Human Pentraxin 3 Binds to the Complement Regulator C4b-Binding Protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1–3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage

Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

<div><p>Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)⁺PI (Propidium Iodide)⁺ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM⁺AnnV⁺PI⁺ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM^-AnnV⁺PI⁺ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM <i>in vivo</i> in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration.</p></div

Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites

C-reactive protein (CRP) concentrations rise in response to tissue injury or infection. Circulating pentameric CRP (pCRP) localizes to damaged tissue where it leads to complement activation and further tissue damage. In-depth knowledge of the pCRP activation mechanism is essential to develop therapeutic strategies to minimize tissue injury. Here we demonstrate that pCRP by binding to cell-derived microvesicles undergoes a structural change without disrupting the pentameric symmetry (pCRP*). pCRP* constitutes the major CRP species in human-inflamed tissue and allows binding of complement factor 1q (C1q) and activation of the classical complement pathway. pCRP*–microvesicle complexes lead to enhanced recruitment of leukocytes to inflamed tissue. A small-molecule inhibitor of pCRP (1,6-bis(phosphocholine)-hexane), which blocks the pCRP–microvesicle interactions, abrogates these proinflammatory effects. Reducing inflammation-mediated tissue injury by therapeutic inhibition might improve the outcome of myocardial infarction, stroke and other inflammatory conditions