditions (*P<0.05, **P<0.01, ***P<0.001 vs control) but significantly reduced with C1q(−) serum and C4(−) serum compared to normal serum (# P<0.001).

FIGURE 4.. The roles of factor I and factor B in regulating complement activation on endothelial cells.

HMEC-1 cells were exposed to W6/32 antibody followed by medium with 10% normal human serum for one hour. A monoclonal antibody that inhibits complement factor I (fI) was added to some of the cells to block factor I mediated cleavage of C3b and C4b. A monoclonal antibody that inhibits complement factor B (fB) was added to some of the cells to block alternative pathway activation. C3 and C4 fragments were analyzed by flow cytometry. Data are plotted as mean fluorescence intensity (MFI) ± SEM (*P<0.05, **P<0.01, ***P<0.001 vs control). No differences between normal serum and serum + fI inhibitor antibody were observed. Serum + fB inhibitor was associated with decreased deposition of C4c compared to fI inhibitor (# P<0.05), decreased C4d deposition compared to normal serum alone (& P<0.05), and decreased C3dg deposition compared to normal serum (@ P<0.01) or fI inhibitor (^ P<0.001).

FIGURE 5.. Antibody mediated complement activation on HMEC-1 cells generates soluble complement fragments.

A) C4a was generated in the supernatants of HMEC-1 cells treated with W6/32 antibody and then exposed to normal serum. C4a was also generated when C1q-deficient (C1q(−)) serum was used (***P<0.001 vs control), although the levels were lower than those seen using normal serum (^P<0.001). B) Ba was generated after treatment of HMEC-1 cells with W6/32 antibody followed by exposure to normal serum (***P<0.001 vs control). Ba levels were slightly lower with normal serum vs C1q(−) serum ($P<0.05). C) sC5b-9 was also generated after treatment of HMEC-1 cells with W6/32 antibody followed by exposure to normal serum. sC5b-9 levels were increased in cells exposed to sera deficient in C1q or C4 (***P<0.001 vs control), although the levels were significantly lower than for cells exposed to normal serum (&P<0.001 vs normal serum).

FIGURE 6.. Representative example of gating used to detect HMEC-1 microvesicles by flow cytometry.

Microvesicles were initially gated by size using forward and side scatter, then by fluorescence of PKH26 to identify microvesicles originating from the labelled HMEC-1 cells.

FIGURE 7.. Anti-HLA antibodies and complement fragments on endothelial microvesicles (MV) generated in vitro.

HMEC-1 cells were labelled with PKH26 fluorescent dye and exposed to W6/32 antibody. Cells were then incubated with medium containing 10% normal human serum or serum deficient in C1q or C4. Microparticles in the supernatants were purified by ultracentrifugation and analyzed by flow cytometry. The number of MVs was standardized using fluorescent counting beads and are presented as MV count per 1000 counting beads ± SEM (*P<0.05, **P<0.01, ***P<0.001). A) MV release was not elicited by exposure to anti-HLA antibody alone. All serum conditions were associated with an increased release of PKH-26 MVs vs control (**P<0.01, ***P<0.001), but complement deficient sera were associated with an increased release of PKH26-positive MVs compared to normal serum (# P<0.01 for C1q(−) serum and & P<0.05 for C4(−) serum). B) MVs with anti-HLA antibody bound were detected in similar quantities after exposure to the various sera (***P<0.001). C) MVs with C4 bound were detected with all serum conditions (*P<0.05, ***P<0.001 vs control), but significantly more were detected after exposure to normal serum than the complement deficient sera (@P<0.001). D) MVs with C3 were also detected with all sera exposures (*P<0.05, ***P<0.001 vs control), but C4(−) serum elicited reduced release of MVs bound with C3 compared to normal serum ($ P<0.001) or C1(−) serum (^ P<0.05).

FIGURE 8.. Antibody-mediated rejection (AbMR) is associated with elevation of plasma complement activation fragments.

Plasma C4a and plasma Ba were both elevated in patients with biopsy-proven AbMR compared to clinically stable renal transplant recipients (**P<0.01, ***P<0.001). These results indicate that classical and alternative pathways are activated in these patients. Plasma sC5b-9 levels were not different between groups.

FIGURE 9.. Representative example of gating used to detect plasma microvesicles by flow cytometry.

Microvesicles were initially gated by size using forward and side scatter. They were next gated by fluorescence of the detection antibody of interest, such as C4 as is shown here.

FIGURE 10.. Anti-HLA antibodies and complement fragments on endothelial microvesicles (MV) in plasma of patients with antibody-mediated rejection (AbMR).

Microvesicles were purified from the plasma of patients with stable kidney transplants or biopsy-proven antibody-mediated rejection. The microparticles were then analyzed by flow cytometry. MV counts are standardized using fluorescent counting beads and presented as MV count per microliter of plasma ± SEM. Total MV and CD41+ MV counts were similar between groups, but MVs with surface-bound IgG, C4, and C3 were significantly lower in the AbMR group compared to control subjects (*P<0.05, **P<0.01, ***P<0.001).