Figure 1.
Complement activation and adherence of complement proteins to Borrelia spirochetes incubated in hirudin plasma.
A) The ability of the Borrelia strains B. afzelii K78 and B.gariini LU59, resistant and sensitive to complement activation, respectively, to activate complement in hirudin plasma (n = 8) was assessed by analyzing the generation of C3a and sC5b-9 using ELISA. Data are normalized against plasma incubated without bacteria and are presented as means ± SEM. B) Borrelia were incubated with hirudin plasma (n = 6), and bound proteins were analyzed using an in-house-developed particle ELISA. Data are shown as absorbance values of bacteria incubated in plasma, normalized against bacteria incubated in PBSCa2+, and are presented as means ± SEM. C) Quantification of bound C3-fragments (using the β-chain that is not proteolytically cleaved) and FH. Results are presented normalized to either K78 or LU59 as mean of six Western blots. D) Silver stained SDS–PAGE of B. afzelii K78 and B.gariini LU59 non-opsonized (PBS) or opsonised (Pl). In addition, C3 and its fragments (E) and FH (F) bound to bacteria incubated with plasma (Pl) were visualized using Western blot with bacteria incubated in PBSCa2+ as controls, and purified C3, C3b and iC3b or FH as references. Insert: loading control, consisting of a 20kDa Borrelia protein that was detected on the membranes after detection with antibodies.
Figure 2.
Importance of complement activation for efficient phagocytosis.
FITC-labeled B. afzelii K78 and B.garinii LU59 were incubated in hirudin blood to study phagocytosis by measuring the mean fluorescence intensity (MFI) for granulocytes. A) Phagocytosis of each bacterial strain was monitored for 30 min. Results are presented as mean ± SEM of two observations. B) The importance of complement activation in phagocytosis was analyzed by incubating Borrelia spirochetes in hirudin blood (n = 3) with the addition of complement inhibitors acting at different stages in the activation cascade. Compstatin selectively inhibits the proteolytic activation of C3 and a C5a receptor antagonist (C5aRa) that blocks the C5a-mediated up-regulation of granulocytes and monocytes. A scrambled peptide of C5aRa was used as a control. The mean fluorescence intensity (MFI) for each bacterial strain incubated in hirudin blood without complement inhibitors was set to 100%. EDTA: ethylene diaminetetraacetic acid. C5aRa: C5a receptor antagonist.
Figure 3.
Cytokine and chemokine levels in plasma as a response to incubation with Borrelia spirochetes.
Spirochetes were incubated in whole blood (n = 3) in duplicate for 4 h, and the cytokine/chemokine production was then measured using a Luminex-based assay. Results are normalized against the values for plasma without the addition of bacteria. (A) The x-fold increase in the panel of cytokines and chemokines analyzed in this report showed no difference between the complement-resistant B. afzelii K78 and -sensitive B. garinii LU59. (B and C) The importance of complement activation was further investigated using two complement inhibitors: compstatin, which selectively inhibits the proteolytic activation of C3, and eculizumab, an anti-C5 antibody blocking the cleavage of C5 and therefore further complement activation and C5a mediated signaling. Data are presented as means ± SEM.