Synergistic activation of PMN with aPL and TLR2 ligation.

<p>Human PMN (2×10⁵ cells per well) were incubated with the TLR2 ligand Pam₃Cys (100 ng/ml, 1 µg/ml, 10 µg/ml). A) Production of radical oxygen species was determined after 170 minutes. B) Phagocytosis rate was analysed defining percentage of PMN taken up PE-labelled microspheres. C) Shedding of L-Selectin was determined by surface staining of expressed molecules. D) Supernatants of stimulated PMN culture were harvested and an IL-8 specific ELISA was performed after 2 hours (left panel) or 6 hours (right panel) of incubation. Data shown are from one representative experiment out of 3 independent with 2 replicates per group. *Indicates significant difference of Pam₃Cys-stimulated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p

A Role for Toll-Like Receptor Mediated Signals in Neutrophils in the Pathogenesis of the Anti-Phospholipid Syndrome

<div><p>The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS.</p> </div

LPS stimulation can be enhanced by facilitating binding to CD14.

<p>Human PMN (2×10⁵ cells per well) were incubated with LPS (100 ng/ml) with or without the addition of aPL (10 µg/ml) or LPS-binding protein (LBP, 10 ng/ml). ROS formation was measured in a fluorescence reading device. Detection of specific fluorescence index (SFI) over time was calculated by subtraction of the background fluorescence of labelled cells incubated in medium. Data from one representative experiment with two replicates is shown.</p

PMN release IL-8 upon stimulation with aPL and LPS.

<p>PMN (2×10⁵ cells per well) were incubated with medium, aPL or hIgG (10 µg/ml) in the presence or absence of LPS. Supernatants were harvested and an IL-8 specific ELISA was performed after A) 2 hours or B) 6 hours of incubation (37°C). Data shown are from one representative experiment out of 3 independent with 2 replicates per group. * Indicates significant difference of LPS-stimulated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p

aPL increase oxidative burst activity in human PMN.

<p>Human PMN (2×10⁵ cells per well) were incubated with medium or LPS in the presence or absence of aPL or human IgG (10 µg/ml). Production of radical oxygen species was determined by oxidation of dichlorofluorescein diacetate to DCF and measured in a fluorescence reading device. A) Detection of specific fluorescence index (SFI) over time was calculated by subtraction of the background fluorescence of labelled cells incubated in medium. LPS stimulation was performed using 100 ng/ml. B) Titration of LPS concentrations (1 ng/ml, 10 ng/ml, 100 ng/ml) as indicated was analysed after 170 minutes as fold induction. Data shown are from one representative experiment out of 3 independent with 2 replicates per group. *Indicates significant difference of aPL treated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p

aPL stimulate PMN for phagocytosis.

<p>Human PMN (2×10⁵ cells per well) were stimulated with medium, aPL or hIgG in the presence or absence of titrated LPS as indicated and analyzed by FACS. A) For quantification of phagocytic activity PE-labelled microspheres were added to the culture. B) Expression of CD11b was determined via surface staining. Data from one representative experiment out of 3 independent with two replicates as percent phagocytosis or percent CD11b⁺ cells is shown. *Indicates significant difference of LPS-stimulated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p

Synergistic induction of L-Selectin shedding mediated via aPL and LPS.

<p>PMN (2×10⁵ cells per well) were incubated with medium, aPL or hIgG in the presence or absence of titrated LPS. Shedding of L-Selectin was determined by surface staining of expressed molecules and analyzed by flow cytometry. Data shown are from one representative experiment out of 3 independent with 2 replicates per group. *Indicates significant difference of LPS-stimulated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p