Margreet Hart | PubMed Health

PubMed2006

Mannan-binding lectin (MBL)-mediated opsonization is enhanced by the alternative pathway amplification loop.

The complement system is a humoral effector in the innate immune system. Three activation pathways exist in the complement system, known as the classical pathway, the lectin pathway and the alternative pathway. Dysfunction of lectin pathway activation is caused by MBL deficiency. MBL deficiency in a cohort of healthy Caucasian blood bank donors was investigated with MBL genotyping and MBL plasma concentration. Recognition of the yeast-derived zymosan by MBL was investigated with Western blot. The involvement of the alternative pathway amplification loop in enhancing MBL-mediated opsonization of zymosan was investigated in a novel opsonophagocytosis assay for flow cytometry. Sera deficient for MBL, factor D or properdin were tested, and purified MBL, factor D or properdin were used to recover opsonization. The optimal receiver-operator characteristic (ROC) cut-off value for dividing the Caucasian cohort in MBL-sufficient and MBL-deficient was calculated at 0.7 microg/ml. Thirty-eight percent of the group had concentrations below 0.7 microg/ml. Zymosan eluates opsonized with MBL-sufficient sera contain high oligomers of MBL, while eluates from MBL-deficient donors contained hardly any MBL. The MBL-, factor D- and properdin-deficient sera showed reduced opsonophagocytosis by human control neutrophils, as compared to normal MBL-sufficient sera. This reduction in opsonization was restored to normal levels by addition of purified MBL, factor D and properdin. The absence of opsonization in the factor D- and properdin-deficient sera, but presence in normal serum after blocking with anti-C1q-F(ab)2 and anti-MBL-F(ab)2, demonstrates the involvement of the amplification loop in MBL-initiated zymosan opsonization, even at very low serum concentrations (up to 3%, v/v). In conclusion, our data demonstrate that the MBL-mediated route of complement activation depends on the alternative pathway amplification loop for optimal opsonization of zymosan.

Cohort Studies↗

PubMed2005

Complexes between C1q and C3 or C4: novel and specific markers for classical complement pathway activation.

Classical pathway activation is often assessed by measuring circulating levels of activated C4. However, this parameter does not discriminate between activation through the classical or the lectin pathway. We hypothesized that during classical pathway activation, complexes are formed between C1q and activated C4 or C3. Using ELISA, we investigated whether such complexes constitute specific markers for classical pathway activation. In vitro, C1q-C3d/C4d complexes were generated upon incubation of normal recalcified plasma with aggregated IgG or an anti-C1q mAb that activates C1 (mAb anti-C1q-130). In contrast, during incubation with C1s or trypsin, C1q-C3d/C4d complexes were not generated, which excludes an innocent bystander effect. Additionally, C1q-C3d/C4d complexes were not generated during activation of the alternative or the lectin pathway. Repeated freezing and thawing did not influence levels of C1q-C3d/C4d complexes in recalcified plasma. To measure C1q-complement complexes in plasma samples, we separated unbound complement proteins from C1q-C3d/C4d complexes in the samples prior to testing with ELISA. In samples from patients undergoing cardiopulmonary bypass surgery or suffering from rheumatoid arthritis, we found higher levels of C1q-C4 complexes than in samples from healthy individuals. We conclude that complexes between C1q and C4 or C3 are specific markers of classical complement pathway activation.

Arthritis, Rheumatoid↗