A differential concentration-dependent effect of IVIg on neutrophil functions: relevance for anti-microbial and anti-inflammatory mechanisms

Background Polymorphonuclear neutrophils (PMN) play a key role in host defences against invading microorganisms but can also potentiate detrimental inflammatory reactions in case of excessive or misdirected responses. Intravenous immunoglobulins (IVIg) are used to treat patients with immune deficiencies and, at higher doses, in autoimmune, allergic and systemic inflammatory disorders. Methodology/Principal Findings We used flow cytometry to examine the effects of IVIg on PMN functions and survival, using whole-blood conditions in order to avoid artifacts due to isolation procedures. IVIg at low concentrations induced PMN activation, as reflected by decreased L-selectin and increased CD11b expression at the PMN surface, oxidative burst enhancement, and prolonged cell survival. In contrast, IVIg at higher concentrations inhibited LPS-induced CD11b degranulation and oxidative burst priming, and counteracted LPS-induced PMN lifespan prolongation. Conclusions/Significance IVIg appears to have differential, concentration-dependent effects on PMN, possibly supporting the use of IVIg as either an anti-microbial or an anti-inflammatory agent

Emergence of antibodies endowed with proteolytic activity against High-mobility group box 1 protein (HMGB1) in patients surviving septic shock

International audienceHigh-mobility group box 1 (HMGB1) concentration in serum or plasma has been proposed as an important biological marker in various inflammation-related pathologies. We previously showed that low titer autoantibodies against HMGB1 could emerge during the course of sepsis. Importantly their presence was positively related with patients' survival. In this study, we focused on plasma samples from 2 patients who survived sepsis and exhibited high titer antibodies to HMGB1. These antibodies were proved to be specific for HMGB1 since they did not bind to HMGB2 or to human serum albumin. Following IgG purification, it has shown that both patients secreted HMGB1-hydrolyzing autoantibodies in vitro. These findings suggested that proteolytic antibodies directed against HMGB1 can be produced in patients surviving septic shock

IVIg at low concentrations delays PMN apoptosis.

<p>Whole-blood samples (500 µl) were incubated in 24-well tissue culture plates at 37°C for 20 h with 5% CO₂ with PBS, HSA (1 or 25 mg/ml), or IVIg (1–25 mg/ml) (Panel C) or equimolar concentrations of F(ab′)₂ (Panel D). Cycloheximide (CHX) (10 µg/ml) and LPS (10 ng/ml) were used as proapoptotic and antiapoptotic controls, respectively. Samples (100 µl) were then washed twice in PBS, incubated at 4°C with APC-anti-CD15 for 15 min, and stained with FITC-annexin V for 15 min. After dilution in PBS (500 µl), the samples were incubated with 7-AAD for 15 minutes at room temperature and analysed immediately by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026469#s4" target="_blank">Materials and Methods</a>. Anti-CD15 fluorescence was used to identify PMN as CD15⁺ cells and to gate out other cells, erythrocytes, and debris. A gate was drawn around the PMN population (Panel A). Fluorescence analysis was performed on this gate (Panel B). The combination of FITC-annexin V and 7-AAD was used to distinguish early apoptotic cells (annexin V⁺/7-AAD⁻), from late apoptotic cells (annexin V⁺/7-AAD⁺), necrotic cells (annexin V⁻/7-AAD⁺) and viable cells (unstained) after incubation with PBS, CHX, LPS or IVIg (1–25 mg/ml). Results are expressed as the percentage of total apoptotic cells (early and late apoptotic cells). The proportion of necrotic cells was always below 2%. Values are means ± sem (n = 3). * Significantly different from sample incubated with PBS (p<0.05).</p

IVIg at high concentrations inhibits the LPS-induced prolongation of PMN survival.

<p>Whole-blood samples (500 µl) were incubated in 24-well tissue culture plates at 37°C with 5% CO₂ with PBS, IVIg (1–25 mg/ml) (panel A) or equimolar concentrations of F(ab′)₂ (panel B) for 15 min and then with LPS (10 ng/ml) for 20 h. Samples were then stained with annexin V and 7-AAD as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026469#pone-0026469-g001" target="_blank">Figure 1</a>. Results are expressed as the percentage of total apoptotic cells (early and late apoptotic cells). Values are means ± sem (n = 3). * Significantly different from sample incubated with LPS (p<0.05).</p

IVIg at high concentrations inhibits PMN responses to LPS stimulation.

<p>Panels A, B, C and D: Effect of IVIg and F(ab′)2 fragments on LPS modulation of adhesion molecule expression at the PMN surface. Whole-blood samples were incubated at 37°C for 15 min with PBS, IVIg (1–25 mg/ml) (panel A, C) or equimolar concentrations of F(ab′)₂ (panel B, D) and then with LPS (10 ng/ml) for 30 min. Samples were then stained with PE-anti-human CD11b or anti-L-selectin as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026469#pone-0026469-g001" target="_blank">Figure 1</a>. Results are expressed as mean fluorescence intensity (MFI). Values are means ± sem (n = 3). ° Significantly different from sample incubated with LPS (p<0.05). Panels E and F: Effect of IVIg and F(ab′)₂ fragments on LPS priming of the PMN oxidative burst in response to formyl peptides. Whole-blood samples (500 µl) were pre-treated with hydroethidine HE and incubated at 37°C for 15 min with PBS, IVIg (1–25 mg/ml) (panel E) or equimolar concentrations of F(ab′)₂ (panel F). Samples were then incubated with LPS and fMLP as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026469#pone-0026469-g001" target="_blank">Figure 1</a>. Results are expressed as mean fluorescence intensity (MFI). Values are means ± sem (n = 3). * Significantly different from sample incubated with LPS+fMLP (p<0.05).</p