Figure 2
aHUS serum induces C3 and C5b-9 deposition on microvascular endothelial cells (HMEC-1). (A-B) Endothelial surface area covered by C3 (A) or C5b-9 (B) staining after incubation of unstimulated (resting) or ADP-activated HMEC-1 for 4 hr with serum (diluted 1:2 in test medium) from healthy subjects (Ctr; n = 4) or from aHUS patients (C3: n = 3, 1 with CFH mutation, 1 with anti-CFH antibodies, and 1 without identified mutations/anti-CFH antibodies; C5b-9: n = 7, 3 with CFH mutation, 1 with anti-CFH antibodies, and 3 without identified mutations/anti-CFH antibodies) studied both during the acute phase of the disease (Acute) and at remission (Rem) or from 7 aHUS patients studied in the acute phase only (panel B, C5b-9, acute only, all without identified mutations/anti-CFH antibodies). Data are mean ± standard error (SE). ^P < .01 vs control resting; °°P < .01, °°°P < .05 vs control ADP-activated; &P < .001, &&P < .01, &&&P < .05 vs remission resting. (C,E) Endothelial surface area covered by C3 (C) or C5b-9 (E) staining after incubation of ADP-activated HMEC-1 for 4 hr with serum from aHUS patients studied in remission (C3: n = 25, CFH mutations: n = 10; CFI mutations: n = 4; C3 mutations: n = 3; CFB mutation: n = 1; anti-CFH antibodies: n = 2; without identified mutations/anti-CFH antibodies: n = 5 ; C5b-9: n = 29, CFH mutations or CFHR1/CFH hybrid gene: n = 12; anti-CFH antibodies: n = 2; CFI mutations: n = 4; C3 mutations: n = 3; CFB mutation: n = 1; without identified mutations/anti-CFH antibodies: n = 7), in the presence or not of the complement inhibitor sCR1 (150 μg/mL). Range of deposits induced by control serum (mean ± SE): dotted horizontal areas. (D,F) Representative confocal microscopy images of C3 (D, in green) or C5b-9 (F, in green) staining of ADP-activated HMEC-1 exposed to serum from an healthy subject (Ctr) or an aHUS patient in remission (aHUS) (original magnification ×400). Additional images are shown in supplemental Figure 11. Data are mean ± SE. °P < .001, °°°P < .05 vs control serum; *P < .001, **P < .01 vs aHUS serum without sCR1. Both C3 and C5b-9 deposits were prevented by addition of sCR1 (an inhibitor of all the 3 complement pathways) to patient serum, indicating that the staining was specifically related to complement activation products. mut, mutation.

aHUS serum induces C3 and C5b-9 deposition on microvascular endothelial cells (HMEC-1). (A-B) Endothelial surface area covered by C3 (A) or C5b-9 (B) staining after incubation of unstimulated (resting) or ADP-activated HMEC-1 for 4 hr with serum (diluted 1:2 in test medium) from healthy subjects (Ctr; n = 4) or from aHUS patients (C3: n = 3, 1 with CFH mutation, 1 with anti-CFH antibodies, and 1 without identified mutations/anti-CFH antibodies; C5b-9: n = 7, 3 with CFH mutation, 1 with anti-CFH antibodies, and 3 without identified mutations/anti-CFH antibodies) studied both during the acute phase of the disease (Acute) and at remission (Rem) or from 7 aHUS patients studied in the acute phase only (panel B, C5b-9, acute only, all without identified mutations/anti-CFH antibodies). Data are mean ± standard error (SE). ^P < .01 vs control resting; °°P < .01, °°°P < .05 vs control ADP-activated; &P < .001, &&P < .01, &&&P < .05 vs remission resting. (C,E) Endothelial surface area covered by C3 (C) or C5b-9 (E) staining after incubation of ADP-activated HMEC-1 for 4 hr with serum from aHUS patients studied in remission (C3: n = 25, CFH mutations: n = 10; CFI mutations: n = 4; C3 mutations: n = 3; CFB mutation: n = 1; anti-CFH antibodies: n = 2; without identified mutations/anti-CFH antibodies: n = 5 ; C5b-9: n = 29, CFH mutations or CFHR1/CFH hybrid gene: n = 12; anti-CFH antibodies: n = 2; CFI mutations: n = 4; C3 mutations: n = 3; CFB mutation: n = 1; without identified mutations/anti-CFH antibodies: n = 7), in the presence or not of the complement inhibitor sCR1 (150 μg/mL). Range of deposits induced by control serum (mean ± SE): dotted horizontal areas. (D,F) Representative confocal microscopy images of C3 (D, in green) or C5b-9 (F, in green) staining of ADP-activated HMEC-1 exposed to serum from an healthy subject (Ctr) or an aHUS patient in remission (aHUS) (original magnification ×400). Additional images are shown in supplemental Figure 11. Data are mean ± SE. °P < .001, °°°P < .05 vs control serum; *P < .001, **P < .01 vs aHUS serum without sCR1. Both C3 and C5b-9 deposits were prevented by addition of sCR1 (an inhibitor of all the 3 complement pathways) to patient serum, indicating that the staining was specifically related to complement activation products. mut, mutation.

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