Activation of the human contact system on neutrophil extracellular traps

Abstract

Pattern recognition is an integral part of the innate immune system. The human contact system has been shown to interact with the surface of many bacterial and fungal pathogens, and once activated leads to the generation of antimicrobial peptides and the proinflammatory mediator bradykinin. Here we show that apart from these surfaces also neutrophil extracellular traps (NETs) provide a surface that allows the binding and activation of the contact system. In addition, we present evidence that M1 protein, a streptococcal surface protein, in concert with human fibrinogen triggers polymorphonuclear neutrophils to form NETs.

Fig. 1

Purified DNA binds and activates the contact system. a Transmission electron microscopy of negative-stained DNA (yellow pseudo color) HK (red pseudo color), FXII (green pseudo color) or a mixture of all 3 molecules. b Measurement of PK activity after incubation of plasma with purified DNA (10 μg/ml). As a control, DNA was pretreated with 200 mU DNase I for 30 min at 37°C before incubating in plasma. c BK release after incubation of DNA (10 or 100 μg/ml) and the DNase I-pretreated controls in human plasma for 15 min at 37°C. All Data represent the mean + SD (n = 3) of 1 representative of 3 independently performed experiments. ** p < 0.01; *** p < 0.0001. Scale bar = 10 nm.

Fig. 2

NET-DNA binds contact system proteins, which is followed by activation of the contact system. a Light and scanning electron microscopy of nonactivated PMNs and PMNs activated with 100 mU/ml GO for 60 min. As a control, PMNs were incubated with 200 mU DNase I for 30 min after activation. Scale bar = 10 μm. b Transmission electron microscopy of nonactivated PMNs as well as PMNs stimulated with 100 mU/ml GO and incubated with gold-labeled HK (small gold particles) and FXII (larger gold particles). Scale bar = 1 μm. A higher magnification of 2 areas (marked with * and **) showing the binding of gold-labeled contact factors to NETs is shown on the lower right (Scale bar = 100 nm). Arrowheads point to gold-labeled proteins. PMNs incubated with 200 mU DNase I after stimulation and before adding to gold-labeled proteins are shown on the lower left. c PK activity after incubation of GO- and IL-8-stimulated PMNs in human plasma. Results shown are a representative of at least 3 experiments with PMNs from 3 different donors.

Fig. 3

NET formation after stimulating the PMNs with M1 protein/fibrinogen complexes. a, b PMNs were incubated with M1 protein/fibrinogen complexes for 60 min and investigated by light and scanning electron microscopy. c, d For light microscopy, the PMNs were stained with DAPI. As a control, the stimulated cells were treated with 200 mU DNase I for 30 min. Arrowheads point to PMNs. Scale bar = 10 μm.