Continual low-level activation of the classical complement pathway - PubMed (original) (raw)

Continual low-level activation of the classical complement pathway

A P Manderson et al. J Exp Med. 2001.

Abstract

There is evidence that the classical complement pathway may be activated via a "C1-tickover" mechanism, analogous to the C3-tickover of the alternative pathway. We have quantitated and characterized this pathway of complement activation. Analysis of freshly collected mouse and human plasma revealed that spontaneous C3 activation rapidly occurred with the generation of C3 fragments in the plasma. By the use of complement- and Ig-deficient mice it was found that C1q, C4, C2, and plasma Ig were all required for this spontaneous C3 activation, with the alternative complement pathway further amplifying C3 fragment generation. Study of plasma from a human with C1q deficiency before and after therapeutic C1q infusion confirmed the existence of a similar pathway for complement activation in humans. Elevated levels of plasma C3 were detected in mice deficient in complement components required for activation of either the classical or alternative complement pathways, supporting the hypothesis that there is continuous complement activation and C3 consumption through both these pathways in vivo. Blood stasis was found to stimulate C3 activation by classical pathway tick-over. This antigen-independent mechanism for classical pathway activation may augment activation of the complement system at sites of inflammation and infarction.

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Figures

Figure 1

Detection of C3 fragments in mouse and human blood following incubation ex vivo at 37°C. (A) The level of C3 fragments in mouse blood collected directly into heparin and EDTA (T0) or collected into heparin and incubated at 37°C for 10 min before addition of EDTA (T10). The histograms represent the amount of C3 fragments detected on B220+ B cells following incubation of splenic lymphocytes with T0 plasma (filled histogram) or T10 plasma (open histogram), as measured by immunofluorescence flow cytometry. (B) Western blot analysis of C3 fragments in mouse plasma, as collected in A. C3 was immunoprecipitated from plasma samples with polyclonal anti–mouse C3 antibody conjugated Sepharose beads, and the Western blot was probed with biotin-labeled polyclonal anti–mouse C3 Ab and developed using streptavidin-HRP. Position of intact C3α and C3β chains and the C3dg fragments, that bind to CR1/2 on B cells (A), is indicated. (C) The level of C3 fragments in human blood collected directly into heparin and EDTA (T0) or into heparin and incubated at 37°C for 30 min before addition of EDTA (T30). The histograms represent the amount of C3 (solid line) or C3d (broken line) fragments detected on Raji cells after incubation with T0 plasma (filled histogram) or T30 plasma (open histograms), as measured by immunofluorescence flow cytometry. (D) The rate of formation of C3 fragments in blood from C57BL/6 mice collected into either heparin (□) or hirudin (▪) is depicted, as measured by the binding of C3 fragments to B220+ spleen cells using immunofluorescence flow cytometry, as shown in A. Error bars represent ± SD of means of triplicate assays.

Figure 1

Figure 2

Spontaneous formation of C3 fragments in mouse plasma requires the classical complement pathway. The rate of formation of C3 fragments in heparin-containing plasma from C57BL/6 mice (▪), and C1qa−/− mice (□) is depicted, as measured by the binding of C3 fragments to B220+ spleen cells using immunofluorescence flow cytometry, as shown in Fig. 1 A. Error bars represent ± SD of means of triplicate assays.

Figure 3

Spontaneous formation of C3 fragments in human plasma is C1q dependent. (A) Plasma from normal and C1q-deficient patients, containing heparin and EDTA, was recalcified and incubated at 37°C for up to 30 min. To some C1q-deficient plasma exogenous purified human C1q (20 μg/ml) was added before incubation. The rate of formation of C3 fragments in normal (▴, n = 8), C1q-deficient (□, n = 3), and reconstituted C1q-deficient (▪, n = 3) plasma is depicted, as measured by the binding of C3 fragments to Raji cells using immunofluorescence flow cytometry, as in Fig. 1 C. (B) The rate of formation of C3 fragments in plasma from normal individuals (▴, n = 8), and a C1q-deficient patient before (□) and immediately after (▪) infusion of Octaplas. All results are represented as means ± SD.

Figure 3

Figure 4

Formation of C3 fragments in mouse blood in vivo after vascular stasis requires the classical complement pathway. After injection of heparin (20 U) mice were killed by CO2 exposure, blood was collected from the mice at appropriate times after death of the mice, and the plasma analyzed for C3 fragments by immunofluorescence flow cytometry, as shown in Fig. 1 A. The rate of C3 fragment formation was examined in the plasma of C57BL/6 mice (▪) and C1qa−/− mice (□). Error bars represent ± SD of mean of triplicate assays. Data are representative of two separate experiments.

Figure 5

Elevated plasma C3 levels in mice deficient in molecules essential for activation of both the classical and alternative complement pathways. Blood was collected from mice directly into EDTA and the level of plasma C3 determined by rocket immunoelectrophoresis. The plasma C3 levels from individual male C57BL/6 (▪, n = 14), Bf−/− (○, n = 20), C1qa−/− (♦, n = 12), H2-Bf,C2−/− (□, n = 9), and RAG−/− (▴, n = 12) mice is depicted, with horizontal bars denoting means. Similar trends in C3 levels were observed in female mice (data not shown).

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Continual low-level activation of the classical complement pathway - PubMed (original) (raw)