Properdin: New roles in pattern recognition and target clearance

Abstract

Properdin was first described over 50 years ago by Louis Pillemer and his collaborators as a vital component of an antibody-independent complement activation pathway. In the 1970s properdin was shown to be a stabilizing component of the alternative pathway convertases, the central enzymes of the complement cascade. Recently we have reported that properdin can also bind to target cells and microbes, provide a platform for convertase assembly and function, and promote target phagocytosis. Evidence is emerging that suggests that properdin interacts with a network of target ligands, phagocyte receptors, and serum regulators. Here we review the new findings and their possible implications.

Fig. 1. Two models are presented for the initiation and assembly of the AP C3 convertase on a target surface

(A) Assembly is initiated via nascent C3b (C3b*) which is generated by non-specific fluid phase activation of C3 and then covalently binds to nearby surfaces. (B) Assembly is initiated via properdin, which binds targets directly and then recruits fluid phase C3b (nascent C3b is not required). In each case, the Ba fragment is released when factor B is cleaved by factor D. (Modified from (Spitzer et al., 2007); Copyright 2007 The American Association of Immunologists, Inc.).

Fig. 2. Properdin directs AP C3 convertase assembly and function on Neisseria surfaces

(A) N. gonorrhoeae was incubated with properdin (black line) or buffer alone (gray shaded area) and analyzed by FACS with anti-P mAb. (B) N. gonorrhoeae was incubated with properdin and C3b (black line), or buffer alone (gray shaded area) and analyzed by FACS with an anti-C3d mAb. Bacteria incubated with C3b alone (gray line) served as a negative control. (C) N. gonorrhoeae-C3bP complexes generated as in (B) (black line) or bacteria alone (dark gray line) were incubated with factor B plus factor D and analyzed by FACS with anti-Bb mAb. Bacteria-C3bP incubated with buffer served as a negative control (gray shaded area). (D) N. gonorrhoeae-P complexes generated as in (A) (black shaded area) or N. gonorrhoeae alone (dark gray line) were incubated in 5% properdin-deficient human serum under conditions that permit AP activation only, and were analyzed by FACS with an anti-C3d mAb. N. gonorrhoeae-P complexes incubated in buffer served as a negative control (gray shaded area). (Figure from reference (Spitzer et al., 2007); Copyright 2007 The American Association of Immunologists, Inc.).

Fig. 3. Properdin bound to apoptotic T cells promotes their phagocytosis by macrophages

Apoptotic T cells were either pre-incubated with purified properdin (d) and (e) or left untreated (a), (b) and (c). T cells were washed and then added to human macrophages grown on slides in media containing normal human serum (NHS) or heat-inactivated human serum (ΔHS). Slides with cell mixtures were incubated for 1 hr, washed, stained with Hematoxylin and analyzed microscopically. As seen, macrophages ingested apoptotic T cells more readily than non-apoptotic control cells [Compare (a) and (b)] and the uptake of apoptotic T cells by macrophages was increased by about 100% in the presence of normal human serum [Compare (b) and (c)]. Uptake was greatest when apoptotic T cells were first pretreated with properdin [Compare (b) and (d)]. Pretreatment of apoptotic T cells with properdin alone increased their ingestion even in the absence of complement activation substantially [Compare (c) and (e)]. Results shown are representative of four independent experiments and were confirmed by a quantitative FACS assay (Kemper et al., 2008). Red *, indicates ingested T cells. Original magnification, × 100. Bar in panel (e) = 50 µm. (Figure derived from (Kemper et al., 2008)).

Fig. 4. Putative thrombospondin repeat 2 GAG-binding face

The conserved tryptophan residues (orange) and disulfide bridges (yellow) position the two arginine residues (blue) ~9 Angstroms apart. The positively-charged arginine side chains have been proposed to interact electrostatically with a similarly positioned pair of negatively-charged GAG sulfate moieties (Tossavainen et al., 2006). The structure is of thrombospondin repeat 2 (Tossavainen et al., 2006) derived with the Protein Data Bank accession code 1LSL coordinates using the program PyMole (DeLano, 2002).

Fig. 5

Properdin released from activated neutrophils binds to “dangerous self” (apoptotic cells) and “dangerous non-self” (microbial pathogens), marking them for complement activation and phagocytosis.

Fig. 6. Properdin, C1q and MBL recognize distinct target cell populations

(A) Analysis of properdin, C1q and MBL binding to either apoptotic or necrotic T cells (shown as mean ± s.d.) of three separate experiments. Statistical difference between the binding of properdin and C1q and properdin and MBL to either apoptotic or necrotic T cells was p < 0.005 in all cases as determined by the paired Student’s t test. (B) Summary/model of the contributions of the three complement activation pathways in the recognition of human apoptotic and necrotic primary CD4⁺ T cells. (Figure derived from (Kemper et al., 2008)).